Development of a Diagnostic Test for Mutations in NPM1 Exon 12 in Cytogenetically Normal Acute Myeloid Leukaemia (AML) Patients

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 GCTATTCAAGATCTCTGGCAGTGGAGGAAGTCTCTTTA
Agaaaatag -A--I--Q--D--L--W--Q--W--R--K--S--L---
--------
Mutation A GCTATTCAAGATCTCTGTCTGGCAGTGGAGGAAGTCT
CTTTAAgaaaatag -A--I--Q--D--L--C--L--A--V--E--E--
V--S--L--R--K---
Mutation B GCTATTCAAGATCTCTGCATGGCAGTGGAGGAAGTCT
CTTTAAgaaaatag -A--I--Q--D--L--C--M--A--V--E--E--
V--S--L--R--K---
Mutation D GCTATTCAAGATCTCTGCCTGGCAGTGGAGGAAGTCT
CTTTAAgaaaatag -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