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

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

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%)

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

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.

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

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

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

Samples received Bronchial brushings 23 NSCLC samples 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

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.

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)

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

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

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

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.

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

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

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