FLT3 and NPM1 Testing in Acute Myeloid

Leukaemia

Louise StanleyNorthern 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

Bad Prognostic IndicatorComplex karyotype

Monosomy 7

deletion of 7q

FLT3 ITD

Good Prognostic Indicatort(15;17)

t(8;21)

inv(16)

NPM1 4bp insertion

Non-specific treatments e.g. BMT, Chemotherapy

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

Adaptive Treatment Strategies

Prognostic Markers Treatment RegimeFit for intensive

treatmentBad 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

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) 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

Taken from: Gale et al. (2008) Blood, 111, 2776_2784.

NPM1-ve/FLT3 ITD+ve

NPM1+ve/FLT3 ITD-ve

NPM1-ve/FLT3 ITD-veNPM1+ve/FLT3 ITD+ve

In Normal Karyotype Leukaemia (~40% of AML)

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 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 Cases1 24

2 15

3 0

4 1

Number of ITDs has no significant influence on survival

FLT3 results

WT allele

ITD ~ 72bp

Single ITD

FLT3 results

WT allele

Multiple ITDs

ITDs ~ 20, 23, 48 and 81bp

NPM1 results 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 casesNPM1 mutation only (i.e. no cytogenetic or FLT3 abnormality

13

FLT3 positive 13Abnormal Karyotype 1

FLT3 and NPM1 NPM1 FLT3

WT ITD

15 FLT3 +ve cases

14 NPM1 +ve cases

13 FLT3 and NPM1 +ve cases

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

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 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• 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)