The role of Minimal Residual Disease in the Management of Acute Leukemia Francesco Lo-Coco Università Tor Vergata, Roma Corso Nazionale di Aggiornamento in Ematologia Clinica Catania, 6-7 Novembre 2008

leukemic cell n. sensitivity RELAPSE Flow cytometry PCR CLINICAL DIAGNOSIS RELAPSE 1012 MORPHOLOGIC & CYTOGENETIC REMISSION 1010 102 Flow cytometry 108 104 PCR 106 106 MOLECULAR REMISSION 104 108 INDUCT. CONSOLID. THERAPY CURE ? 10 2 10 0 2 4 6 8 10 12 14 16 18 20 24 36 48 MOS.

Relevant issues in MRD studies - Disease & therapeutic context - MRD techniques and targets Standardization Timing/source of sampling clinicians’ compliance

Methods for studying MRD Technique Targets Sensitivity Flow cytometry Antigens 10-3 FISH DNA 10-3/10-4 PCR/Q-PCR DNA/RNA 10-4/10-6

Leukemia-Associated Immunophenotypes (LAIP) in AML Incidence Examples Asynchronous antigen expression 60-70% CD34+ CD14+ CD117+CD15+ CD33+ CD13-CD15+ Cross-lineage antigen expression 30-40% CD19+ C2+ CD7+D Antigen overexpression 20-30% CD34+CD13+ CD33+ CD15+CD14+ Overall 60-90%

Genetic markers detectable by PCR and useful for MRD detection in acute leukemia Clono-specific markers -Ig gene rearrangements -TCR gene rearrangements and  Tumor specific markers -new hybrid genes produced by reciprocal chromosome translocations -gene mutations

Genetic defects useful for MRD detection by PCR DISEASE Chrom. abnormality Molecular Target Frequency% ALL B-lineage t (9:22) BCR/ABL 25-30% t (1;19) E2A/PBX1 5-6% t (4;11) MLL/AF4 5% T- Lineage del 1p32 TAL1/SCL 5% AML t (15;17) PML/RAR 10% t ( 8;21) AML1/ETO 8-10%  inv (16) CBF/MYH11 6-8% t (6;9) DEK/CAN 1% 11q23 MLL/? 5-10%

‘Europe Against Cancer’ Network • • ‘Europe Against Cancer’ Network Fusion Transcripts (n=9) & RQ PCR For leukemias Standardization Quality Controls • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • •

Is molecular remission a therapeutic goal in acute leukemia ? t(15;17) APL yes inv(16) AML yes t(8;21) AML ? t(9;22) ALL yes t(4;11) ALL yes Other AML/ALL ?

Real Time PCR in inv(16) AML CBFβ/MYH11 X 104 copies CCR pts. R ABL Relapsed pts. 10000 R R R R R 1000 R 100 10 1 2 4 6 8 10 12 14 16 18 20 24 26 28 MONTHS

Distinct kinetics of molecular remission in AML AML1-ETO PML-RARa CBFb-MYH11 0 3 6 9 12 15 18 21 24 m. 2-3 log 4-5 log PCR + ve PCR - ve Jaeger et al, 2003

Int. BFM study for MRD in childhood pre-B ALLs Detection of MRD in patients remaining in CCR PCR + % THERAPY INDUCTION MAINTENANCE END

Molecular heterogeneity of ALL Hypodiploidy 1% Hyperdiploidy 7% TEL-AML1 t(12;21) 2% Myc t(8;14), t(2;8), t(8;22) 4% E2a-PBX1 t(1;19) 3% Lyl1 19p13 Tal1 1p32 12% MLL-ENL 0% Hox11 10q24 8% Hox11L2 5p35 MLL t(4;11) t(11;19) t(9;11) 10% Other 23% Bcr-Abl t(9;22) 26%

CIR according to MRD status in Ph’+ve ALL MRD at transplant MRD at day +100 Spinelli, Haematologica 2007

DFS and OS by Early MRD Response to Imatinib 100 early MRD neg.: 15/29 90 OS earlyCRmol OS 80 70 60 % p=0.0002 50 40 persisting MRD+: 14/29 DFS 30 OS OS - persistent MRD+ 20 p=0.0001 DFS 10 DFS - persistent MRD+ 3 6 9 12 15 18 21 24 27 30 33 Months since Imatinib start Wassmann et al, Blood 2005

Randomized Study of Pre-emptive versus MRD- Triggered Imatinib after SCT for Ph+ALL (EBMT) MRD monitoring Imatinib Dasatinib* Randomisation (<6 wks. post SCT) Mutation analysis Pre- registr. SCT R DLI (optional) MRD+ MRD MRD monitoring Imatinib Dasatinib* Mutation analysis * If MRD by >2log or BCR-ABL transcripts >10-3 or BCR-ABL Mutation

Molecular heterogeneity of NK-AML Döhner et al, ASH 2007

ASO-RT-PCR to detect NPM1 type A mutation NPM1-mutA NPM1-w/t NPM1-mutA 320 bp ABL 258 bp 10-1 10-2 10-3 M 10-1 10-2 10-3 10-4 10-5 10-6 M ctrl+ ctrl- M ASO-RT-PCR semi-nested-ASO-PCR Ottone et al, J Mol Diag 2008

Early assessment of MRD by optimized RQ-PCR of WT1 provides an independent predictor of DFS IN AML (Cilloni et al, EHA 2008) 20 Background: WT1 overexpressed in >90% AMLs Suitable “universal” MRD marker for AML Methods: Comparison of sensitivity & specifitiy of 9 different R-Q-PCR assays 729 diagnostic & 106 f-up samples 11 European labs (LeukemiaNet)

Early assessment of MRD by optimized RQ-PCR of WT1 provides an independent predictor of DFS IN AML (Cilloni et al, EHA 2008) 21 Results: Failure to normalize WT1 transcripts post-induction correlates with relapse in 100% of cases (independent predictive factor) Decreased levels post-treatment not fully informative on outcome Comments: Important early information, most relevant (if confirmed) to adjust post-induction therapy

RFS analysis in 80 adult patients with NK-AML 1,0 MRD negative FLT3 negative (80%) MRD positive FLT3 negative (29%) 0,9 FLT3 positive (10%) 0,8 0,7 0,6 0,5 0,4 0,3 Multimediale normali, FLT3+ FLT3-, NPM-FLT3 4 CATEGORIE, flt3 mdr 0,2 0,1 0,0 365 730 1095 1460 1825 2190 2555 2920 3285 3650 4015 4380 4745 Time (Days) Buccisano et al, unpublished

RFS analysis in 80 adult patients with NK-AML 1,0 MRD positive NPM positive (24%) MRD negative NPM positive (60%) MRD negative (75%) MRD positive (23%) 0,9 0,8 0,7 0,6 0,5 0,4 0,3 Multimediale normali, FLT3+ FLT3-, NPM-FLT3 4 CATEGORIE, flt3 mdr 0,2 0,1 0,0 365 730 1095 1460 1825 2190 2555 2920 3285 3650 4015 4380 4745 Time (Days) Buccisano et al, unpublished

Clinical relevance of PCR monitoring in APL Assessment of response to therapy (molecular remission) at the end of consolidation as an early surrogate of improved survival Identification of molecular relapse. Benefit in early salvage Provides sensitive surveillance of disease status in experimental clinical trials

J Clin Oncol 2003

Suggested timing of marrow sampling for RT-PCR in patients receiving ATRA and chemotherapy Induction Consolidation Follow-up Mos. from dx 3 6 9 12 15 18 21 24 27 30

Kinetics of molecular/frank relapse in APL revealed by RQ-PCR: Implications for optimal frequency of MRD monitoring Grimwade et al. (submitted)

Evaluation of MRD monitoring & pre-emptive ATO therapy to reduce rates of frank relapse in PML-RARA+ APL in MRC AML15 trial Burnett AK (unpublished)

Summary Well standardised PCR-based MRD evaluation drives therapy in APL and Ph’ ALL MRD assessment using “universal” markers (LAIP, WT1) appears promising and may provide early prognostic information (WT1) Need of better standardization of flow cytometry and of its comparison/integration with PCR data Need of reference labs and standardised MRD evaluation in prospective large trials *

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