Presentation on theme: "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,"— Presentation transcript:
1Analysis of the Epidermal Growth Factor Receptor and K-Ras genes in patients with Non-small Cell Lung CancerH. Mugalaasi1, J. Davies2, L Medley2, D Talbot2, R. Brito1, R. Butler11All Wales Molecular Genetics Laboratory, Cardiff2 Oxford Radcliffe Hospitals Trust
2Overview Lung Cancer Epidermal growth factor receptor (EGFR) Non-small Cell Lung Cancer (NSCLC)Epidermal growth factor receptor (EGFR)Gefitinib/ ErlotinibBroncoscopy protein studyProject aimsResultsFuture work
3LUNG 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%)
4Non-small Cell Lung Carcinoma NSCLC (adenocarcinoma) most common in ‘never smokers’Current treatmentEarly detection – surgery and radiotherapyMetastatic disease - combined cytotoxic chemotherapyDeveloping therapiesTargeted inhibition of the Epidermal Growth Factor Receptor (EGFR)Monoclonal antibodies – e.g. CetuximabTyrosine kinase inhibitors – e.g. Gefitinib/ Erlotinib
5Epidermal Growth Factor Receptor (EGFR) EGFR/Erb1 - Tyrosine kinase receptor1 of 4 homologous TKs in the EGF/erb growth factor familyRegulates 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.
6EGFR 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 relapseAcquisition of EGFR resistance mutation – T790MAcquisition of K-Ras mutations
7Bronchoscopy Protein Screening (BPS) study Oxford Radcliffe Hospitals NHS trustBPS studyProtein expression as a patient selection criteria for treatment with erlotinibEntry into the study is based on EGFR over-expressionDoes drug response correlate with EGFR mutation status?Molecular analysis is currently a retrospective studySamples obtained by fibre optic bronchoscopyBronchial biopsiesDetermine tumour subtype2 Bronchial brushings1 brushing for protein study1 brushing for molecular analysis
8Project AimsCompare EGFR over-expression to TK mutation analysis as a patient selection criterionTest 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
9Samples received Bronchial brushings 23 NSCLC samples 4 Non-malignant4 Miscellaneous (1 undefined & 3 failed at extraction)Samples extracted on the day of receipt using the EZ-1 tissue protocol23 NSCLC samples10 Adenocarcinomas6 Squamous cell carcinomas1 Large cell carcinoma6 UnknownParaffin fixed biopsies11 Adenocarcinomas
10Sequencing 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 biopsiesp.Leu858Arg mutation detected.
11Pyrosequencing analysis of K-Ras Wildtype for codon 12Pyrosequencing 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)
12Mutation frequencies observed Mutations observed in similar frequencies to published data.EGFR mutations present in 2/23 (8.7%) NSCLC patientsPublished data – ~10%K-Ras mutations present in 4/23 (17%) NSCLC patients and in 3/10 (30%) adenocarcinomasPublished data – 10-30%No patient had both EGFR and K-Ras mutationsResults from bronchial brushings concordant with those obtained from macro-dissected paraffin fixed biopsies.Bronchial brushings are a reasonable source of tumour tissue
13Other observations Mutations more common in adenocarcinomas All EGFR mutations and ¾ K-Ras mutations¼ K-Ras mutations found in the large cell subtypeK-Ras mutation identified in 1 brushing sample with no detectable tumour cellsEGFR mutations found only in non-smokersInsufficient data relating K-Ras mutations to smokers
14Mutation status Vs. Drug response Rapid disease progression in 4 patients.All were negative for EGFR TK domain mutations2/4 found to have K-Ras mutationsBut stable disease in 3 patients without EGFR mutations
15EGFR over-expression Vs. Mutation analysis for patient selection Protein over-expressionEGFR over-expressed in all 23 NSCLC tumour samples studiedK-Ras mutations found in 4/23 tumours showing EGFR over expressionHence at least 17% of patients would not benefit from treatmentMutation analysisOnly 2 patients found to have EGFR mutations3 patients without EGFR mutations responded to treatmentBut 4/23 patients prevented from unnecessary treatmentGiven that erlotinib is effective in only 10-20% of NSCLC patients selection on the basis of EGFR over-expression alone would be wasteful.
16ConclusionsDesigned 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 geneBronchial brushings can be used as source for tumour tissue for mutation analysisConcerns remain with regards to the heterogeneity of these samplesMutation analysis is a better tool for patient selection criteriaExcludes patients with K-Ras mutationsTargets patients with EGFR mutations
17Future work How can we improve the sensitivity of our tests? Alternative sources of tumour DNABrushingsBiopsiesCell free tumour DNAAlternative assaysTheraScreen: EGFR29 Mutation test kitCan detect less than 1% of mutant in a background of wt genomic DNA
18Acknowledgements Institute of Medical Genetics Rachel ButlerRose BritoOxford Radcliffe Hospitals NHS TrustDenis TalbotJo DaviesLouise Medley