1. Mutation testing in the EGFR gene
Dr Ann Curtis
NewGene Ltd
A partnership between Newcastle Hospitals NHS Foundation Trust and Newcastle University

2. Meeting Agenda Introduction to NewGene EGFR testing
The technical basis of the assay
Mutations covered by the assay
Types of samples NewGene can handle
User feedback
Close

3. NewGene
Partnership between Newcastle University and the Newcastle Hospitals NHS Foundation Trust
Established in 2008.
Employ latest technologies to deliver molecular diagnostics.
Expertise from Northern Genetics Service and Newcastle University.
Two principal analysis platforms
Roche GS-FLX Next Generation Sequencer
Sequenom™ Mass spectrometer
EGFR is being delivered on the MALDI TOF platform
NewGene have been successfully delivering a KRAS service on this platform for the past 12 months.

4. EGFR Testing Technology Mutations included in the assay
Sample requirements

5. EGFR Mutations – two classes Exons 18 to 24 of EGFR gene encode the tyrosine kinase domain Important activating mutations cluster in exons 18 to 21
Mutations associated with drug sensitivity: 5%
45%
<1%
40-45%
V689M
N700D
E709K
G719A/C/S/R
Various
deletions
involving
codons
V765A
T783A
L858R
L861Q
N826S
A839T
K846R
G863D
Exon 18
Exon 19
Exon 20
Exon 21
Mutations associated with drug resistance:
<1%
<5%
D761Y
T790M
Various insertions
at codons 770 &771
Sharma et al. (2007) Nat Rev Cancer 7:

6. Technology PCR and capillary electrophoresis on AB3730
Size difference
Exon 19 deletions
Exon 20 insertions
Mass spectrometry
All single base mutations

7. Exon 19 deletions - ~45% of all mutationsB A B
~15%
~30% A B
~40%
~50% A B A B
~70%
A = deleted allele
B = normal allele

8. Technology SEQUENOM iPLEX reaction for point mutation detection
Wild type allele (T)
Mutant allele (C)
‘Extension’ primer of specific mass anneals immediately upstream of mutation
extension primer (5500Da)
extension primer (5500Da) T C
+Polymerase enzyme
+ddATP/ddCTP/
ddTTP/ddGTP
extended primer (5800Da)
extended primer (6100Da)
If supplied with all 4 ddNTPs, the primer is extended by one nucleotide generating a product of specific mass. A G T C

9. SEQUENOMTM MALDI TOF MASS SPECTROMETER Matrix Assisted Laser Desorption/Ionisation Time of Flight mass spectrometry
Detector
Flight path
Time of flight
Laser
Sequenom chip

10. SEQUENOM iPLEX output Wild type allele only (TT)
5500Da
7000Da
5500Da
7000Da
5500Da
7000Da
Wild type allele only (TT)
Wild type and mutant allele present (TC)
Mutant allele only (CC)

11. Power of SEQUENOM iPLEX for EGFR genotyping
Step 1: Multiplex PCR using multiple sets of primers per reaction
Step 2: Extension reaction
PLEX group
D761Y L858R K846R G863D G719C
V765A T783A N700D G719A G719S
T790M A839T G719R
E709K
L861Q
N826S
V689M

12. Validation Phase 1 Phase 2 Phase 3 Phase 4
All assays performed on control DNA from blood
All wild type genotypes reproducibly detected
Phase 2
DNA extracted from FFPE tissue (anonymised tumour tissue curls)
Some minor modifications and redesign of the assay
Reproducible
Phase 3
Mutation control samples from other laboratories
Quantitation controls – pick up low levels of mutant
Phase 4
Parallel testing (6 samples)

13. Development of EGFR assay DNA from paraffin embedded tissue
Plex 1 – identifies 7 mutations
WT-V689M
WT-T790M
WT-N826S
WT-L861Q
WT-V765A
WT-D761Y
WT-E709K/Q

14. Development of EGFR assay
Plex 2 – identifies 3 mutations, including L858R
WT-A839T
WT-L858R
WT-T783T

15. Development of EGFR assay L858R pos
Plex 2 –positive for L858R mutation
Mutant
Wild type

16. Development of EGFR assay Plex 3
Plex 3 – identifies 2 mutations
Plex 4 – identifies 2 mutations
Plex 5 – identifies 3 mutations at
codon 719

17. Sensitivity and specificity
Assuming sample contains at least 10% tumour cells
NewGene test will pick up mutations in 95% of mutation positive samples
From other assays (eg KRAS), specificity of technique is close to 100%

18. Samples for EGFR testing
Histology samples
Biopsies
Resections
Metastatic deposits
Samples on slides
Cytology samples
Fine Needle Aspirates
Bronchial washings
Pleural effusions
Paraffin embedded tissue FFPE
Cut in curls from blocks (10µm) – NewGene can do this if
required
Tumour content as high as possible – ideal >30% but min
10%
NewGene can perform H&E assessment of sample for tumour
cell quality and quantity.
We aim for a 5 working day turnaround, however for tissue blocks and slides this will vary between 5 to7 working days.

19. External Quality Assessment
Pilot scheme through UK NEQAS for Molecular Genetics
3 samples distributed 4 times per year
Mounted slides or curls
FFPE and “fixed” cell-lines
Mutation analysis and interpretation
NewGene have received a set of 3 samples in May.

20. EGFR mutation testing -the referral pathway
1° referrer - oncologist
instruct
Pathology lab
where sample is stored
send sample
NewGene
Centre for Life
testing
5-7 working days
Result to oncologist (fax)
Written report to oncologist and pathologist

21. Referral form

22. EGFR testing to date Since March 2011 NewGene have tested 196 samples.
Positive mutation rate has been 5%.
Average turnaround time has been 5 working days.
We have developed the service to include all types of samples including slides.
We offer testing at a very competitive price and we bill each PCT directly.

23. Acknowledgements Technical Management www.newgene.org.uk Ria Chaston
Jon Coxhead
Charlotte Masters
Dayne Bromley
Claire Bourn
Laura Boyd
Management
David Allison (Chairman)
David Huntley (CEO)
Jonathan Robinson (Business manager)
Lesley Gooding (Admin)