Development of an EGFR/KRAS
testing service for Non-Small
Cell Lung Cancer (NSCLC)
Joel Tracey1, Caroline Clark1, Christine Bell1, Keith Kerr2,
Marianne Nicholson3, Aileen Osborne1, Zosia Miedzybrodzka1,
Kevin Kelly1
1Department
of Medical Genetics, Polwarth Building, Aberdeen Royal Infirmary, Aberdeen
2Department of Pathology, Aberdeen Royal Infirmary, Aberdeen
3Clinical Oncology, Aberdeen Royal Infirmary, Aberdeen
Non Small Cell Lung Cancer (NSCLC)
 Lung cancer is one of the most commonly diagnosed types of
cancer in the UK
 Leading cause of cancer-related death in both men and women
 Non-Small Cell Lung Cancer ~ 80% (3)



Percentage of
NSCLC
subtypes in UK
Adenocarcinoma (ADC)
Squamous cell carcinoma (SCC)
14%
Large cell carcinoma (LCC)
38%
ADC
48%
SCC
LCC
EGFR and KRAS in NSCLC
 Acquired mutations in the EGFR and KRAS genes are important in
the development of NSCLC
 Mutations result in inappropriately activated proteins – tumour cells
become ‘addicted’ to growth signals
 EGFR and KRAS mutations most common in adenocarcinoma
 EGFR and KRAS mutations are mutually exclusive
Treatment of NSCLC
 Surgery - possible in about 20% of cases
(4)
 Cytotoxic chemotherapy and/or radiotherapy - mostly ineffective
 Survival rate poor (7% alive 5 years after diagnosis)
(4)
 Tyrosine Kinase Inhibitors (TKI) – new type of
chemotherapeutic agent – fewer side-effects than cytotoxic
chemotherapy
 Target and block growth factor signals – e.g. Epidermal
Growth Factor Receptor (EGFR)
EGFR Tyrosine Kinase Inhibitors
 Erlotinib (Tarceva) and Gefitinib
(Irresa)
 EGFR targeted TKIs can be used
for treatment of NSCLC patients
with somatic activating EGFR
mutations
TKI
 Mutations within EGFR TK domain
enable TKIs to bind with greater
affinity
 Patients with activating EGFR
mutations have a better
response to TKI therapy and
improved survival
 Patients with KRAS mutations
show little or no response to
TKI treatment
Clinical Trial – IPASS Study
EGFR mutation +ve (M+) patients respond better to TKI therapy than
chemotherapy but....
EGFR mutation –ve patients (M-) have a poorer response to TKI therapy
than chemotherapy!!!
Probability
of
Progression
Free
Survival
Time (months)
EGFR mutations
 Mutations in the EGFR gene found in 10-15% of NSCLC patients (5, 6)
 Exon 19 deletions & L858R (Exon 21) make up 85-90% of all
mutation +ve cases (7)
 Exon 20 mutations (e.g. T790M) commonly resistance mutations
KRAS mutations
12
Codons
Wt seq
G
G
Multivariable
mutations
A
A
C
C
T
T
13
T
G
G
A
61
C
C
A
A
A
A
C
C
C
G
G
T
T
 KRAS mutations occur in ~30% of NSCLC tumours (8)
 Codon 12 most common mutation site
Project Aims
1.
Develop and set-up methods for EGFR and KRAS
analysis
2.
Determine best methods for analysis of EGFR and
KRAS mutations
3.
Develop and validate appropriate methodologies for
testing
Samples

48 Adenocarcinoma patient samples (ARI Pathology Dept)

4 EGFR +ve control samples (Holland)

All were FFPE lung tumour samples (cores, slides & rolls)

14 DNA samples for KRAS analysis (Transgenomic Inc)

Mutations in codon 12, 13 and 61

Varied mutation levels (3% to 33%)
Challenges with NSCLC testing using
FFPE samples
• Frequently low sample quantity = Low DNA yield
• Variable tumour content within sample (<5% to 100%)
• Poor DNA quality - Degradation (<300bp), PCR inhibitors
• Genetic heterogeneity – inter- and intra-tumour variation
• Pathology departments involvement at this stage
important to maximise % tumour – macrodissection
Methodology Plan
Extract DNA from FFPE lung tumour samples (Dewax, phenol/chloroform)
Quantify all DNA samples (Nanodrop)
PCR amplification using specific primers (in-house/published)
Quantify all DNA samples
PCR amplification using specific primers
EGFR
KRAS
Direct Sequencing
Direct Sequencing
WAVE HS dHPLC + fragment
collection
WAVE Surveyor
WAVE Surveyor
Ex19 Fragment Length Analysis
Ex21 Pyrosequencing
Pyrosequencing
Principles of Methods Used
WAVE HS dHPLC – Partially denaturing, High sensitivity by
fluorescent detection (x100), mutation identified by presence of
mutant/WT heteroduplex peaks
Fragment collection – After passing through detector
eluted DNA fragments were collected in vials at 30s intervals
WAVE Surveyor – Enzymatic
method, detects DNA mismatches,
WAVE size separation, mutation
identified by presence of cleavage
products
Principles of Methods Used
Fragment Length Analysis – FAM labelled PCR products,
size separation on ABI 3130, analysis using Gene Marker software
Pyrosequencing – Real-time
sequence data, Pyrophosphate (PPi)
substrate for reaction cascade, light
produced measured – relative to
nucleotides incorporated
Summary of KRAS Results
Blind study (Transgenomic samples)

Pyrosequencer detected all mutations in Transgenomic samples (lowest = 3%)

2 samples not detected by the WAVE Surveyor method (3%)

6 samples were below the detection limit of sequencing (<10%)

33% (16/48) of patient samples positive for KRAS mutations – tested by
both pyrosequencing and direct sequencing
Percentage of KRAS
mutations identified
(by codon)
Codon 12 (9)
8%
8%
Codon13 (1)
Codon 61 (1)
84%
EGFR Results
Sample: EGFR +ve Control
Mutation: Exon 19 Del (c.2240-2254del; p.L747 – T751del)
+ve control
Sequencing
WT
257
174
WAVE Surveyor
80
102
Uncleaved
product
267 298
Size control
+ve control
WT
+ve control
WAVE HS
dHPLC
WT
Enrichment of EGFR mutant by WAVE dHPLC
+ fragment collection
Ex 19 WT
Ex 19 del
(direct seq)
Ex 19 del
(enriched by
fragment
collection +
repeat PCR)
Sequences analysed with Mutation Surveyor software (Soft Genetics)
Additional methods
Ex 19 del
Ex 19
Fragment
length analysis
Ex 19 WT
G863D
Ex 21 Pyrosequencing
L861Q
L858R
L858R
mutant
Summary of EGFR Results
 12.5% (6/48) of patient samples positive for EGFR mutation
(Ex 19 - 3 Deletions; Ex 20 - 1 Insertion; Ex 21 - 2 L858R)
 All methods detected Ex19 del mutations in 4 EGFR +ve control samples
 WAVE Surveyor confirmed all mutations found by direct sequence
analysis
 One Ex19 del mutant too low to report by direct sequence analysis but
clear by WAVE Surveyor and Fragment length analysis
 Pyrosequencer – successfully detected Ex21 mutants
 Confident no false positive results
All EGFR +ve samples were KRAS –ve
Mutations confirmed by multiple methods
Comparison of EGFR methods
WAVE HS
dHPLC
SURVEYOR
Sequencing
Fragment
analysis (Ex19
only)
Pyrosequencing
(Ex 21 only)
Hands on time *
2hr 30min
2hr 45min
2hr 45min
1hr 30min
2hr 15min
Cost (per
sample)
£16.50
£15.60
£26.70
£0.57
£8.04
Results
analysis time*
45min
45min
1hr 30min
30min
30min
Total Time to
result *
40hr 40min
24hr 45min
11hr 15min
6hr
4hr 35min
Sample
required
120ng
120ng
120ng
20ng
20ng
Detection Limit
?
~4-5%
~10%
?
3-5%
 Times based on analysis of 15 samples
 Cost per sample does not include staff costs
Conclusions
 Pick-up rate of EGFR mutations consistent with published data
 Direct sequencing pick-up rate higher than expected. This likely
to be due to enrichment of samples for tumour tissue by macrodissection
 WAVE Surveyor, fragment analysis and pyrosequencing
methods may be useful as a higher sensitivity screen in
conjunction with direct sequencing
 Fragment collection is a viable method for enrichment of low
level mutations
Current Testing Strategy
NSCLC Patient (M/F,
smoker/non-smoker)
SCC / LCC
Adenocarcinoma
Not Tested
Assessment of
tumour content and
macrodissection
Pathology
Molecular Genetics
EGFR Ex1821 PCR
KRAS codons
12, 13 and 61
PCR
Direct Sequencing
/Pyrosequencing
Direct Sequencing/WAVE
Surveyor/Fragment
Length Analysis
Report
Acknowledgements
 Aberdeen Lab
Caroline Clark
Christine Bell
Aileen Osborne
Louise Carnegie
Heather Greig
Kevin Kelly
 Transgenomic
Gerald Martin
 Clinical/Pathology
Keith Kerr
Marianne Nicholson
Zosia Miedzybrodzka
 Astra Zeneca
For providing funding
References
1
Ferlay J. et al. Estimates of the cancer incidence and mortality in Europe in 2006. Annals of
Oncology (2007) 18: 581-5923
2
Harkness E.F. et al. Changing trends in incidence of lung cancer by histologic type in Scotland.
Int. J. Cancer (2002) 102: 179-183
3
D’Addario G. & Felip E. Non-small-cell lung cancer: ESMO Clinical Recommendations for
diagnosis, treatment and follow-up. Annals of Oncology (2008) 19 (Sup 2): ii39 - ii40
4
Scottish Executive Health Department. Cancer scenarios: an aid to planning cancer services in
Scotland in the next decade.. 2001 The Scottish Executive: Edinburgh.
5
Janne P.A. et al. A rapid and sensitive enzymatic method for epidermal growth factor receptor
mutation screening. Clin Cancer Res (2006); 12 (3): 751 - 758
6
Sequist L.V. et al. Epidermal Growth Factor Receptor mutation testing in the care of lung cancer
patients. Clin Cancer Res (2006); 12 (Sup 14): 4403s – 4408s
7
Sequist L.V. & Lynch T.J. EGFR Tyrosine Kinase Inhibitors in lung cancer: an evolving story. Ann
Rev Med (2008) 59: 429-42
8
Do, H. et al. High resolution melting analysis for rapid and sensitive EGFR and KRAS detection
in formalin fixed paraffin embedded biopsies. BMC Cancer (2008) 8:142