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FLT3 and NPM1 Testing in Acute Myeloid Leukaemia (AML) PowerPoint Presentation

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FLT3 and NPM1 Testing in Acute Myeloid Leukaemia (AML) Presentation Transcript

Slide 1 - FLT3 and NPM1 Testing in Acute Myeloid Leukaemia Louise Stanley Northern Genetics Service April 2010
Slide 2 - Acute Myeloid Leukaemia (AML) Uncontrolled proliferation of immature myeloid cells (blast) Median age of onset ~60 years Analysis to look for ACQUIRED abnormalities in leukaemic clone (i.e. not constitutional) Abnormalities can evolve during disease progression For diagnosis and prognosis
Slide 3 - Prognostic Indicators Cytogenetic markers and molecularly determined mutation status of FLT3 and NPM1 Allows a risk-adapted treatment approach
Slide 4 - Non-specific treatments e.g. BMT, Chemotherapy Target specific inhibitors – e.g. anti-FLT3 drugs (CEP-701) Adaptive Treatment Strategies
Slide 5 - 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
Slide 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
Slide 7 - Prognostic Stratification Taken from: Gale et al. (2008) Blood, 111, 2776_2784. In Normal Karyotype Leukaemia (~40% of AML)
Slide 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
Slide 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
Slide 10 - FLT3 results WT allele ITD ~ 72bp Single ITD
Slide 11 - FLT3 results WT allele Multiple ITDs ITDs ~ 20, 23, 48 and 81bp
Slide 12 - NPM1 results From September 2008 to February 2010 tested 137 cases for NPM1 status (four base pair insertion) 27 cases (~20%) positive
Slide 13 - NPM1 results From September 2008 to February 2010 tested 137 cases for NPM1 status (four base pair insertion) 27 cases (~20%) positive
Slide 14 - FLT3 and NPM1 95 FLT3 and NPM1 –ve cases
Slide 15 - Presentation vs Relapse
Slide 16 - Case 1 – Recurrence of the presentation clone Presentation – ITD ~ 23bp (NPM1–ve) WT ~23bp ITD
Slide 17 - Case 1 – Recurrence of the presentation clone Relapse – Same 23bp ITD present (NPM1-ve) WT ~23bp ITD
Slide 18 - Case 2 – importance of detecting low levels of ITD Presentation – very low levels of ~49bp ITD (NPM1+ve) WT ~49bp ITD
Slide 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
Slide 20 - Case 3 – loss of ITD Presentation – low level ~54bp ITD (NPM1-ve) WT ~54bp ITD
Slide 21 - Case 3 – loss of ITD Relapse – No evidence of FLT3 ITD (NPM1-ve) WT
Slide 22 - Case 4 – apparent change in ITD size/loss of WT allele Presentation - ~72bp ITD (NPM1+ve) WT ~72bp ITD
Slide 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
Slide 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
Slide 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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