Transcript Rachel Coleman

Implementation of nucleophosmin
(NPM1) screening in acute myeloid
leukaemia (AML) patients.
Trainee Clinical Scientist Project
Rachel Coleman
CMGS 2010
Acute Myeloid Leukaemia (AML)
• Phenotypically and genetically heterogenous disease
• 70% of all acute leukaemia cases
• Incidence of 4 in 100,000 - ↑ cases: ↑ age
• Disease progression is rapid and typically fatal within
weeks to months if left untreated
• Depending upon the sub-type of the disease:
 Disease free survival rates 15-70%
 Relapse rates 33-78%
Classification of AML
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40-50% of AML cases are cytogenetically normal (CN-AML)
Largely poorly understood
Have substantially different clinical courses
Genetic classification → further subdivide → stratify treatment
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 FLT3
 NPM1
a
Nucleophosmin (NPM1) and CN-AML
• Ubiquitously expressed, multi functional phosphoprotein
• Chaperone protein – nuclear localisation
• Falini et al (2005) NEJM, 352(3), 254-66
 Aberrant cytoplasmic localisation of NPM1 in CN-AML
 Heterozygous frameshift mutations in exon 12
 Disrupts conserved NLS and creates a new NES
NPM1: prognostic indicator
• 50-60% of CN-AML cases = 1/3 of all adult AML cases
• One of the most frequently mutated genes in AML
• Associated with a good prognosis without concomitant FLT3
Schnittger et al (2005) Blood, 106(12), 3733-3739
• Valuable and important prognostic indicator in AML
Project testing strategy
AIM: To validate diagnostic screening and minimal residual disease
monitoring assays for the detection of NPM1 mutations in AML.
Define patient cohort
Validate diagnostic screen by fragment analysis
Confirm and characterise NPM1 mutation by sequencing
Evaluate an MRD assay suitable for monitoring NPM1 patients
Correlate NPM1 and FLT3 status to define prognostic subgroups
Patient cohort
• AML-15 trial → 97 CN-AML patients
• 30 normal controls
• Mutation A (c.860_3dupTCTG; p.Trp288CysfsX12) positive control
• cDNA template of choice
Diagnostic screening
Fragment analysis
• Frameshift mutations – fragment size shift
• Two sets of primers used for validation
 Published primers – amplifying exon 12
 Newly designed primers – amplifying exons 9 – 12
Sequencing
• Same primer sequences – M13 tag
Fragment analysis results
• 52/97 CN-AML pts NPM1+ = pick up rate 55%
• One discrepant result
Sequencing results
• 51/94 CN-AML pts NPM1+ = pick up rate 54%
Minimal residual disease (MRD) monitoring
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Real-time Quantitative PCR (RQ-PCR) on cDNA
Common exon 12 mutations only
Common forward primer/mutation specific reverse primers
MGB probe
Commercial NPM1+ plasmid standards
Results normalised to ABL1 gene
MRD monitoring - results
• 46 NPM1+ patients throughout clinical course of the disease
Key
Marrow
Blood
Sensitivity of
detection
NPM1 and FLT3
• FLT3 routinely screened – clinical service
• 24% of cohort were NPM1+/FLT3+
NPM1/FLT3 status at diagnosis associated
with prognostically significant risk groups
↑NPM1+/FLT3-
↑NPM1+/FLT3+
Key
Marrow
Blood
Sensitivity of
detection
Conclusions
• NPM1 is one of the most frequently mutated genes in AML
• Proven to be an important and prognostically significant molecular
marker in AML
• Shown to be associated with a favourable outcome
• Mutation detection quick and easy
• Stable and reliable marker for long-term MRD monitoring
• Clinical service:
 NPM1/FLT3 status at diagnosis will have prognostic value predictive of
overall survival
 Can aid in risk stratification and patient treatment
 MRD monitoring can follow the dynamics of the leukaemic clone making
it possible for early intervention if a predicted relapse is detected by a
rise in transcript levels.
Acknowledgements
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Susanna Akiki
Mike Griffiths
Joanne Mason
Fiona Macdonald
Jennie Bell
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Danielle Crompton
Lindsey Bradley
Molecular Oncology
Fragment analysis team
Sequencing team