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FLT3 and NPM1 Testing in Acute Myeloid Leukaemia

FLT3 and NPM1 Testing in Acute Myeloid Leukaemia. Louise Stanley Northern Genetics Service April 2010. Acute Myeloid Leukaemia (AML). Uncontrolled proliferation of immature myeloid cells (blast) Median age of onset ~60 years

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FLT3 and NPM1 Testing in Acute Myeloid Leukaemia

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  1. FLT3 and NPM1 Testing in Acute Myeloid Leukaemia Louise Stanley Northern Genetics Service April 2010

  2. Acute Myeloid Leukaemia (AML) • Uncontrolled proliferation of immature myeloid cells (blast) • Median age of onset ~60 years • Analysis to look for ACQUIREDabnormalities in leukaemic clone (i.e. not constitutional) • Abnormalities can evolve during disease progression • For diagnosis and prognosis

  3. Prognostic Indicators • Cytogenetic markers and molecularly determined mutation status of FLT3 and NPM1 • Allows a risk-adapted treatment approach

  4. Adaptive Treatment Strategies • Non-specific treatments e.g. BMT, Chemotherapy • Target specific inhibitors – e.g. anti-FLT3 drugs (CEP-701)

  5. Exon 14 Fms-related tyrosine kinase (FLT3) • Encodes a tyrosine kinase receptor (13q12) – involved in regulation of stem cell proliferation • Internal Tandem Duplications (ITDs) cause constitutive activation of receptor • Associated with elevated risk relapse and reduced overall survival • Bad prognostic indicator

  6. NPM1 (nucleophosmin) • Encodes a ubiquitously expressed nuclear protein (5q35) • Involved in nuclear-cytoplasmic shuttling facilitating transport of ribosomal proteins • 4bp insertion in exon 12 of the NPM1 gene • Loss of the nucleolar-localisation signal and gain of a nuclear export signal motif at the C-terminus. Abnormal cytoplasmic accumulation • Good prognostic indicator in AML

  7. NPM1-ve/FLT3 ITD+ve NPM1+ve/FLT3 ITD+ve NPM1-ve/FLT3 ITD-ve NPM1+ve/FLT3 ITD-ve Prognostic Stratification • In Normal Karyotype Leukaemia (~40% of AML) Taken from: Gale et al. (2008) Blood, 111, 2776_2784.

  8. Testing Strategy • DNA extracted from fixed cell pellets using the automated EZ1 machine (time consuming part of process). • PCR: uniplex reaction examining FLT3 only and multiplex examining FLT3 and NPM1 mutation status • Analysis on the ABI3130 and Genemarker software (Softgenetics) • Information on blast cell count can be important for interpretation

  9. FLT3 results • From January 2007 to February 2010 tested 267 cases for FLT3 ITDs • 40 cases (~15%) positive • ~70 % of FLT3 +ve samples identified in Normal Karyotype Leukaemia • ITD range in size from 17bp to 182bp • Number of ITDs has no significant influence on survival

  10. FLT3 results Single ITD WT allele ITD ~ 72bp

  11. FLT3 results Multiple ITDs WT allele ITDs ~ 20, 23, 48 and 81bp

  12. 4bp insertion WT allele NPM1 results • From September 2008 to February 2010 tested 137 cases for NPM1 status (four base pair insertion) • 27 cases (~20%) positive

  13. NPM1 results • From September 2008 to February 2010 tested 137 cases for NPM1 status (four base pair insertion) • 27 cases (~20%) positive

  14. NPM1 FLT3 WT ITD 13 FLT3 and NPM1 +ve cases 15 FLT3 +ve cases 14 NPM1 +ve cases FLT3 and NPM1 95 FLT3 and NPM1 –ve cases

  15. Presentation vs Relapse

  16. Case 1 – Recurrence of the presentation clone • Presentation – ITD ~ 23bp (NPM1–ve) WT ~23bp ITD

  17. Case 1 – Recurrence of the presentation clone • Relapse – Same 23bp ITD present (NPM1-ve) WT ~23bp ITD

  18. Case 2– importance of detecting low levels of ITD • Presentation – very low levels of ~49bp ITD (NPM1+ve) WT ~49bp ITD

  19. Case 2– importance of detecting low levels of ITD • Relapse - ~49bp ITD and loss of WT allele: usually by acquired UPD of mutated Chr13 (NPM1+ve) WT ~49bp ITD

  20. Case 3– loss of ITD • Presentation – low level ~54bp ITD (NPM1-ve) WT ~54bp ITD

  21. Case 3– loss of ITD • Relapse – No evidence of FLT3 ITD (NPM1-ve) WT

  22. Case 4 – apparent change in ITD size/loss of WT allele • Presentation - ~72bp ITD (NPM1+ve) WT ~72bp ITD

  23. Case 4 – apparent change in ITD size/loss of WT allele • Relapse - ~33bp ITD and loss of WT allele: usually by acquired UPD of mutated Chr13 (NPM1+ve) WT ~33bp ITD ~72bp ITD absent

  24. Conclusions/Future Directions • FLT3 and NPM1 useful prognostic indicators in cases of AML • ITDs in FLT3 and 4bp insertion in NPM1 predominantly identified in patients with normal karyotype leukaemia • Testing of other molecularly determined markers to aid stratification of patients in the “intermediate” prognosis group (e.g. WT1 and CEBPA) • Introduction of assays to assess minimal residual disease

  25. Acknowledgements • Nick Bown – Cytogenetics, Northern Genetics Service (NGS) • Helen Powell • Ruth Sutton • Ottie O’Brien • David Bourn • Dr G Jones – Consultant Haematologist, Freeman Hospital, Newcastle Molecular Genetics (NGS)

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