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

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Presentation on theme: "Mutation testing in the EGFR gene Dr Ann Curtis NewGene Ltd A partnership between Newcastle Hospitals NHS Foundation Trust and Newcastle University."— Presentation transcript:

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 Partnership between Newcastle University and the Newcastle Hospitals NHS Foundation Trust Established in 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

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 Exon 18Exon 19Exon 20Exon 21 Mutations associated with drug sensitivity: 5%45%<1%40-45% Sharma et al. (2007) Nat Rev Cancer 7: V689M N700D E709K G719A/C/S/R Various deletions involving codons V765A T783A L858R L861Q N826S A839T K846R G863D Mutations associated with drug resistance: <1% <5% D761Y T790M Various insertions at codons 770 &771

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 mutations A = deleted allele B = normal allele A B A B A B A B A B ~15% ~30% ~40% ~50% ~70%

8 Technology SEQUENOM 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) extended primer (5800Da) +Polymerase enzyme +ddATP/ddCTP/ ddTTP/ddGTP Wild type allele (T) A G

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

10 SEQUENOM iPLEX output 5500Da 7000Da5500Da 7000Da 5500Da 7000Da Wild type allele only (TT) Mutant allele only (CC)Wild type and mutant allele present (TC)

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 D761YL858RK846RG863DG719C V765AT783AN700DG719AG719S T790MA839TG719R E709K L861Q N826S V689M

12 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)

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

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

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

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 Cytology 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.

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 Pathology lab where sample is stored instruct 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 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)

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