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Molecular monitoring in AML: Impact on Therapy

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.

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Molecular monitoring in AML: Impact on Therapy

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  1. Molecular monitoring in AML: Impact on Therapy Hwei-Fang Tien, National Taiwan University Hospital West part of NTUH

  2. Outline • Molecular markers for monitoring • Prognostic implications • Pre-emptive therapy on molecular relapse • Samples and timing for monitoring

  3. Outline • Molecular markers for monitoring • Prognostic implications • Pre-emptive therapy on molecular relapse • Samples and timing for monitoring

  4. 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

  5. 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

  6. NPM1 mutant as a molecular marker for MRD monitoring A NPM1 mutations: 4-nucleotide insertions 951 960 964 No. patients Wild type …... GCAG ……. TGGAGGA AGATCTCTG Type I TGGAGGA 26 AGATCTCTG TCTG GCAG …… Type II AGATCTCTG CCTG GCAG …… TGGAGGA 5 Type III AGATCTCTG CATG GCAG …… TGGAGGA 2 Type IV AGATCTCTG TCGG GCAG …… TGGAGGA 1 Type V AGATCTCTG CCAG GCAG …… TGGAGGA 2 Type VI AGATCTCTG CCGG GCAG …… TGGAGGA 1 Type VII AGATCTCTG …... GCAG CGGC TGGAGGA 1 B Quantitative real-time PCR 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

  7. NPM1 mutant monitoring during the clinical course Patient 3 Patient 31 Relapse Relapse CR Relapse CR CR CR CR CR CR CR CR CR CR Mon. Mon. intensive chemotherapy NTUH, Leukemia, 2007, 21998

  8. 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

  9. Correlation between marrow blast percentage and mutant load of NUP98-HOXA9 Real-time PCR for NUP98–HOXA9 Log NUP98-HOXA9/ 106 HUPO 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

  10. MRD monitoring of t(7;11)/NUP98-HOXA9 A Patient No. 11 Patient No. 7 B CR CR relapse Sibling HSCT (reduced intensity) CR CR CR Log NUP98-HOXA9/106 HUPO MUD HSCT (myeloablative) Log NUP98-HOXA9/106 HUPO CR CR CR CR CR Months from diagnosis Months from diagnosis Patient No. 6 Patient No. 8 D C 5 PR Relapse CR CR CR 4 Relapse CR CR CR CR MUD HSCT myeloablative 3 PR Log NUP98-HOXA9/106 HUPO CR Sibling HSCT (myeloablative) Log NUP98-HOXA9/106 HUPO 2 CR CR CR 1 DLI CR CR AutoBMT 0 DLI 0 10 20 30 40 50 Months from diagnosis 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

  11. WT1overexpression 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

  12. Outline • Molecular markers for monitoring • Prognostic implications • Pre-emptive therapy on molecular relapse • Samples and timing for monitoring

  13. Prognostic implication of MRD in patients withNPM1 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

  14. 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

  15. 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

  16. 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

  17. Detection of MRD by ELN Standardized WT1 Assay 91 AML patients: with significant high WT1 expression (>2x 104WT1 copies/104 ABL copies) After induction chemotherapy Cilloni et al, a European LeukemiaNet Study, JCO, 2009, 27:5195

  18. Outline • Molecular markers for monitoring • Prognostic implications • Pre-emptive therapy on molecular relapse • Samples and timing for monitoring

  19. MRD Monitoring of PML/RARαFusion 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

  20. MRD: WT1 expression for AML, IgH and TCR for ALL DLI Improve the Outcome of Patients with MRD after allo-HSCT Dominietto et al, Blood, 2007, 109:5063

  21. Outline • Molecular markers for monitoring • Prognostic implications • Pre-emptive therapy on molecular relapse • Samples and timing for monitoring

  22. Proposed guidelines for MRD 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 Figure 1. Kaplan-Meier curves of overall survival by two factors (FLT3ITD and AML1/RUNX1) Non Allo-HSCT group Allo-HSCT group 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 FLT3ITD

  23. 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.

  24. Future Prospective Standardize the method and timing of molecular monitoring. • Incorporate MRD detection into pretreatmentrisk 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.

  25. 台灣藍鵲（ Formosan Blue Magpie )

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