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