Mutation testing in the EGFR gene
Dr Ann Curtis
NewGene LtdA partnership between Newcastle Hospitals NHS Foundation
Trust and Newcastle University
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
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.
NewGene
EGFR Testing
Technology
Mutations included in the assay
Sample requirements
EGFR Mutations – two classesExons 18 to 24 of EGFR gene encode the tyrosine kinase domainImportant activating mutations cluster in exons 18 to 21
Exon 18 Exon 19 Exon 20 Exon 21
Mutations associated with drug sensitivity:
5% 45% <1% 40-45%
Sharma et al. (2007) Nat Rev Cancer 7: 169-181
V689MN700DE709KG719A/C/S/R
Various deletions involving codons 746-759
V765AT783A
L858RL861QN826SA839TK846RG863D
Mutations associated with drug resistance:<1% <5%
D761Y T790MVarious insertionsat codons 770 &771
Technology
PCR and capillary electrophoresis on AB3730 Size difference Exon 19 deletions Exon 20 insertions
Mass spectrometry All single base mutations
Exon 19 deletions - ~45% of all mutations
A = deleted alleleB = normal allele
A B
A B A BA B
A B
~15% ~30% ~40%
~50% ~70%
TechnologySEQUENOM iPLEX reaction for point mutation detection
‘Extension’ primer of specific mass anneals immediately upstream of mutation
If supplied with all 4 ddNTPs, the primer is extended by one nucleotide generating a product of specific mass.
extended primer (6100Da)
T
C
C
T
Mutant allele (C)
extension primer (5500Da) extension primer (5500Da)
extended primer (5800Da)
+Polymerase enzyme+ddATP/ddCTP/ ddTTP/ddGTP
Wild type allele (T)
A G
SEQUENOMTM MALDI TOF MASS SPECTROMETERMatrix Assisted Laser Desorption/Ionisation Time of Flight mass spectrometry
Laser
Flight path
Detector
Time of flight
Sequenom chip
SEQUENOM iPLEX output
5500Da 7000Da 5500Da 7000Da 5500Da 7000Da
Wild type allele only (TT)
Mutant allele only (CC)Wild type and mutant allele present (TC)
Power of SEQUENOM iPLEX for EGFR genotyping
Step 1: Multiplex PCR using multiple sets of primers per reaction
Step 2: Extension reaction
PLEX group 1 2 3 4 5D761Y L858R K846R G863D G719CV765A T783A N700D G719A G719ST790M A839T G719RE709KL861QN826SV689M
Validation
Phase 1 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)
Development of EGFR assayDNA from paraffin embedded tissue
WT-V689M
WT-T790M
WT-N826SWT-L861Q WT-V765A
WT-D761Y
WT-E709K/Q
Plex 1 – identifies 7 mutations
Development of EGFR assay
WT-A839T
WT-L858R
WT-T783T
Plex 2 – identifies 3 mutations, including L858R
Development of EGFR assay L858R pos
Plex 2 –positive for L858R mutation
Wild type
Mutant
Development of EGFR assay Plex 3Plex 3 – identifies 2 mutations
Plex 4 – identifies 2 mutations
Plex 5 – identifies 3 mutations at codon 719
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%
Samples for EGFR testingCytology samples
•Fine Needle Aspirates•Bronchial washings•Pleural effusions
Histology samples•Biopsies•Resections •Metastatic deposits•Samples on slides
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.
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.
EGFR mutation testing-the referral pathway
1° referrer - oncologist
Pathology lab where sample is stored
instruct
send sample
NewGeneCentre for Life
testing5-7 working days
•Result to oncologist (fax)•Written report to oncologist and pathologist
Referral form
EGFR testing to date
www.newgene.org.uk
• 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.
Acknowledgements
Technical 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)
www.newgene.org.uk