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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

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implementation of nucleophosmin npm1 screening in acute myeloid leukaemia aml patients

Implementation of nucleophosmin (NPM1) screening in acute myeloid leukaemia (AML) patients.

Trainee Clinical Scientist ProjectRachel ColemanCMGS 2010

acute myeloid leukaemia aml
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
Classification of AML
  • 40-50% of AML cases are cytogenetically normal (CN-AML)
  • Largely poorly understood
  • Have substantially different clinical courses
  • Genetic classification → further subdivide → stratify treatment
        • 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

Nucleophosmin (NPM1) and CN-AML
npm1 prognostic indicator
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
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
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
slide8
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

Diagnostic screening

fragment analysis results
Fragment analysis results
  • 52/97 CN-AML pts NPM1+ = pick up rate 55%
  • One discrepant result
minimal residual disease mrd monitoring
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

Minimal residual disease (MRD) monitoring
mrd monitoring results
MRD monitoring - results
  • 46 NPM1+ patients throughout clinical course of the disease

Key

Marrow

Blood

Sensitivity of detection

npm1 and flt3
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 status at diagnosis associated with prognostically significant risk groups

↑NPM1+/FLT3-

↑NPM1+/FLT3+

Key

Marrow

Blood

Sensitivity of detection

conclusions
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
Acknowledgements
  • Susanna Akiki
  • Mike Griffiths
  • Joanne Mason
  • Fiona Macdonald
  • Jennie Bell
  • Danielle Crompton
  • Lindsey Bradley
  • Molecular Oncology
  • Fragment analysis team
  • Sequencing team