Alison Skinner Wessex Regional Genetics Laboratory

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 A Mutation B Mutation D

9. Further Analysis of the Pyrosequencing Results

10. Examples of Results Using Analysis Spreadsheet

11. Validation – 1Retesting 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 – 2Normal 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 – 3Titration • 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