H. Mugalaasi 1 , J. Davies 2 , L Medley 2 , D Talbot 2 , R. Brito 1 , R. Butler 1
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Transcript of H. Mugalaasi 1 , J. Davies 2 , L Medley 2 , D Talbot 2 , R. Brito 1 , R. Butler 1
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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
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Overview Lung Cancer
Non-small Cell Lung Cancer (NSCLC) Epidermal growth factor receptor (EGFR) Gefitinib/ Erlotinib Broncoscopy protein study Project aims Results Future work
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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%)
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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
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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.
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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
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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
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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
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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
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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.
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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)
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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
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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
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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
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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.
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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
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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
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Acknowledgements
Institute of Medical Genetics Rachel Butler Rose Brito
Oxford Radcliffe Hospitals NHS Trust Denis Talbot Jo Davies Louise Medley