Molecular monitoring in AML: Impact on Therapy

Hwei-Fang Tien, National Taiwan University Hospital

West part of NTUH

• 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

Outline

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

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

NPM1 mutant as a molecular marker for MRD monitoring

0

1

2

3

4

5

6

7

0 10 20 30 40 50 60 70 80 90 100

Log

Mut

ants

Blast % in BM

NTUH, Leukemia, 2007, 21998

　 … ...

　 TCTG

　 CCTG

　 CATG

　 TCGG

　 CCAG

　 CCGG

　 … ...

951 960 964

TGGAGGA

TGGAGGA

TGGAGGA

TGGAGGA

TGGAGGA

TGGAGGA

TGGAGGA

TGGAGGACGGC

…….

……

……

……

……

……

……

AGATCTCTG

AGATCTCTG

AGATCTCTG

AGATCTCTG

AGATCTCTG

AGATCTCTG

AGATCTCTG

AGATCTCTG

GCAG

GCAG

GCAG

GCAG

GCAG

GCAG

GCAG

GCAG

Wild type

Type VII

Type VI

Type V

Type IV

Type III

Type II

Type I

No. patients

26　5

1

2

1

1

2

A NPM1 mutations: 4-nucleotide insertions

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

B Quantitative real-time PCR

0

1

2

3

4

5

6

0 5 10 15 20 25 30

0

1

2

3

4

5

6

0 5 10 15 20 25 30 35

Patient 3 Patient 31

Mon. Mon.

CR CR

CR

CR

Relapse Relapse

Relapse

CR

CR

CR CR CR CR CR

NPM1 mutant monitoring during the clinical course

intensive chemotherapy

t(7;11)/NUP98-HOXA9 as a marker

More common in Asia and is associated with poor prognosis

#11

#7

fusion

NTUH, Leukemia, 2009, 23:1303

Blast percentage in bone marrow

Lo

g N

UP

98

-HO

XA

9/

10

6 H

UP

O

0

1

2

3

4

5

6

0 10 20 30 40 50 60

Correlation between marrow blast percentage and mutant load of NUP98-HOXA9

The mutant signals always remain high even when BM blasts <5% and are rarely undetectable.

NTUH, Leukemia, 2009, 23:1303

Real-time PCR for NUP98–HOXA9

0

1

2

3

4

5

6

0 1 2 3 4 5 6 7 8

MUD HSCT (myeloablative)

relapse

0

1

2

3

4

5

6

0 2 4 6 8 10 12

Sibling HSCT (reduced intensity)

0

12

34

5

0 10 20 30 40 50

Sibling HSCT (myeloablative)

DLIDLI

Relapse

Lo

g N

UP

98-H

OX

A9/

106 H

UP

O

Lo

g N

UP

98-H

OX

A9/

106 H

UP

O

Lo

g N

UP

98-H

OX

A9/

106 H

UP

O

Months from diagnosis

Months from diagnosis

Months from diagnosis

A B

C D

Patient No. 7 Patient No. 11

Patient No. 6

0 10 20 30 40 50

0

5

4

3

1

CR

CR

CR

CR

CR

CR

CR

CRCR

CR

CR

CR

CR

CR

CRCR

0

1

2

3

4

5

6

0 5 10 15 20

Months from diagnosis

Lo

g N

UP

98-H

OX

A9/

106 H

UP

OAutoBMT

Patient No. 8

MUD HSCTmyeloablative

2

PR

PR

CR Relapse

CR CR

CR

CR

CR

CR

MRD monitoring of t(7;11)/NUP98-HOXA9

NTUH, Leukemia, 2009, 23:1303

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.

WT1 overexpression as a marker to monitor MRD

European LeukemiaNet, JCO, 2009, 27:5195

Overexpression of WT1 can be found in >80% of AML patients

European LeukemiaNet (ELN) has standardized the assay method for WT1 expression

• Molecular markers for monitoring

• Prognostic implications

• Pre-emptive therapy on molecular relapse

• Samples and timing for monitoring

Outline

Prognostic implication of MRD in patients with NPM1 mutation

More aggressive treatment may be needed for this group of patients.

NPM1-mutated patients with less than 2 logs reduction of NPM1 mutant after consolidation C/T had shorter OS and RFS

OS RFS

Reduction <2 logs, n=6

Reduction <2 logs, n=6

P=0.010 P=0.001

Month Month

Others, n=16 Others, n=16

NTUH 2006

NTUH: Leukemia 21:998, 2007

After consolidation

P=0.002

Any > 1.5%, n=18

OS

Month

n=13

RFS

Any > 1.5%, n=18

Month

n=13

P<0.001

Prognostic Significance of MRD Levels of NPM1 mutants during Follow-ups

NTUH, : Leukemia 21:998, 2007

Early intervention at this moment may be needed for these patients.

During sequential follow-ups, patients who had >1.5% of NPM1 mutant in any sample had poorer prognosis

Gene mutations as biomarkers for MRD monitoring

CR patients

after doubleinduction C/T

after completion of therapy

JCO, 2011, 29:2709

NPM1 mutation: German-Austrian AML Study Group

MRD Monitoring in patients with CBF-AML

Jourdan et al, Blood , prepublished online January 15, 2013; DOI 10.1182

French AML Intergroup198 patients , aged 18 to 60 yearold and with newly diagnosed CBF-AML with t(8;21)/RUNX1-RUNX1T1 or inv(16)/CBFB-MYH11

A more than 3-log MRD reduction after the second consolidation C/T was associated with better prognosis

Relapse rate

reduction<3 log

reduction>=3 log

P<0.001

Overall survival

reduction>=3 log

reduction<3 log

P=0.066

Same for PFS, p<0.001

After 2nd consolidation C/T

Detection of MRD by ELN Standardized WT1 Assay

91 AML patients: with significant high WT1 expression (>2x 104 WT1 copies/104 ABL copies)

Cilloni et al, a European LeukemiaNet Study, JCO, 2009, 27:5195

After induction chemotherapy

• Molecular markers for monitoring

• Prognostic implications

• Pre-emptive therapy on molecular relapse

• Samples and timing for monitoring

Outline

MRC, UK 1. Detection of MRD at the end of consolidation in APL patients could predict RFS.

MRD Monitoring of PML/RARα Fusion Transcript by Real-Time PCR in APL patients

AML15: with pre-emptive therapy

Grimwade et al, JCO 2009 27:3650

AML12: without pre-emptive therapy

2. Pre-emptive therapy with As2O3 on molecular relapse reduced rate of clinical relapse.

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

• Molecular markers for monitoring

• Prognostic implications

• Pre-emptive therapy on molecular relapse

• Samples and timing for monitoring

Outline

Sampling interval (mo)

CBFB-MYH11 PB 6* BM AvoidRUNX1-RUNX1T1 PB 3 BM 4PML-RARA PB 1 BM 2NPM1c/FLT3-ITD-

PB 4 BM 6NPM1c/FLT3-ITD+

PB 3 BM 4WT1 expression PB 2 BM 4

Proposed guidelines for MRD

Modified from Hokland & Ommen, Blood 2011, 117:2577

*One additional MRD sampling recommended 3 months after end of C/T.＃ incidence in NTUH **In normal karyotype

During follow-ups: according to the relapse kinetics of different molecular alterations During treatment:

according to the prognostic significance of MRD at different time points

PML-RARA, CBFB-MYH11, RUNX1-RUNX1T1: after consolidation

NPM1 mutation: after double induction and consolidation

WT1 expression: after induction

Summary

• Molecular monitoring after treatment is helpful for

risk-stratification.

• The optimal timing for MRD assessment varies with

molecular groups.

• Pre-emptive therapy at the time of molecular relapse during follow-ups may improve the clinical outcome.

• Molecular monitoring by quantitative real-time PCR

is sensitive and specific for MRD detection in AML.

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 )