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CANCER RESEARCH CENTER, UNIVERSITY & UNIVERSITY HOSPITAL of SALAMANCA (SPAIN)

PLASMA CELLS (MORMAL AND NEOPLASTIC). CANCER RESEARCH CENTER, UNIVERSITY & UNIVERSITY HOSPITAL of SALAMANCA (SPAIN) Multicolor Immunophenotyping: Standardization and Applications March 9-11, 2012 TMH, Mumbay (India). MRD MONITORING IN HAEMATOLOGICAL MALIGNANCIES. Tumor micro-environment.

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CANCER RESEARCH CENTER, UNIVERSITY & UNIVERSITY HOSPITAL of SALAMANCA (SPAIN)

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

  2. MRD MONITORING IN HAEMATOLOGICAL MALIGNANCIES Tumor micro-environment In vivo drug kinetics Tumor cell features Treatment compliance - CML - APL - Childhood ALL - … Therapeutic decisions Therapy Morphology, Cytogenetics Southern-Blot, FCM DNA aneuploidy Resistance 1011 1010 10-2 Complete remission 109 10-3 Sensitivity F.I.S.H 108 10-4 Immunological CR Molecular CR 107 10-5 Flow cytometry N. of tumor cells 106 10-6 P.C.R. 105 104 103 102 101 100

  3. MRD TECHNIQUES FOR HAEMATOPOIETIC MALIGNANCIES FCM immunophenotyping PCR/RT-PCR analyses (sensitivity) (sensitivity) Disease category LAIP sIgk/sIgl Junctional Reg Chromosomal or TCRVb Ig/TCR genes aberrations (10-3-10-4) (10-2-10-3) (10-3-10-6) (10-4-10-6) 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% From: Szczepanski, Orfao et al, Lancet Oncol, 2001; 2: 409-417

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

  5. CD138 PerCP/Cy5 -> TRANSFORMED SSC -> 0 1 2 3 4 10 10 10 10 10 CD38 FITC -> 0 1 2 3 4 10 10 10 10 10 CD38 FITC -> Plasma cell quantification (BM infiltration) • Morphological PC count : • - area of BM smear • - infiltration pattern Variability • Immunophenotyping: • - precise identification by CD38/CD138 Co-expression of CD38/CD138 High-intensity Specific expression = + TRANSFORMED SSC -> 0 1 2 3 4 10 10 10 10 10 CD138 PerCP/Cy5-> • - but…..diluted sample  lower numbers

  6. Correlation between Immunophenotyping & Morphology: 100 100 2 2 R R = 0,4 = 0,39 W W W W W W W W W W W W W W W W W W W W W W 75 75 W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W Proportion of plasma cell by flow cytometry Proportion of plasma cell by flow cytometry W W W W W W W W W W W W W W 50 50 W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W 25 25 W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W W 0 0 0 0 25 25 50 50 75 75 100 100 Proportion of plasma cell by morphology Proportion of plasma cell by morphology • Prognostic influence of the number of BMPC:

  7. BACKGROUND • High-dose chemotherapy and Novel Drugs • Complete remission (CR): 25%-75% • Relapse-free survival (RFS) at 5 year: 40%-70% • 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. Pan-leuc. Ag: CD45+...20-40% B-cell-associated Ags: CD19+..........3-8% CD20+..........2-25% CD22+..........20-30% CD10+..........6-20% HLA-DR+het….. 10% CD23+.......... 0% FMC7+......... 0% PC-associated Ags: CD38++/+++.... 100% CD138 +....... 98% MM PLASMA CELL HPC-associated Ags: CD34+..........0% CD117 +......27% CyIg+ Co-stimulatory Ags: CD28+/++....... 30-40% CD40 +....... 100% CD81,CD27-/lo.40-50% CD52……………….10-50% Adhesion molecules: CD56+/++.....60-70% b1/b2 integrins 98% CD54….……….50-70% Myeloid-associated Ags: CD13+......... 28% CD33 +/++..... 24% Rawstron et al, Haematologica 2008

  11. Incidence of aberrant phenotypes in PC from MM 96% 100 92% 80% 90 73% 80 70 60% 60 50 36% 32% 40 30 17% 18% 20 10 0 CD19 CD38 CD45 CD56 CD28 CD33 CD117 CD20 Asynchronous expression Infra-expression Over-expression Mateo et al. J Clin Oncol; 2008;26:2737

  12. MOST USEFUL ANTIGENS FOR THE DETECTION OF ABERRANT PC IN MM Antigen Expression % MM with Requirement for Normal Altered 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 + -/dim N.A. Recommended CD27 ++ -/dim 40-50% Recommended N.A.: not analyzed/not reported. Rawstron et al, EMN consensus, Haematologica, 2008

  13. 60 120 80 100 50 100 80 CD126 CD86 CD95 CD56 60 40 80 60 p0.001 p0.001 p0.001 p=0.72 40 30 60 40 20 40 20 20 20 10 Normal PC Clonal PC Normal PC Clonal PC 0 0 0 0 Normal PC Clonal PC Normal PC Clonal PC 10000 10000 3000 4000 8000 8000 3000 2000 6000 6000 2000 4000 4000 1000 1000 2000 2000 0 0 0 0 Normal PC Clonal PC Clonal PC Normal PC Normal PC Clonal PC Normal PC Clonal PC Immmunophenotype of normalvsclonal PC % of positive PC CD38 HLA-I 2-microglobulin CD40 p=0.002 p=0.21 p=0.005 Mean FL Intensity p0.001 Perez-Andres et al, Leukemia, 2005; Perez-Andres et al, Int J Cancer, 2009

  14. MGUS vs MM: IMMUNOPHENOTYPIC PANELS N.of PBAMCAFITCPEPerCPCy5.5APCPE-Cy7APCH7 colors PO 3 CD38 CD56 CD19 4 CD38 CD56 CD19 CD45 6 CD38 CD56CD45 cyIgk CD19 cyIgl 8 CD45 CD138 CD38 CD56 CD117 cyIgk CD19 cyIgl

  15. Characterization markers B cell homing Normal B lymphopoiesis CD11a, CD11c, CD31, CD49d, CD62L, CXCR5, CCR6, CD303 CD10, CD20, CD22 CD24, CD27, CD38 CD39, CD43, CD63 CD81, CD95, CD138 Bcl-2, HLA-DR, CyIg Known to differentiate CD13, CD15, CD28, CD33, CD56, CD45, CD117, b2M 7 informative markers

  16. NORMAL vs NEOPLASTIC PC: IMMUNOPHENOTYPIC PANELS N.of PBHV500FITCPEPerCPCy5.5APCPE-Cy7Alexa700 colors HV450POAPC-H7 3 CD38 CD56 CD19 4 CD38 CD56 CD19 CD45 6 cyIgL cyIgk CD19 CD45 CD56 CD38 CD138 CD117 CD19 CD45 CD56 CD38 8 CD45 CD138 cyIgL cyIgk CD138 CD117 CD56 CD38

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

  18. EuroFlow PANEL: Plasma cell dyscrasias Abnormal PC detection /classification in MGUS & MM (APS view) Normal PCs Abnormal PCs Most informative markers Responsible scientist: J.Flores

  19. PCD panel: Backbone markers Responsible scientists: Juan Flores

  20. PCD panel: Backbone markers Responsible scientists: Juan Flores

  21. Normal PC Clonal PC CD19-PcpCy5 C D 1 9 CD138 PerCP/Cy5 -> C C D CD45-APC P 0 1 2 3 4 10 10 10 10 10 5 A CD56-PE TRANSFORMED SSC -> 6 5 CD38 FITC -> P 4 E D C 0 1 2 3 4 10 10 10 10 10 CD38 FITC -> MONOCLONAL GAMMOPATHIES: IDENTIFICATION OF CLONAL PLASMA CELLS CD38-FITC gated PC T-SSC CD138-PerCP/Cy5.5 CD38-FITC Perez-Andres, J Biol Reg, 2004

  22. MRD TECHNIQUES FOR HAEMATOPOIETIC MALIGNANCIES FCM immunophenotyping PCR/RT-PCR analyses (sensitivity) (sensitivity) Disease category LAIP sIgk/sIgl Junctional Reg Chromosomal or TCRVb Ig/TCR genes aberrations (10-3-10-4) (10-2-10-3) (10-3-10-6) (10-4-10-6) 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 50% 50% 0,35% 10 0 10 1 10 2 10 3 10 4 10 10 10 10 10 0 1 2 3 4 0 256 512 768 1024 CD38 -> CD38 -> FSC-Height -> JR67635.001 JR67635.002 JR67635.002 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 CD38 -> CD45 -> CD38 -> JR67635.002 JR67635.002 JR67635.002

  24. Risk of MGUS transformation2 Cases with predominantly (>95%) CD19- ve PC.... High risk (26% transformed in 31 months) 2. Rawstron A, Blood 2003, 102, 36 a (Abstr.116) Differential diagnosis MGUS MM Clonal Poly-Clonal versus Only 20% of MM patients showed poly-PC and constantly <5% (median: 0.25%)1 >5% poly-PC: 98% MGUS The most powerful single criteria for differential diagnosis (even in stage I MM) 1. Ocqueteau M, Am J Pathol 1998, 152: 1655

  25. CD56 & CD117 1.0 1.0 CD56+CD28- n= 1116 41 m +/+ or -/- n=266 36 m CD56-CD28+ n=116 29 m CD56+CD117+ n= 130 45 m +/- or +/- n=267 36 m CD56-CD117- n=186 31 m .9 .9 .8 .8 .7 .7 .6 .6 .5 .5 .4 .4 .3 .3 .2 p=0.01 p=0.001 .2 .1 .1 Months from diagnosis 0.0 0.0 0 6 12 18 24 30 36 42 48 54 60 66 72 0 6 12 18 24 30 36 42 48 54 60 66 72 Months from diagnosis Months from diagnosis CD28 & CD117 CD28-CD117+ n= 142 45 m +/- or +/- n=327 37 m CD28+CD117- n=114 29 m 1.0 .9 .8 .7 .6 PFS .5 .4 .3 .2 p=0.0005 .1 0.0 0 6 12 18 24 30 36 42 48 54 60 66 72 Prognostic influence of phenotypic profiles CD56 & CD28 PFS

  26. Kyle & Alexanian 1980a. Estimated incidence: 15% of newly diagnosed MMb. Estimated Risk of progression: 10% per yearc vs. 1% on MGUS IntroductionSmoldering Multiple Myeloma a Kyle 1980, Alexanian 1980; bRajkumar 05; cKyle 05

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

  28. Flow Cytometry Results % Total PC in BM* 2.8 (0.9-22.0) % of aPC / BMPC compartment 97 (35-100) < 95% aPC / BMPC 36 (40%) > 95% aPC / BMPC 53 (60%) * Median (range)

  29. Impact of % aPC/BMPC by FC on Progression Free Survival Median Not reached 1,0 0,8 0,6 Median 40 months % of Progression Free Survival 0,4 0,2 p=0.0000 0,0 0 20 40 60 80 100 120 Months <95% aPC/BMPC n= 36 (4 progressions) 37% 92% >95% aPC/BMPC n= 53 (34 progressions) 5 years

  30. Multivariate analysis for PFS p HR % a PC /BMPC0.004 4.9 Immunoparesis 0.007 2.6

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

  32. Impact of prognostic index on PFS Median not reached 1,0 0,8 Median 75 months 0,6 % of Progression Free Survival 0,4 0,2 Median 20 months p= 0.003 0,0 0 20 40 60 80 100 120 Months 91% 58% No adverse factors n= 32 (3 progressions) >95% aPC/BMPC or paresis n= 27 (12 progressions) 18% >95% aPC/BMPC + paresis n= 27 (22 progressions) 5 years

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

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

  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. Impact of CR in the ASCT setting 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) - Significant correlation between maximal response and outcome prospective studies (<0.00001) & rétrospective studies (< 0.00001) Is it the same CR & VGPR ??

  37. PD, n=25 CR and nCR are not the same: “depth of response” PETHEMA-GEM 2000: Outcome according to post-transplant response EFS OS 1,0 CR vs nCR P=0.01 1,0 CR vs nCR or PR P<10-5 CR vs PR P<10-6 0,9 0,9 nCR vs PR P=0.07 nCR vs PR P=0.04 0,8 0,8 0,7 0,7 0,6 0,6 0,5 Cumulative Proportion Event Free Surviving Cumulative Proportion Surviving 0,5 0,4 0,4 0,3 0,3 0,2 0,1 0,2 0,0 0,1 0 12 24 36 48 60 72 84 96 0 12 24 36 48 60 72 84 96 Months from diagnosis Months from diagnosis CR, n=278 nCR, n=124 PR, n=280 Lahuerta et al. JCO 2008;26:5775–5782

  38. CR correlates with long-term PFS and OS in elderly patients treated with novel agents OS PFS CR CR VGPR VGPR PR PR P<0.001 P<0.001 • 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 Gay et al. Blood 2011

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

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

  41. Correlation between immunophenotyping & electrophoretic responses at three months post-ASCT (GEM 2000 trial, n=295) Partial Response Complete remission EF + n=108 P IFx + n=40 (21%) IFx - n=147 (79%) MRD evaluation #MM-PC 0.76 0.21 0.1 <.001 *MRD+ cases 86% 62% 36% <.001 # % N-PC/BMPC 44 73 85 <.001 #Results expressed as medians *≥0.001% MM-PC Paiva et al; Blood. 2008, 112: 4017-4023

  42. GEM2000 & GEM2005: Impact on survival of achieving an Immunophenotypic CR after HDT/ASCT independent of the induction regimen PFS OS 100 100 P =.132 80 P =.640 80 P =.091 60 60 40 40 P =.802 20 20 P <.001 P <.001 0 0 0 20 40 60 80 100 120 0 25 50 75 100 125 GEM2000 GEM2005 (<65y) Paiva et al. Blood 2010. 116; abstr 1910

  43. Kinetics of response: conventional CR vs. immunophenotypic response (IR) Paiva et al, JCO, 2011 Post-Induction Maintenance (months) 1 2 3 4 5 6 7 8 9 10 11 12 // 16 // 20 // 24 // 28 // 32 // 36 // 40 // 44 // 48 Patient no. 1 IgG ---------------------------------------------------------------------- 2 B-J ------------------------------------------------------------------------------------------ 3 IgG ----------------------------------------------------------------------------------------------- 4 IgA ------------------------------------------------------------------------------------------------------ 5 IgG --------------------------------------------------------------------------------------------------------------- 6 B-J ------------------------------------------------------------------------------------------ P ------------------------------- 7 IgG ---------------------------------------------------------------------------------------------------------------------------------- 8 B-J -------------------------------- P ---------------------------- 9 IgG ------------------------------------------------------------------- 10 B-J -------------------------------------------------------------------- 11 IgG ----------------------------------------------------------------------- 12 IgA ------------------------------------------- P ------------------------------ 13 IgA ------------------------------------------- P --------------------------------- 14 IgG -------------------------------------------------------------------------------- 15 IgA --------------------------------------------------------------------------------- 16 IgG -------------------------------------------------------------------------------------------- 17 IgG ------------------------------------------------------------------------------- P ------- 18 IgG ------------------------------------------------------------------------------------------ 19 IgA --------------------------------------------------------------------- P -------------------- 20 IgG ------------------------------------------------------------------------------------------------------ 21 IgA --------------------------------------------------------------------------------------------------------------- 22 IgA ---------------------------------------------------------------------------------------------------- P ----------- 23 IgA ------------------------------------------------------------------------------------------------------------------------- 24 IgA ---------------------------------------------------------------------------------------------------- P ------------------------- 25 IgA ------------------------------------------------------------------------------------------- P ---------------------------------------- 26 IgG ------------------------------------------------------------------------------------------------------------------------------------------- 27 IgA ------------------------------------------------------------------------------------------------------------------------------------------- 7/7 (100%) patients turned IFx- IR / non-CR 10/20 (50%) patients turned IFx+ non-IR /CR M-component positive M-component negative ------- Follow-up P Progression Death Treatment interruption

  44. GEM2000: Impact on survival of achieving an immunophenotypic CR vs. conventional CR after HDT/ASCT PFS At 5 years: 59% 49% 24% 17% 100 80 60 MRD- IFE- 71 m (n= 94) 40 MRD- IFE+ 65 m (n=31) MRD+ IFE- 37 m (n=53) 20 MRD+ IFE+ 37 m (n=117) p= 0.002 0 0 25 50 75 100 125 Months

  45. GEM 2000 trial: Multivariate Analysis PFS OS p risk p risk MRD+ at day +100 0.002 3.6 0.02 2.0 High Risk Cytog*. 0.006 1.79 ns Age >60y. ns0.04 1.6 IF+ at dey +100 ns ns t(4;14), t(4;16), del (17p) Paiva et al; Blood. 2008

  46. GEM 2000+2005: Immunophenotypic response & FISH for the prediction of early relapse in CR patients after HDT/ASCT (n=241) PFS OS 100 @ 1y after ASCT 100 93% Medians: NR 80 80% Median: 97m 80 60 60 Median: 43m Median: 64m 0% 40 40 Median: 35m 20 20 P <.001 Median: 17m P <.001 0 Months Months 0 0 20 40 60 80 100 120 0 20 40 60 80 100 120 MRD negative + Standard risk FISH (n=58) MRD positive OR High-risk FISH (n=45) MRD positive + High-risk FISH (n=7)

  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) PFS OS 100 100 80 80 60 60 40 40 20 20 P <.001 P =.353 0 0 0 10 20 30 40 50 60 0 10 20 30 40 50 60 Months Months

  48. Updated results from the MRC myeloma IX trial • 711 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 Owen et al. IMW Paris 2011 abstr O-09

  49. MM: Flow cytometry immunophenotyping vs. molecular monitoring of MRD ? Molecular techniques Flow cytometry Speed 2-3 days (up to weeks)fast: 1-2 hours Target DNA or RNAprotein/cells (RNA is an instable target) (“end-product”) Applicability 70-75% >95% Sensitivity 10-5-10-6 10-4-10-5 Multiplexing technically demanding relatively easy(even 25 to 100 tests per tube) Accuracy semi-quantitative quantitative Focus all cells in sample any subpopulation(or: prior purification) (FACSorted for further analyses) Facilitiesspecial laboratories needed only 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)

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