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H. Mugalaasi 1 , J. Davies 2 , L Medley 2 , D Talbot 2 , R. Brito 1 , R. Butler 1

Analysis of the Epidermal Growth Factor Receptor and K-Ras genes in patients with Non-small Cell Lung Cancer. H. Mugalaasi 1 , J. Davies 2 , L Medley 2 , D Talbot 2 , R. Brito 1 , R. Butler 1 1 All Wales Molecular Genetics Laboratory, Cardiff 2 Oxford Radcliffe Hospitals Trust. Overview.

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H. Mugalaasi 1 , J. Davies 2 , L Medley 2 , D Talbot 2 , R. Brito 1 , R. Butler 1

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  1. Analysis of the Epidermal Growth Factor Receptor and K-Ras genes in patients with Non-small Cell Lung Cancer H. Mugalaasi1, J. Davies2, L Medley2, D Talbot2, R. Brito1, R. Butler1 1All Wales Molecular Genetics Laboratory, Cardiff 2 Oxford Radcliffe Hospitals Trust

  2. Overview • Lung Cancer • Non-small Cell Lung Cancer (NSCLC) • Epidermal growth factor receptor (EGFR) • Gefitinib/ Erlotinib • Broncoscopy protein study • Project aims • Results • Future work

  3. LUNG CANCER • Types of Lung Cancer • Small Cell Lung Cancer (SCLC) – 15% • Non-small Cell Lung Cancer (NSCLC) – 85% • Squamous cell carcinoma (25-30%) • Adenocarcinoma (40%) • Large cell cancer (10-15%)

  4. Non-small Cell Lung Carcinoma • NSCLC (adenocarcinoma) most common in ‘never smokers’ • Current treatment • Early detection – surgery and radiotherapy • Metastatic disease - combined cytotoxic chemotherapy • Developing therapies • Targeted inhibition of the Epidermal Growth Factor Receptor (EGFR) • Monoclonal antibodies – e.g. Cetuximab • Tyrosine kinase inhibitors – e.g. Gefitinib/ Erlotinib

  5. Epidermal Growth Factor Receptor (EGFR) • EGFR/Erb1 - Tyrosine kinase receptor • 1 of 4 homologous TKs in the EGF/erb growth factor family • Regulates numerous transcription factors involved in cell proliferation through various pathways. • Disregulation of the EGFR pathway is key in tumourigenesis. • Over-expressed in numerous cancers but particularly in 40-80% of NSCLC – hence ideal target for drug inhibition.

  6. EGFR Tyrosine Kinase Inhibitors • Gefitinib (& Erlotinib) • Reversible EGFR tyrosine kinase inhibitor (TKI) • Competitively binds to the ATP cleft within the EGFR TK domain. • Dramatic response observed in 10-19% of NSCLC patients. • Especially in women, ‘never smokers’, East Asians (Japanese) and in patients with adenocarcinomas. • 88% of responders harboured acquired mutations within the EGFR TK domain (exons 18-21). • Most responders eventually relapse • Acquisition of EGFR resistance mutation – T790M • Acquisition of K-Ras mutations

  7. Bronchoscopy Protein Screening (BPS) study Oxford Radcliffe Hospitals NHS trust • BPS study • Protein expression as a patient selection criteria for treatment with erlotinib • Entry into the study is based on EGFR over-expression • Does drug response correlate with EGFR mutation status? • Molecular analysis is currently a retrospective study • Samples obtained by fibre optic bronchoscopy • Bronchial biopsies • Determine tumour subtype • 2 Bronchial brushings • 1 brushing for protein study • 1 brushing for molecular analysis

  8. Project Aims • Compare EGFR over-expression to TK mutation analysis as a patient selection criterion • Test the validity of bronchial brushings as a suitable sample type for sequencing analysis – heterogeneity. • Design sequencing assay for the EGFR TK domain (exons 18-21) • Design pyrosequencing assay for the analysis of codons 12, 13 and 61 of the K-Ras gene

  9. Bronchial brushings 35 samples received 4 SCLC 4 Non-malignant 4 Miscellaneous (1 undefined & 3 failed at extraction) Samples extracted on the day of receipt using the EZ-1 tissue protocol 23 NSCLC samples 10 Adenocarcinomas 6 Squamous cell carcinomas 1 Large cell carcinoma 6 Unknown Paraffin fixed biopsies 11 Adenocarcinomas Samples received

  10. Sequencing analysis of EGFR • Sequence assay successfully designed for the analysis of the TK domain of the EGFR gene (exons 18-21 inclusive). • Nested PCR was required for sequence analysis of paraffin fixed biopsies • p.Leu858Arg mutation detected.

  11. Pyrosequencing analysis of K-Ras Wildtype for codon 12 • Pyrosequencing assay designed to interrogate codons 12, 13 and 61 of the K-Ras gene. • Detects the various mutation combinations within the 3 codons. c.34G>T (p.Gly12Cys) c.35G>A (p.Gly12Tyr)

  12. Mutation frequencies observed • Mutations observed in similar frequencies to published data. • EGFR mutations present in 2/23 (8.7%) NSCLC patients • Published data – ~10% • K-Ras mutations present in 4/23 (17%) NSCLC patients and in 3/10 (30%) adenocarcinomas • Published data – 10-30% • No patient had both EGFR and K-Ras mutations • Results from bronchial brushings concordant with those obtained from macro-dissected paraffin fixed biopsies. • Bronchial brushings are a reasonable source of tumour tissue

  13. Other observations • Mutations more common in adenocarcinomas • All EGFR mutations and ¾ K-Ras mutations • ¼ K-Ras mutations found in the large cell subtype • K-Ras mutation identified in 1 brushing sample with no detectable tumour cells • EGFR mutations found only in non-smokers • Insufficient data relating K-Ras mutations to smokers

  14. Mutation status Vs. Drug response • Rapid disease progression in 4 patients. • All were negative for EGFR TK domain mutations • 2/4 found to have K-Ras mutations • But stable disease in 3 patients without EGFR mutations

  15. EGFR over-expression Vs. Mutation analysis for patient selection • Protein over-expression • EGFR over-expressed in all 23 NSCLC tumour samples studied • K-Ras mutations found in 4/23 tumours showing EGFR over expression • Hence at least 17% of patients would not benefit from treatment • Mutation analysis • Only 2 patients found to have EGFR mutations • 3 patients without EGFR mutations responded to treatment • But 4/23 patients prevented from unnecessary treatment • Given that erlotinib is effective in only 10-20% of NSCLC patients selection on the basis of EGFR over-expression alone would be wasteful.

  16. Conclusions • Designed assay for the analysis of exons 18-21 of the EGFR gene (TK domain). • Designed assay for the analysis of codons 12, 13 and 61 of the K-Ras gene • Bronchial brushings can be used as source for tumour tissue for mutation analysis • Concerns remain with regards to the heterogeneity of these samples • Mutation analysis is a better tool for patient selection criteria • Excludes patients with K-Ras mutations • Targets patients with EGFR mutations

  17. Future work • How can we improve the sensitivity of our tests? • Alternative sources of tumour DNA • Brushings • Biopsies • Cell free tumour DNA • Alternative assays • TheraScreen: EGFR29 Mutation test kit • Can detect less than 1% of mutant in a background of wt genomic DNA

  18. Acknowledgements • Institute of Medical Genetics • Rachel Butler • Rose Brito • Oxford Radcliffe Hospitals NHS Trust • Denis Talbot • Jo Davies • Louise Medley

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