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Development of an EGFR/KRAS testing service for Non-Small Cell Lung Cancer (NSCLC)

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

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Development of an EGFR/KRAS testing service for Non-Small Cell Lung Cancer (NSCLC)

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  1. Development of an EGFR/KRAS testing service for Non-Small Cell Lung Cancer (NSCLC) Joel Tracey1, Caroline Clark1, Christine Bell1, Keith Kerr2, Marianne Nicholson3, Aileen Osborne1, Zosia Miedzybrodzka1,Kevin Kelly1 1Department of Medical Genetics, Polwarth Building, Aberdeen Royal Infirmary, Aberdeen 2Department of Pathology, Aberdeen Royal Infirmary, Aberdeen 3Clinical Oncology, Aberdeen Royal Infirmary, Aberdeen

  2. 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) Non Small Cell Lung Cancer (NSCLC) Percentage of NSCLC subtypes in UK

  3. 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 EGFR and KRAS in NSCLC

  4. Treatment of NSCLC • 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)

  5. TKI 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 • 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. Codons 12 13 61 Wt seq C A A G G T G G C A A A A A C C Multi-variable mutations C C C G T G T T T KRAS mutations • KRAS mutations occur in ~30% of NSCLC tumours (8) • Codon 12 most common mutation site

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

  10. Samples • 48 Adenocarcinoma patient samples (ARI Pathology Dept) • 4EGFR +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 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 WAVE Surveyor – Enzymatic method, detects DNA mismatches, WAVE size separation, mutation identified by presence of cleavage products

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

  15. 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%) 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)

  16. Size control +ve control WT EGFR Results Sample: EGFR +ve Control Mutation: Exon 19 Del (c.2240-2254del; p.L747 – T751del) +ve control Sequencing WT 267 298 257 174 Uncleaved product WAVE Surveyor 102 80 +ve control WT WAVE HS dHPLC

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

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

  19. Summary of EGFR Results • 12.5% (6/48) of patient samples positive for EGFR mutation (Ex 19 - 3 Deletions; Ex 20 - 1 Insertion; Ex 21 - 2 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

  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 NSCLC Patient (M/F, smoker/non-smoker) SCC / LCC Adenocarcinoma Not Tested Assessment of tumour content and macrodissection Pathology Molecular Genetics EGFR Ex18-21 PCR KRAS codons 12, 13 and 61 PCR Direct Sequencing /Pyrosequencing Direct Sequencing/WAVE Surveyor/Fragment Length Analysis Report

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

  24. References 1 Ferlay J. et al. Estimates of the cancer incidence and mortality in Europe in 2006. Annals of Oncology (2007) 18: 581-5923 2 Harkness E.F. et al. Changing trends in incidence of lung cancer by histologic type in Scotland.Int. J. Cancer (2002) 102: 179-183 3 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.. 2001 The Scottish Executive: Edinburgh. 5 Janne P.A. et al.A rapid and sensitive enzymatic method for epidermal growth factor receptor mutation screening. Clin Cancer Res (2006); 12 (3): 751 - 758 6 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 7 Sequist L.V. & Lynch T.J. EGFR Tyrosine Kinase Inhibitors in lung cancer: an evolving story. Ann Rev Med (2008) 59: 429-42 8 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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