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MOLECULAR MONITORING
- 1. Molecular monitoring in AML: Impact on Therapy Hwei-Fang Tien, National Taiwan University Hospital West part of NTUH
- 2. • Molecular markers for monitoring • Prognostic implications • Pre-emptive therapy on molecular relapse • Samples and timing for monitoring Outline
- 3. • Molecular markers for monitoring • Prognostic implications • Pre-emptive therapy on molecular relapse • Samples and timing for monitoring Outline
- 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. 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
- 6. 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 LogMutants 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
- 7. 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
- 8. 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
- 9. Blast percentage in bone marrow LogNUP98-HOXA9/106HUPO 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
- 10. 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 1 2 3 4 5 0 10 20 30 40 50 Sibling HSCT (myeloablative) DLI DLI Relapse LogNUP98-HOXA9/106HUPO LogNUP98-HOXA9/106HUPO LogNUP98-HOXA9/106HUPO 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 CR CR CR CR CR CR CR CR CR 0 1 2 3 4 5 6 0 5 10 15 20 Months from diagnosis LogNUP98-HOXA9/106HUPO AutoBMT Patient No. 8 MUD HSCT myeloablative 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.
- 11. 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
- 12. • Molecular markers for monitoring • Prognostic implications • Pre-emptive therapy on molecular relapse • Samples and timing for monitoring Outline
- 13. 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
- 14. 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
- 15. Gene mutations as biomarkers for MRD monitoring CR patients after double induction C/T after completion of therapy JCO, 2011, 29:2709 NPM1 mutation: German-Austrian AML Study Group
- 16. MRD Monitoring in patients with CBF-AML Jourdan et al, Blood , prepublished online January 15, 2013; DOI 10.1182 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 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
- 17. 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
- 18. • Molecular markers for monitoring • Prognostic implications • Pre-emptive therapy on molecular relapse • Samples and timing for monitoring Outline
- 19. 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.
- 20. 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
- 21. • Molecular markers for monitoring • Prognostic implications • Pre-emptive therapy on molecular relapse • Samples and timing for monitoring Outline
- 22. Sampling interval (mo) CBFB-MYH11 PB 6* BM Avoid RUNX1-RUNX1T1 PB 3 BM 4 PML-RARA PB 1 BM 2 NPM1c/FLT3-ITD- PB 4 BM 6 NPM1c/FLT3-ITD+ PB 3 BM 4 WT1 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 alterationsDuring 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
- 23. 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.
- 24. 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.
- 25. 台灣藍鵲（ Formosan Blue Magpie )