1. 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

2. 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

3. 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%)

4. 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

5. 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.

6. 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

7. 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

8. 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

9. 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

10. Sequencing 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 biopsies
p.Leu858Arg mutation detected.

11. 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)

12. 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

13. 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

14. 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

15. 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.

16. Conclusions
Designed 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 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

17. 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

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