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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.

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Presentation on theme: "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."— Presentation transcript:

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

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  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 Oxford Radcliffe Hospitals NHS trust

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 Samples received  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

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

11 Pyrosequencing analysis of K-Ras  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) Wildtype for codon 12 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 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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