1. 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

2. 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
MOS.

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

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

5. 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%

6. 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

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

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

10. 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 ?

11. 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
MONTHS

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

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

14. 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%

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

16. 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

17. 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

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

19. ASO-RT-PCR to detect NPM1 type A mutation
NPM1-mutA
NPM1-w/t
NPM1-mutA
320 bp
ABL
258 bp
M M ctrl+ ctrl- M
ASO-RT-PCR semi-nested-ASO-PCR
Ottone et al,
J Mol Diag 2008

20. 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)

21. 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

22. 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

23. 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

24. 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

25. J Clin Oncol 2003

26. Suggested timing of marrow sampling for RT-PCR in patients receiving ATRA and chemotherapy
Induction
Consolidation
Follow-up
Mos. from dx

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

28. 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)

29. 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 *

30. *