Transcript Epidermal Growth Factor Receptor and K

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
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
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.

Wildtype for codon 12
c.34G>T (p.Gly12Cys)
Detects the various mutation
combinations within the 3 codons.
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
Drug response
Mutation status Vs. Drug response
Stable Disease
Disease
progression
0
1
2
3
4
5
No. of patients
EGFR -ve
K-Ras +ve
 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