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Development of an EGFR/KRAS testing service for Non-Small Cell Lung Cancer (NSCLC) Joel Tracey 1, Caroline Clark 1, Christine Bell 1, Keith Kerr 2, Marianne.

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Presentation on theme: "Development of an EGFR/KRAS testing service for Non-Small Cell Lung Cancer (NSCLC) Joel Tracey 1, Caroline Clark 1, Christine Bell 1, Keith Kerr 2, Marianne."— Presentation transcript:

1 Development of an EGFR/KRAS testing service for Non-Small Cell Lung Cancer (NSCLC) Joel Tracey 1, Caroline Clark 1, Christine Bell 1, Keith Kerr 2, Marianne Nicholson 3, Aileen Osborne 1, Zosia Miedzybrodzka 1, Kevin Kelly 1 1 Department of Medical Genetics, Polwarth Building, Aberdeen Royal Infirmary, Aberdeen 2 Department of Pathology, Aberdeen Royal Infirmary, Aberdeen 3 Clinical Oncology, Aberdeen Royal Infirmary, Aberdeen

2 Non Small Cell Lung Cancer (NSCLC)  Lung cancer is one of the most commonly diagnosed types of cancer in the UK  Leading cause of cancer-related death in both men and women  Non-Small Cell Lung Cancer ~ 80% (3)  Adenocarcinoma (ADC)  Squamous cell carcinoma (SCC)  Large cell carcinoma (LCC) Percentage of NSCLC subtypes in UK

3 EGFR and KRAS in NSCLC  Acquired mutations in the EGFR and KRAS genes are important in the development of NSCLC  Mutations result in inappropriately activated proteins – tumour cells become ‘addicted’ to growth signals  EGFR and KRAS mutations most common in adenocarcinoma  EGFR and KRAS mutations are mutually exclusive

4  Surgery - possible in about 20% of cases (4)  Cytotoxic chemotherapy and/or radiotherapy - mostly ineffective  Survival rate poor (7% alive 5 years after diagnosis) (4)  Tyrosine Kinase Inhibitors (TKI) – new type of chemotherapeutic agent – fewer side-effects than cytotoxic chemotherapy  Target and block growth factor signals – e.g. Epidermal Growth Factor Receptor (EGFR) Treatment of NSCLC

5 EGFR Tyrosine Kinase Inhibitors  Erlotinib (Tarceva) and Gefitinib (Irresa)  EGFR targeted TKIs can be used for treatment of NSCLC patients with somatic activating EGFR mutations  Mutations within EGFR TK domain enable TKIs to bind with greater affinity  Patients with activating EGFR mutations have a better response to TKI therapy and improved survival TKI  Patients with KRAS mutations show little or no response to TKI treatment

6 Clinical Trial – IPASS Study EGFR mutation +ve (M+) patients respond better to TKI therapy than chemotherapy but.... EGFR mutation –ve patients (M-) have a poorer response to TKI therapy than chemotherapy!!! Probability of Progression Free Survival Time (months)

7 EGFR mutations  Mutations in the EGFR gene found in 10-15% of NSCLC patients (5, 6)  Exon 19 deletions & L858R (Exon 21) make up 85-90% of all mutation +ve cases (7)  Exon 20 mutations (e.g. T790M) commonly resistance mutations

8 CAAGCGGTG ACCAAAA G G T CCC TT Codons Wt seq Multi- variable mutations T  KRAS mutations occur in ~30% of NSCLC tumours (8)  Codon 12 most common mutation site KRAS mutations

9 Project Aims 1.Develop and set-up methods for EGFR and KRAS analysis 2.Determine best methods for analysis of EGFR and KRAS mutations 3.Develop and validate appropriate methodologies for testing

10 Samples  48 Adenocarcinoma patient samples (ARI Pathology Dept)  4 EGFR +ve control samples (Holland)  All were FFPE lung tumour samples (cores, slides & rolls)  14 DNA samples for KRAS analysis (Transgenomic Inc)  Mutations in codon 12, 13 and 61  Varied mutation levels (3% to 33%)

11 Challenges with NSCLC testing using FFPE samples Frequently low sample quantity = Low DNA yield Variable tumour content within sample (<5% to 100%) Poor DNA quality - Degradation (<300bp), PCR inhibitors Genetic heterogeneity – inter- and intra-tumour variation Pathology departments involvement at this stage important to maximise % tumour – macro- dissection

12 Methodology Plan Extract DNA from FFPE lung tumour samples (Dewax, phenol/chloroform) Quantify all DNA samples (Nanodrop) PCR amplification using specific primers (in-house/published) Quantify all DNA samples PCR amplification using specific primers EGFR Direct Sequencing WAVE HS dHPLC + fragment collection WAVE Surveyor Ex19 Fragment Length Analysis Ex21 Pyrosequencing KRAS Direct Sequencing WAVE Surveyor Pyrosequencing

13 Principles of Methods Used WAVE Surveyor – Enzymatic method, detects DNA mismatches, WAVE size separation, mutation identified by presence of cleavage products WAVE HS dHPLC – Partially denaturing, High sensitivity by fluorescent detection (x100), mutation identified by presence of mutant/WT heteroduplex peaks Fragment collection – After passing through detector eluted DNA fragments were collected in vials at 30s intervals

14 Principles of Methods Used Fragment Length Analysis – FAM labelled PCR products, size separation on ABI 3130, analysis using Gene Marker software Pyrosequencing – Real-time sequence data, Pyrophosphate (PPi) substrate for reaction cascade, light produced measured – relative to nucleotides incorporated

15 Summary of KRAS Results  33% (16/48) of patient samples positive for KRAS mutations – tested by both pyrosequencing and direct sequencing Percentage of KRAS mutations identified (by codon) Blind study (Transgenomic samples)  Pyrosequencer detected all mutations in Transgenomic samples (lowest = 3%)  2 samples not detected by the WAVE Surveyor method (3%)  6 samples were below the detection limit of sequencing (<10%)

16 EGFR Results Sample: EGFR +ve Control Mutation: Exon 19 Del (c del; p.L747 – T751del) WAVE HS dHPLC Sequencing +ve control WT +ve control WT WAVE Surveyor Uncleaved product Size control +ve control WT

17 Enrichment of EGFR mutant by WAVE dHPLC + fragment collection Ex 19 WT Ex 19 del (enriched by fragment collection + repeat PCR) Ex 19 del (direct seq) Sequences analysed with Mutation Surveyor software (Soft Genetics)

18 Additional methods G863D L861Q L858R Ex 19 del Ex 19 WT L858R mutant Ex 19 Fragment length analysis Ex 21 Pyro- sequencing

19 Summary of EGFR Results  12.5% (6/48) of patient samples positive for EGFR mutation (Ex Deletions; Ex Insertion; Ex L858R)  All methods detected Ex19 del mutations in 4 EGFR +ve control samples  WAVE Surveyor confirmed all mutations found by direct sequence analysis  One Ex19 del mutant too low to report by direct sequence analysis but clear by WAVE Surveyor and Fragment length analysis  Pyrosequencer – successfully detected Ex21 mutants  Confident no false positive results All EGFR +ve samples were KRAS –ve Mutations confirmed by multiple methods

20 Comparison of EGFR methods  Times based on analysis of 15 samples  Cost per sample does not include staff costs WAVE HS dHPLC SURVEYORSequencing Fragment analysis (Ex19 only) Pyrosequencing (Ex 21 only) Hands on time *2hr 30min2hr 45min 1hr 30min2hr 15min Cost (per sample) £16.50£15.60£26.70£0.57£8.04 Results analysis time* 45min 1hr 30min30min Total Time to result * 40hr 40min24hr 45min11hr 15min6hr4hr 35min Sample required 120ng 20ng Detection Limit?~4-5%~10%?3-5%

21 Conclusions  Pick-up rate of EGFR mutations consistent with published data  Direct sequencing pick-up rate higher than expected. This likely to be due to enrichment of samples for tumour tissue by macro- dissection  WAVE Surveyor, fragment analysis and pyrosequencing methods may be useful as a higher sensitivity screen in conjunction with direct sequencing  Fragment collection is a viable method for enrichment of low level mutations

22 Current Testing Strategy Assessment of tumour content and macrodissection SCC / LCC NSCLC Patient (M/F, smoker/non-smoker) Adenocarcinoma EGFR Ex PCR KRAS codons 12, 13 and 61 PCR Pathology Molecular Genetics Direct Sequencing /Pyrosequencing Direct Sequencing/WAVE Surveyor/Fragment Length Analysis Not Tested Report

23 Acknowledgements  Aberdeen Lab Caroline Clark Christine Bell Aileen Osborne Louise Carnegie Heather Greig Kevin Kelly  Transgenomic Gerald Martin  Clinical/Pathology Keith Kerr Marianne Nicholson Zosia Miedzybrodzka  Astra Zeneca For providing funding

24 References 1 Ferlay J. et al. Estimates of the cancer incidence and mortality in Europe in Annals of Oncology (2007) 18: Harkness E.F. et al. Changing trends in incidence of lung cancer by histologic type in Scotland. Int. J. Cancer (2002) 102: D’Addario G. & Felip E. Non-small-cell lung cancer: ESMO Clinical Recommendations for diagnosis, treatment and follow-up. Annals of Oncology (2008) 19 (Sup 2): ii39 - ii40 4 Scottish Executive Health Department. Cancer scenarios: an aid to planning cancer services in Scotland in the next decade The Scottish Executive: Edinburgh.. 5Janne P.A. et al. A rapid and sensitive enzymatic method for epidermal growth factor receptor mutation screening. Clin Cancer Res (2006); 12 (3): Sequist L.V. et al. Epidermal Growth Factor Receptor mutation testing in the care of lung cancer patients. Clin Cancer Res (2006); 12 (Sup 14): 4403s – 4408s 7Sequist L.V. & Lynch T.J. EGFR Tyrosine Kinase Inhibitors in lung cancer: an evolving story. Ann Rev Med (2008) 59: Do, H. et al. High resolution melting analysis for rapid and sensitive EGFR and KRAS detection in formalin fixed paraffin embedded biopsies. BMC Cancer (2008) 8:142


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