Transcript File

Molecular monitoring in AML:
Impact on Therapy
Hwei-Fang Tien,
National Taiwan University Hospital
West part of NTUH
Outline
• Molecular markers for monitoring
• Prognostic implications
• Pre-emptive therapy on molecular
relapse
• Samples and timing for monitoring
Outline
• Molecular markers for monitoring
• Prognostic implications
• Pre-emptive therapy on molecular
relapse
• Samples and timing for monitoring
Molecular markers for monitoring in AML
Abnormal fusion genes:～30%
t(8;21)/RUNX1-RUNX1T1
inv(16)/CBFβ-MYH11
t(15;17)/PML-RARα
t(7;11)/NUP98-HOXA9
t(11;v)/MLL-partner gene
Molecular mutations that are stable during
disease progression
NPM1, IDH1/2, DNMT3A ,CEBPA, MLL-PTD
Gene overexpression
WT1
Molecular markers for monitoring in AML
Abnormal fusion genes:～30%
t(8;21)/RUNX1-RUNX1T1
inv(16)/CBFβ-MYH11
t(15;17)/PML-RARα
t(7;11)/NUP98-HOXA9
t(11;v)/MLL-partner gene
Molecular mutations that are stable during
disease progression
NPM1, IDH1/2, DNMT3A ,CEBPA, MLL-PTD
Gene overexpression
WT1
NPM1 mutant as a molecular marker for MRD monitoring
A NPM1 mutations: 4-nucleotide insertions
951
Wild type
Type I
Type II
Type III
Type IV
Type V
Type VI
Type VII
960
AGATCTCTG
AGATCTCTG
AGATCTCTG
AGATCTCTG
AGATCTCTG
AGATCTCTG
AGATCTCTG
AGATCTCTG
964
No. patients
…...
GCAG
…….
TGGAGGA
TCTG
CCTG
CATG
TCGG
CCAG
CCGG
…...
GCAG
GCAG
GCAG
GCAG
GCAG
GCAG
GCAG
……
……
……
……
……
……
CGGC
TGGAGGA
26
TGGAGGA
TGGAGGA
TGGAGGA
TGGAGGA
TGGAGGA
TGGAGGA
5
2
1
2
1
1
B Quantitative real-time PCR
7
6
Log Mutants
5
4
3
2
1
0
0
10
20
30
40
50
60
70
80
90
100
Blast % in BM
The copy number of mutants correlate well with BM blast %, but frequently
in BM samples with blasts <5%, NPM1 mutant levels can be high.
＊sensitivity: 1/105
NTUH, Leukemia, 2007, 21998
NPM1 mutant monitoring during the clinical course
Patient 3
Patient 31
6
6
5
CR
Relapse
5
Relapse
4
Relapse
4
3
3
2
2
CR
1
CR
CR
CR
CR
1
0
CR
0
0
5
10
15
20
25
30
35
0
5
Mon.
intensive chemotherapy
CR
10
CR
15
CR
20
25
CR
30
Mon.
t(7;11)/NUP98-HOXA9 as a marker
More common in Asia and is associated with poor prognosis
#11
fusion
#7
NTUH, Leukemia, 2009, 23:1303
Correlation between marrow blast percentage
and mutant load of NUP98-HOXA9
Log NUP98-HOXA9/ 106 HUPO
Real-time PCR for NUP98–HOXA9
6
5
4
3
2
1
0
0
10
20
30
40
50
60
Blast percentage in bone marrow
The mutant signals always remain high even when
BM blasts <5% and are rarely undetectable.
NTUH, Leukemia, 2009, 23:1303
Patient No. 7
B
6
CR
5
CR
4
relapse
CR
3
MUD HSCT
(myeloablative) CR
2
1
0
0
1
2
3
4
5
6
7
8
Log NUP98-HOXA9/106 HUPO
A
Log NUP98-HOXA9/106 HUPO
MRD monitoring of t(7;11)/NUP98-HOXA9
Patient No. 11
6
5
Sibling HSCT
(reduced intensity)
CR
4
CR
3
CR
2
1
CR
0
0
2
4
Patient No. 6
55
44
33
22
11
00
D
Relapse
CR
CR
Sibling HSCT
(myeloablative)
CR
CR
CR
CR
DLI
CR
DLI
0
0
10
10
20
30
40
20
30
40
Months from diagnosis
6
8
10
12
Months from diagnosis
50
50
Log NUP98-HOXA9/106 HUPO
Log NUP98-HOXA9/106 HUPO
Months from diagnosis
C
CR
CR
Patient No. 8
6
PR
CR
5
CR
4
3
CR
Relapse
CR
MUD HSCT
myeloablative
PR
2
CR
1
CR
0
0
5
AutoBMT
10
15
20
Months from diagnosis
In general, chemotherapy and even HSCT can only partially reduce the mutant
signals which are always detectable even at CR. Most pts died of disease.
The leukemic cells are very resistant to the present treatment and novel therapy
is needed for these patients.
NTUH, Leukemia, 2009, 23:1303
WT1 overexpression as a marker
to monitor MRD
Overexpression of WT1 can be found in >80% of AML patients
European LeukemiaNet (ELN) has standardized the assay method for WT1 expression
European LeukemiaNet, JCO, 2009, 27:5195
Outline
• Molecular markers for monitoring
• Prognostic implications
• Pre-emptive therapy on molecular
relapse
• Samples and timing for monitoring
Prognostic implication of MRD in
patients with NPM1 mutation
After consolidation
OS
RFS
Others, n=16
Others, n=16
Reduction <2 logs, n=6
Reduction <2 logs, n=6
P=0.010
P=0.001
Month
Month
NTUH 2006
NPM1-mutated patients with less than 2 logs reduction of NPM1 mutant
after consolidation C/T had shorter OS and RFS
More aggressive treatment may be needed for this group of patients.
NTUH: Leukemia 21:998, 2007
Prognostic Significance of MRD Levels
of NPM1 mutants during Follow-ups
OS
RFS
n=13
n=13
P=0.002
P<0.001
Any > 1.5%, n=18
Any > 1.5%, n=18
Month
Month
During sequential follow-ups, patients who had >1.5% of NPM1 mutant
in any sample had poorer prognosis
Early intervention at this moment may be needed for these patients.
NTUH, : Leukemia 21:998, 2007
Gene mutations as biomarkers for MRD monitoring
NPM1 mutation: German-Austrian AML Study Group
CR patients
after double
induction C/T
after completion
of therapy
JCO, 2011, 29:2709
MRD Monitoring in patients with CBF-AML
French AML Intergroup
198 patients , aged 18 to 60 yearold and with newly diagnosed CBF-AML
with t(8;21)/RUNX1-RUNX1T1 or inv(16)/CBFB-MYH11
After 2nd consolidation C/T
Overall survival
Relapse rate
reduction>=3 log
P<0.001
reduction<3 log
reduction<3 log
reduction>=3 log
P=0.066
Same for PFS, p<0.001
A more than 3-log MRD reduction after the second consolidation C/T
was associated with better prognosis
Jourdan et al, Blood , prepublished online January 15, 2013; DOI 10.1182
Detection of MRD by ELN Standardized WT1 Assay
91 AML patients: with significant high WT1 expression
(>2x 104 WT1 copies/104 ABL copies)
After induction chemotherapy
Cilloni et al, a European LeukemiaNet Study, JCO, 2009, 27:5195
Outline
• Molecular markers for monitoring
• Prognostic implications
• Pre-emptive therapy on molecular
relapse
• Samples and timing for monitoring
MRD Monitoring of PML/RARα Fusion
Transcript by Real-Time PCR in APL patients
MRC, UK
1. Detection of MRD at the end of consolidation in APL patients could predict RFS.
2. Pre-emptive therapy with As2O3 on molecular relapse reduced rate of clinical
relapse.
AML12: without pre-emptive therapy
AML15: with pre-emptive therapy
Grimwade et al, JCO 2009 27:3650
DLI Improve the Outcome of Patients
with MRD after allo-HSCT
MRD: WT1 expression for AML, IgH and TCR for ALL
Dominietto et al, Blood, 2007, 109:5063
Outline
• Molecular markers for monitoring
• Prognostic implications
• Pre-emptive therapy on molecular
relapse
• Samples and timing for monitoring
Proposed guidelines for MRD
During treatment:
according to the prognostic
significance of MRD at
different time points
During follow-ups: according to the relapse
kinetics of different molecular alterations
Sampling
interval (mo)
CBFB-MYH11
PB
BM
RUNX1-RUNX1T1
PB
BM
PML-RARA
PB
BM
NPM1c/FLT3-ITDPB
BM
NPM1c/FLT3-ITD+
PB
BM
WT1 expression
PB
BM
PML-RARA, CBFB-MYH11,
RUNX1-RUNX1T1:
after consolidation
NPM1 mutation:
after double induction
and consolidation
WT1 expression:
after induction
Modified from Hokland & Ommen,
Blood 2011, 117:2577
6*
Avoid
3
4
1
2
4
6
3
4
2
4
*One additional MRD sampling recommended 3 months after end of C/T.
in NTUH
**In normal karyotype
＃incidence
Summary
• Molecular monitoring by quantitative real-time PCR
is sensitive and specific for MRD detection in AML.
• Molecular monitoring after treatment is helpful for
risk-stratification.
• Pre-emptive therapy at the time of molecular relapse
during follow-ups may improve the clinical outcome.
• The optimal timing for MRD assessment varies with
molecular groups.
Future Prospective
Standardize the method and timing of molecular
monitoring.
Incorporate MRD detection into pretreatment risk
factors in clinical trials to evaluate more clearly the
prognostic significance of MRD and to design
preemptive measurement on a molecular relapse.
Apply next generation deep sequencing of targeted
genes for MRD detection.
台灣藍鵲（ Formosan Blue Magpie )