The role of Minimal Residual Disease in the

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. 10 2 sensitivity CLINICAL DIAGNOSIS 1 RELAPSE MORPHOLOGIC & CYTOGENETIC REMISSION 1010 Flow cytometry 108 104 PCR 106 104 102 106 MOLECULAR REMISSION INDUCT. 108 CONSOLID. CURE ? THERAPY 10 2 10 0 2 MOS. 4 6 8 10 12 14 16 18 20 24 36 48

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

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

Leukemia-Associated Immunophenotypes (LAIP) in AML Phenotype Incidence Examples Asynchronous antigen expression 60 -70% CD 34+ CD 117+CD 15+ CD 33+ CD 13 -CD 15+ Cross-lineage antigen expression 30 -40% CD 19+ C 2+ CD 7+D Antigen overexpression 20 -30% Overall 60 -90% CD 34+CD 13+ CD 33+ CD 15+CD 14+

Genetic markers detectable by PCR and useful for MRD detection in acute leukemia l Clono-specific markers -Ig gene rearrangements -TCR gene rearrangements and l 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% t (9: 22) BCR/ABL t (1; 19) E 2 A/PBX 1 5 -6% t (4; 11) MLL/AF 4 5% del 1 p 32 TAL 1/SCL 5% ALL B-lineage T- Lineage AML t (15; 17) 25 -30% PML/RAR 10% t ( 8; 21) AML 1/ETO 8 -10% inv (16) CBF /MYH 11 6 -8% t (6; 9) DEK/CAN 1% 11 q 23 MLL/? 5 -10%

‘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/MYH 11 ABL X 104 copies CCR pts. R Relapsed pts. 10000 R 1000 R R R 100 10 1 0 2 28 MONTHS 4 6 8 10 12 14 16 18 20 24 26

Distinct kinetics of molecular remission in AML 1 -ETO PML-RARa CBFb-MYH 11 0 2 -3 log PCR + ve 4 -5 log PCR - ve 0 3 6 9 12 15 18 21 24 m. 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 Bcr-Abl t(9; 22) 26% Other 23% MLL t(4; 11) t(11; 19) t(9; 11) 10% Hypodiploidy 1% Hyperdiploidy 7% TEL-AML 1 t(12; 21) 2% Lyl 1 19 p 13 2% Tal 1 1 p 32 12% Hox 11 L 2 5 p 35 1% Hox 11 10 q 24 8% MLL-ENL 0% Myc t(8; 14), t(2; 8), t(8; 22) 4% E 2 a-PBX 1 t(1; 19) 3%

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 OS early. CR 80 70 mol % 60 p=0. 0002 50 40 persisting MRD+: 14/29 DFS 30 OS OS - persistent MRD+ 20 DFS - persistent MRD+ DFS 10 0 p=0. 0001 0 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 MRDTriggered Imatinib after SCT for Ph+ALL (EBMT) MRD monitoring Randomisation (<6 wks. post SCT) Preregistr. Dasatinib* Imatinib Mutation analysis SCT R DLI (optional) MRD+ MRD monitoring Imatinib * If MRD by >2 log or BCR-ABL transcripts >10 -3 or BCR -ABL Mutation analysis Dasatinib*

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

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

20 Early assessment of MRD by optimized RQ-PCR of WT 1 provides an independent predictor of DFS IN AML (Cilloni et al, EHA 2008) Background: WT 1 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 (Leukemia. Net)

21 Early assessment of MRD by optimized RQ-PCR of WT 1 provides an independent predictor of DFS IN AML (Cilloni et al, EHA 2008) Results: Failure to normalize WT 1 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 0, 9 MRD negative FLT 3 negative (80%) 0, 8 0, 7 0, 6 0, 5 0, 4 0, 3 MRD positive FLT 3 negative (29%) 0, 2 0, 1 0, 0 FLT 3 positive (10%) 0 365 730 1095 1460 1825 2190 2555 Time (Days) 2920 3285 3650 4015 4380 4745 Buccisano et al, unpublished

RFS analysis in 80 adult patients with NK-AML 1, 0 0, 9 0, 8 0, 7 MRD negative (75%) 0, 6 MRD negative NPM positive (60%) 0, 5 0, 4 0, 3 0, 2 MRD positive NPM positive (24%) MRD positive (23%) 0, 1 0, 0 0 365 730 1095 1460 1825 2190 2555 Time (Days) 2920 3285 3650 4015 4380 4745 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 Mos. from dx 3 27 Follow-up 6 30 9 12 15 18 21 24

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 AML 15 trial Burnett AK (unpublished)

Summary • Well standardised PCR-based MRD evaluation drives therapy in APL and Ph’ ALL • MRD assessment using “universal” markers (LAIP, WT 1) appears promising and may provide early prognostic information (WT 1) • 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