Presentation on theme: "Mutation testing in the EGFR gene"— Presentation transcript:
1Mutation testing in the EGFR gene Dr Ann CurtisNewGene LtdA partnership between Newcastle Hospitals NHS Foundation Trust and Newcastle University
2Meeting Agenda Introduction to NewGene EGFR testing The technical basis of the assayMutations covered by the assayTypes of samples NewGene can handleUser feedbackClose
3NewGenePartnership between Newcastle University and the Newcastle Hospitals NHS Foundation TrustEstablished in 2008.Employ latest technologies to deliver molecular diagnostics.Expertise from Northern Genetics Service and Newcastle University.Two principal analysis platformsRoche GS-FLX Next Generation SequencerSequenom™ Mass spectrometerEGFR is being delivered on the MALDI TOF platformNewGene have been successfully delivering a KRAS service on this platform for the past 12 months.
4EGFR Testing Technology Mutations included in the assay Sample requirements
5EGFR Mutations – two classes Exons 18 to 24 of EGFR gene encode the tyrosine kinase domain Important activating mutations cluster in exons 18 to 21Mutations associated with drug sensitivity:5%45%<1%40-45%V689MN700DE709KG719A/C/S/RVariousdeletionsinvolvingcodonsV765AT783AL858RL861QN826SA839TK846RG863DExon 18Exon 19Exon 20Exon 21Mutations associated with drug resistance:<1%<5%D761YT790MVarious insertionsat codons 770 &771Sharma et al. (2007) Nat Rev Cancer 7:
6Technology PCR and capillary electrophoresis on AB3730 Size differenceExon 19 deletionsExon 20 insertionsMass spectrometryAll single base mutations
7Exon 19 deletions - ~45% of all mutations BAB~15%~30%AB~40%~50%ABAB~70%A = deleted alleleB = normal allele
8Technology SEQUENOM iPLEX reaction for point mutation detection Wild type allele (T)Mutant allele (C)‘Extension’ primer of specific mass anneals immediately upstream of mutationextension primer (5500Da)extension primer (5500Da)TC+Polymerase enzyme+ddATP/ddCTP/ddTTP/ddGTPextended primer (5800Da)extended primer (6100Da)If supplied with all 4 ddNTPs, the primer is extended by one nucleotide generating a product of specific mass.AGTC
9SEQUENOMTM MALDI TOF MASS SPECTROMETER Matrix Assisted Laser Desorption/Ionisation Time of Flight mass spectrometryDetectorFlight pathTime of flightLaserSequenom chip
10SEQUENOM iPLEX output Wild type allele only (TT) 5500Da7000Da5500Da7000Da5500Da7000DaWild type allele only (TT)Wild type and mutant allele present (TC)Mutant allele only (CC)
11Power of SEQUENOM iPLEX for EGFR genotyping Step 1: Multiplex PCR using multiple sets of primers per reactionStep 2: Extension reactionPLEX groupD761Y L858R K846R G863D G719CV765A T783A N700D G719A G719ST790M A839T G719RE709KL861QN826SV689M
12Validation Phase 1 Phase 2 Phase 3 Phase 4 All assays performed on control DNA from bloodAll wild type genotypes reproducibly detectedPhase 2DNA extracted from FFPE tissue (anonymised tumour tissue curls)Some minor modifications and redesign of the assayReproduciblePhase 3Mutation control samples from other laboratoriesQuantitation controls – pick up low levels of mutantPhase 4Parallel testing (6 samples)
13Development of EGFR assay DNA from paraffin embedded tissue Plex 1 – identifies 7 mutationsWT-V689MWT-T790MWT-N826SWT-L861QWT-V765AWT-D761YWT-E709K/Q
14Development of EGFR assay Plex 2 – identifies 3 mutations, including L858RWT-A839TWT-L858RWT-T783T
15Development of EGFR assay L858R pos Plex 2 –positive for L858R mutationMutantWild type
17Sensitivity and specificity Assuming sample contains at least 10% tumour cellsNewGene test will pick up mutations in 95% of mutation positive samplesFrom other assays (eg KRAS), specificity of technique is close to 100%
18Samples for EGFR testing Histology samplesBiopsiesResectionsMetastatic depositsSamples on slidesCytology samplesFine Needle AspiratesBronchial washingsPleural effusionsParaffin embedded tissue FFPECut in curls from blocks (10µm) – NewGene can do this ifrequiredTumour content as high as possible – ideal >30% but min10%NewGene can perform H&E assessment of sample for tumourcell 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.
19External Quality Assessment Pilot scheme through UK NEQAS for Molecular Genetics3 samples distributed 4 times per yearMounted slides or curlsFFPE and “fixed” cell-linesMutation analysis and interpretationNewGene have received a set of 3 samples in May.
20EGFR mutation testing -the referral pathway 1° referrer - oncologistinstructPathology labwhere sample is storedsend sampleNewGeneCentre for Lifetesting5-7 working daysResult to oncologist (fax)Written report to oncologist and pathologist
22EGFR 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.