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
Analysis to look for ACQUIRED abnormalities in leukaemic
clone (i.e. not constitutional)
Abnormalities can evolve during disease progression
For diagnosis and prognosis
Prognostic Indicators


Cytogenetic markers and molecularly determined mutation
status of FLT3 and NPM1
Allows a risk-adapted treatment approach
Good Prognostic Indicator
Bad Prognostic Indicator
t(15;17)
Complex karyotype
t(8;21)
Monosomy 7
inv(16)
deletion of 7q
NPM1 4bp insertion
FLT3 ITD
Adaptive Treatment Strategies

Non-specific treatments e.g. BMT, Chemotherapy
Prognostic Markers
Treatment Regime
Fit for intensive
treatment
Bad
Bone Marrow Transplant
Fit for intensive
treatment
Good
Intensive Chemotherapy
Not fit for intensive
treatment
Bad
Reduced Intensity Chemotherapy/
Palliative Care
Not fit for intensive
treatment
Good
Chemotherapy

Target specific inhibitors – e.g. anti-FLT3 drugs (CEP-701)
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
Exon 14
NPM1 (nucleophosmin)


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
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

In Normal Karyotype Leukaemia (~40% of AML)
NPM1-ve/FLT3 ITD+ve
NPM1+ve/FLT3 ITD+ve
NPM1-ve/FLT3 ITD-ve
NPM1+ve/FLT3 ITD-ve
Taken from: Gale et al. (2008) Blood, 111, 2776_2784.
Testing Strategy


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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

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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
No. of ITDs No. of Cases
1
24
2
15
3
0
4
1
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Number of ITDs has no
significant influence on
survival
FLT3 results
Single ITD
WT allele
ITD ~ 72bp
FLT3 results
Multiple ITDs
WT allele
ITDs ~ 20, 23, 48 and 81bp
NPM1 results
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
From September 2008 to February 2010 tested 137 cases for
NPM1 status (four base pair insertion)
27 cases (~20%) positive
WT allele
4bp insertion
NPM1 results


From September 2008 to February 2010 tested 137 cases for
NPM1 status (four base pair insertion)
27 cases (~20%) positive
Prognostic Marker
Number of NPM1 positive cases
NPM1 mutation only (i.e. no
cytogenetic or FLT3
abnormality
13
FLT3 positive
13
Abnormal Karyotype
1
FLT3 and NPM1
NPM1
FLT3
WT
15 FLT3
+ve cases
13 FLT3
and NPM1
+ve cases
14 NPM1
+ve cases
ITD
95 FLT3 and
NPM1 –ve cases
Presentation vs Relapse
Case 1 – Recurrence of the presentation clone

Presentation – ITD ~ 23bp (NPM1–ve)
WT
~23bp ITD
Case 1 – Recurrence of the presentation clone
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Relapse – Same 23bp ITD present (NPM1-ve)
WT
~23bp 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 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
Presentation – low level ~54bp ITD (NPM1-ve)
WT
~54bp
ITD
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

Presentation - ~72bp ITD (NPM1+ve)
WT
~72bp ITD
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
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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
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Nick Bown – Cytogenetics, Northern Genetics Service (NGS)
Helen Powell
Ruth Sutton
Molecular Genetics (NGS)
Ottie O’Brien
David Bourn
Dr G Jones – Consultant Haematologist, Freeman Hospital,
Newcastle