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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
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
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
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 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
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
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)
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
Mutation status Vs. Drug response
0 1 2 3 4 5
Diseaseprogression
Stable Disease
Dru
g r
esp
on
se
No. of patients
EGFR -ve K-Ras +ve
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