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