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

1. Implementation of nucleophosmin (NPM1) screening in acute myeloid leukaemia (AML) patients. Trainee Clinical Scientist ProjectRachel ColemanCMGS 2010

2. 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%

3. 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

4. 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

5. 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

6. 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

7. 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

8. 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

9. Fragment analysis results • 52/97 CN-AML pts NPM1+ = pick up rate 55% • One discrepant result

10. 51/94 CN-AML pts NPM1+ = pick up rate 54% Sequencing results

11. 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

12. MRD monitoring - results • 46 NPM1+ patients throughout clinical course of the disease Key Marrow Blood Sensitivity of detection

13. NPM1 and FLT3 • FLT3 routinely screened – clinical service • 24% of cohort were NPM1+/FLT3+

14. NPM1/FLT3 status at diagnosis associated with prognostically significant risk groups ↑NPM1+/FLT3- ↑NPM1+/FLT3+ Key Marrow Blood Sensitivity of detection

15. 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.

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