MRD detection in AML. Adriano Venditti Hematology Fondazione Policlinico Tor Vergata Rome

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1 MRD detection in AML Adriano Venditti Hematology Fondazione Policlinico Tor Vergata Rome

2 Determinants of Treatment Response Leukemia Tumor burden Growth potential Drug resistance Karyotype Genetics Host Age Pharmacogenomics Therapy Drug dosage Drug interactions RESPONSE OUTCOME

3 Assessing MRD: does it worthwhile? MRD = QoR = CHT-resistance leukemic cells surviving cytotoxic therapy Normal karyotype is molecularly heterogeneous Also patients belonging to the most favorable categories do have relapse Cytogenetic/genetic risk-stratification inadequate for predicting relapse in individual patients Can MRD detection improve outcome prediction?

4 Methods for detecting MRD in AML PCR on chimeric fusion genes mutations gene overexpression Flow cytometry leukemia-associated aberrant phenotypes (LAIP), LAIPs are absent or very infrequent in NBM

5 Potential molecular targets for MRD Fusion genes Mutations Overexpression PML-RARA CBFB-MYH11 RUNX1-RUNX1T1 MLL-fusion partner DEK-NUP % of AMLs stable at relapse NPM1 FLT3 CEBPA MLL-PTD RUNX1 ~ 75% of CN-AML?stability at relapse WT1 BAALC ERG MN1 ~ 30% of AMLs?sensitivity

6 Minimal residual disease assessed by NPM1 specific RQ-PCR (Schnittger, 2009)

7 Cilloni et al, JCO 2009

8 The five attributes of modern FC Lightness Its underlying principle is solid, without any intricate conjecture Quickness AL diagnosis can be provided within 1 h Exactitude Analytical process is highly controlled and data are quantitatively expressed Visibility Dot plot pattern recognition Multiplicity 6-9 color polichromatic analysis Del Vecchio et al, Leukemia 2007

9 FSC-Height -> di pasquo e bm postil2.001 new CD3 FITC -> di pasquo e bm postil2.001 new CD3 FITC -> di pasquo e bm postil2.001 new CD34 APC -> di pasquo e bm postil2.001 new CD4 PE -> di pasquo e bm postil2.001 new CD34 APC -> di pasquo e bm postil2.001 new

10 Designing MRD studies in AL Multiple staining at diagnosis Identification of leukemia-associated phenotypes Definition of a patient s immunologic fingerprint Immunologic fingerprint used during follow-up Venditti et al Blood 2000, Venditti et al Leukemia 2003, Buccisano et al Leukemia Maurillo et al JCO 2008, Buccisano et al Blood 2010

11 RFS after different courses of CHT and MRD % in BM 1 st course 2 nd course 3 rd course MRD% is an independent prognostic factor; use for risk stratification Terwijn M et al. ASH 2010

12 Tor Vergata University Experience 1,0 0,9 0,8 0,7 MRD+ MRD- 1,0 0,9 0,8 MRD+ MRD- 0,6 0,7 0,5 0,6 0,4 0,5 0,3 0,4 Overall Survival 0,2 0,1 0, YEARS Relapse Free S 0,3 0,2 0, pts with de novo AML enrolled in the EORTC/GIMEMA Years protocols MRD levels cells at the end of consolidation predicts relapse Venditti et al, Blood 2000

13 Tor Vergata University Experience Maurillo et al, JCO 2008

14 Tor Vergata University Experience RFS OS Cytogenetics N.S. MDR1 expression 0.03 N.S. Post-Induction MRD status Post-Consolidation MRD status N.S. N.S Maurillo et al, JCO 2008

15 3.5 x 10-4 RLC Post-consolidation time-point Comprehensive risk-assessment Integration of baseline prognosticators (cytogenetics, genetics) and parameters inherent the quality of response (MRD) Optimization of post-remission therapy Development of MRD-oriented therapies More appropriate use of allosct

16 Buccisano et al, Blood 2010 ν ν ν ν TVUH 143 adults with AML in CR (median age 50y, range 18-75; 40 60y) EORTC-GIMEMA AML-10, AML-12, AML-13, AML-17 MRD+: 3.5 x 10-4 (0.035%) at post-consolidation

17 Buccisano et al, Blood 2010

18 Integrated Risk-Score Low-Risk Good K / MRD- Int K / MRD- High-Risk Adverse K FLT3+ Good K / MRD+ Int K / MRD+

19 Integrated Risk-Score Alonzo TA et al, abst 761, ASH 2010 Conventional Cytogenetics, Molecular Profiling, and Flow Cytometric Response Data Allow the Creation of a Two-Tiered Risk-Group System for Risk-Based Therapy Allocation In Childhood AML- a Report From the Children's Oncology Group

20 MRD monitoring!!! MRD monitoring feasible Independent prognostic factor for Risk of relapse Relapse-free survival Event-free survival Overall survival

21 MRD monitoring??? No agreement on relevant thresholds and checkpoints Post-induction? Post-consolidation? Is MRD equally relevant in all subsets of AML? Will MRD translate in new interventions? Can early intervention make a difference?

22 allosct > autosct according to MRD status N=42 MRD+ post-cons N=37 MRD- post-cons B Maurillo et al, JCO 2008

23 allosct > autosct for High-risk AML Low-Risk Good K / MRD- Int K / MRD- High-Risk Adverse K FLT3+ Good K / MRD+ Int K / MRD+ ausct allosct Total L-risk H-risk Total Buccisano et al, Blood 2010

24 allosct > autosct for High-risk AML Disease Free Survival 1,0 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 P= % 44% 20% 0, Time (yrs) AlloSCT (ITT) N=21 AlloSCT N=15 AutoSCT N=53 Buccisano et al, 2010 unpublished

25 GIMEMA AML1310: a study of risk-adapted and MRD-directed therapy for adult AML MRD marker LAIP Risk stratif CG, molecular MRD assess LAIP Diagnosis Low-risk Int-risk Induction (1 or 2 courses) CR Consolidation 1 MRD- MRD+ autosct allosct High-risk No CR FLA-Ida salvage CR Low-risk: CBF/Kit wt ; NPM1+/FLT3- Int-risk: all others High-risk: Adverse K; FLT3+ allosct: MRD, MUD, UCB, HRD

26 Points for consideration Kinetic of MRD fluctuation between IND and CONS Prediction failure for 20-30% MRD neg pts who do relapse PB as alternative source to BM

27 100 patients Buccisano et al, Leukemia 2006

28 Relationship between MRD level after IND and MRD status after CONS

29 Maurillo et al, JCO 2008 Excel: Ind Cons Good: Ind + Cons Poor: Ind + Cons + Ugly: Ind Cons +

30 Prediction failure for some MRD neg patients 20-30% relapse rate in MRD negative. Why don t we predict it? Technical reasons: sensitivity/specificity Biological reasons: role of LSC

31 Role of LSC CD34+ CD38- To find markers and/or properties that discriminate LSC from HSC within the CD34 + CD38 - compartment

32 Strategy to discriminate leukemic and normal CC34+ CD38- stem cell AML marker: CLL-1 Bakker et al Cancer Res 64: , 2004

33 RFS after different courses of CHT and LSC frequency in BM

34 After induction: median value of BMRLC: 5.2x10-3 (range 1x x10-1 ) median value of PBRLC: 2.85x10-3 (range 1x x10-1 ) r=0.84, P<0.001 Maurillo L. et al Haematologica 2007

35 After consolidation: median value of BMRLC: 4.1x10-3 (range 2x x10-2 ) median value of PBRLC: 3.7x10-3 (range 1x x10-1 ) r=0.82, P<0.001 Bone marrow MRD after consolidation Peripheral blood MRD after consolidation Maurillo L. et al Haematologica 2007

36 MRD assessment to individualize therapy of AML Informs appropriate post-remission therapy Informs decision regarding transplantation in first CR Optimizes timing & type of transplant Defines need for additional therapy posttransplant Identifies impending relapse & drives preemptive therapy