Development of a Diagnostic Test for Mutations in NPM1 Exon 12 in

Cytogenetically Normal Acute Myeloid Leukaemia (AML) Patients

Alison SkinnerWessex Regional Genetics Laboratory

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

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

Acquired Mutations in NPM1Wildtype:

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--*-

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

Principles of Pyrosequencing

Image from www.pyrosequencing.com

Designing the Pyrosequencing Assay

Wildtype

Mutation A

Mutation B

Mutation D

Testing the Pyrosequencing Assay

100E S G C T C T G C A T A G T C G C A G A G T C T C

A:100.0%T:0.0%

T:100.0%C:0.0%

5 10 15 20

Normal

100

120

E S G C T C T G C A T A G T C G C A G A G T C T C

A:53.8%T:46.2%

T:53.5%C:46.5%

5 10 15 20

Mutation D

100

120

E S G C T C T G C A T A G T C G C A G A G T C T C

A:59.1%T:40.9%

T:57.8%C:42.2%

5 10 15 20

Mutation A

100

110

120

E S G C T C T G C A T A G T C G C A G A G T C T C

A:71.7%T:28.3%

T:64.2%C:35.8%

5 10 15 20

Mutation B

Further Analysis of the Pyrosequencing Results

Examples of Results Using Analysis Spreadsheet

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

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)

Validation – 3Titration

• A titration of mutational load was set up• Reliably sensitive to around 20% mutation

-10

0

10

20

30

40

50

60

Neat 9:1 8:2 7:3 6:4 5:5 4:6 3:7 2:8 1:9

Dilution

%

A

B

D

Linear (A)

Linear (D)

Linear (B)

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

Acknowledgements

• Dr Helen White – NGRL (Wessex)

• Prof. Nick Cross – WRGL

• Christine Waterman - WRGL