1. Development of a Diagnostic Test for Mutations in NPM1 Exon 12 in Cytogenetically Normal Acute Myeloid Leukaemia (AML) Patients Alison Skinner Wessex Regional Genetics Laboratory

2. NPM1 function Implicated in leukaemia as a translocation partner for various oncogenes Nucleolar phosphoprotein present predominantly in the nucleolus Regulates translational activity of p53 after stress Involved in centrosome duplication in the cell cycle via cyclin E/CDK2 phosphorylation

3. Effect of NPM1 mutations Gives prognostic information to the AML patients with a normal karyotype (phenotypically variable) Favourable prognosis in the absence of the FLT3 ITD Better response to induction therapy and have a better overall survival / longer event free survival

4. Acquired Mutations in NPM1 Wildtype: GCTATTCAAGATCTCTGGCAGTGGAGGAAGTCTCTTTAAgaaaatag -A--I--Q--D--L--W--Q--W--R--K--S--L--*--------- Mutation A: GCTATTCAAGATCTCTGTCTGGCAGTGGAGGAAGTCTCTTTAAgaaaatag -A--I--Q--D--L--C--L--A--V--E--E--V--S--L--R--K--*- Mutation B: GCTATTCAAGATCTCTGCATGGCAGTGGAGGAAGTCTCTTTAAgaaaatag -A--I--Q--D--L--C--M--A--V--E--E--V--S--L--R--K--*- Mutation D: GCTATTCAAGATCTCTGCCTGGCAGTGGAGGAAGTCTCTTTAAgaaaatag -A--I--Q--D--L--C--L--A--V--E--E--V--S--L--R--K--*-

5. Results of Direct Sequencing From the original 66 samples 41 had no visible mutation 1 had an intronic mutation of unknown significance 19 had a frameshift mutation: –13 had mutation ‘A’ –1 had mutation ‘B’ –3 had mutation ‘D’ –2 had novel mutations which still produced the same NES

6. Principles of Pyrosequencing Image from www.pyrosequencing.com

7. Designing the Pyrosequencing Assay Wildtype Mutation A Mutation B Mutation D

8. Testing the Pyrosequencing Assay Normal Mutation D Mutation A Mutation B

9. Further Analysis of the Pyrosequencing Results

10. Examples of Results Using Analysis Spreadsheet

11. Validation – 1 Retesting the Original Cohort All 66 samples that were tested by direct sequencing were re-tested using the pyrosequencing assay 6 / 66 failed –1 sample failed for pyrosequencing but was normal on direct sequencing –1 sample failed for direct sequencing but had mutation ‘A’ on pyrosequencing, –All other failures had failed for both techniques Results of all other samples matched the results for direct sequencing

12. Validation – 2 Normal controls 3 / 96 failed There was no evidence of mutations in the normal test plate A quantification below 10% should be treated as normal / with caution (depending on the quality of the data)

13. Validation – 3 Titration A titration of mutational load was set up Reliably sensitive to around 20% mutation

14. Summary The test is sensitive and relatively high- throughput Identifies and quantifies the common NPM1 exon 12 mutations Is able to identify other ‘novel’ mutations in the region Helpful in identifying patients who have a favourable prognosis

15. Acknowledgements Dr Helen White – NGRL (Wessex) Prof. Nick Cross – WRGL Christine Waterman - WRGL