Flt3 and npm1 testing in acute myeloid leukaemia
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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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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

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


Prognostic indicators

Prognostic Indicators

  • Cytogenetic markers and molecularly determined mutation status of FLT3 and NPM1

  • Allows a risk-adapted treatment approach


Adaptive treatment strategies

Adaptive Treatment Strategies

  • Non-specific treatments e.g. BMT, Chemotherapy

  • Target specific inhibitors – e.g. anti-FLT3 drugs (CEP-701)


Fms related tyrosine kinase flt3

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


Npm1 nucleophosmin

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


Prognostic stratification

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.


Testing strategy

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


Flt3 results

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


Flt3 results1

FLT3 results

Single ITD

WT allele

ITD ~ 72bp


Flt3 results2

FLT3 results

Multiple ITDs

WT allele

ITDs ~ 20, 23, 48 and 81bp


Npm1 results

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


Npm1 results1

NPM1 results

  • From September 2008 to February 2010 tested 137 cases for NPM1 status (four base pair insertion)

  • 27 cases (~20%) positive


Flt3 and npm1

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


Flt3 and npm1 testing in acute myeloid leukaemia

Presentation vs Relapse


Case 1 recurrence of the presentation clone

Case 1 – Recurrence of the presentation clone

  • Presentation – ITD ~ 23bp (NPM1–ve)

WT

~23bp ITD


Case 1 recurrence of the presentation clone1

Case 1 – Recurrence of the presentation clone

  • Relapse – Same 23bp ITD present (NPM1-ve)

WT

~23bp ITD


Case 2 importance of detecting low levels of itd

Case 2– importance of detecting low levels of ITD

  • Presentation – very low levels of ~49bp ITD (NPM1+ve)

WT

~49bp ITD


Case 2 importance of detecting low levels of itd1

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


Case 3 loss of itd

Case 3– loss of ITD

  • Presentation – low level ~54bp ITD (NPM1-ve)

WT

~54bp ITD


Case 3 loss of itd1

Case 3– loss of ITD

  • Relapse – No evidence of FLT3 ITD (NPM1-ve)

WT


Case 4 apparent change in itd size loss of wt allele

Case 4 – apparent change in ITD size/loss of WT allele

  • Presentation - ~72bp ITD (NPM1+ve)

WT

~72bp ITD


Case 4 apparent change in itd size loss of wt allele1

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


Conclusions future directions

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


Acknowledgements

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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