EGFR gene mutation testing in
NSCLC
Rachel Butler
All Wales Molecular Genetics Laboratory
Benefits of stratified medicine

Reduce adverse reactions

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Reduce morbidity and patient
distress
Reduce associated costs
Improve patient response through
correct dose or effective therapy

Reduce associated costs
Benefits of stratified medicine
Non-small cell lung cancer

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Normal
80-90% of cases
Adenocarcinoma and squamous cell
carcinoma
Majority are smoking related
Majority present with incurable advanced
disease
Squamous
Metaplasia
Dysplasia
in situ Cancer
Invasive
Cancer
Mutant EGFR
Pathways active
TKI against EGFR,
blocks pathways
Ref: T Mok IPASS trial. JCO 2009 27 (suppl):408s (abstract 8006)
Ref: T Mok IPASS trial. JCO 2009 27 (suppl):408s (abstract 8006)
Exon 21 L858R mutation case study (courtesy of Dr S Popat)
Iressa- 6 weeks & 4 months
EGFR analysis
Report faxed to referrer
Oncologist
EGFR analysis
Path sample
request
DNA extraction
Sample received
Sample
assessment
Sampling
Sensitivity
Wt
Wt
EGFR EGFR
Mt
Wt
EGFR EGFR
Mutant signal is a % of the
wild-type signal
Sample enrichment: Macrodissection
Essential that Pathologist
assesses specimen for
tumour quantity and quality:

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Selects appropriate tissue
block
Representative block
containing a high number of
tumour cells
Selects and marks tumour
cells
Area should contain >30%
tumour cells
DNA extracted from tumour
cells only, to enrich for
EGFR mutations (if present)
Sample enrichment: COLD-PCR

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Modification of PCR to directly amplify mutation:WT
heteroduplexes
Can improve sensitivity to ~1-3%
Used for samples with low tumour %
Mutations analysed
Molecular technologies
DNA sequencing, Pyrosequencing, Fragment-length analysis,
Real-time PCR (DxS kits), HRM, SNapShot, RFLPS
A few problems…..
And solutions
Reporting times (TaTs)
Total analytical time
5 days
Time of
request
Sample receipt
in molecular lab
5.3 days
Sample rejected
as insufficient
tissue or tumour
Insufficient
information re.
requesting
clinician
Manchester solution
Other
Trust
Request
Central
Path M/Cr Trust
Other
Trust
Path
LOCAL
REVIEW
Onco/MDTs
Sample
Genetic
lab
Path
Christie
Trust
Onco/MDTs
Path
Path
Onco/MDTs
EGFR test
request
Path
Wythenshawe
Path
CENTRAL
REVIEW
Genetic
lab
EGFR sequence variants

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Detected by screening technologies
(sequencing, pyrosequencing etc)
EGFR variants not previously described or
without
clinical data
Benign,
sensitising
or resistant?

External quality assurance

Format:
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3 validated FFPE samples distributed
Labs to analyse and report by usual processes (1 month)
Assessed by 2 independent assessors for
 Correct genotype (result), Interpretation, Clerical accuracy
(i.e. name, dob…)
UK NEQAS
Histopathology Labs
UK Labs
Genetics Labs
non-UK Labs
11 labs
16 labs
2010
23 labs
27%
24 labs
2011 run 1
73%
50%
2010
2010
- 27 labs -- deletion in exon 19 (tumour content 60-70%)
- No mutation (tumour content 40-50%)
- c.2582A>T; p.Leu861Gln (tumour content 40-50%)
- genotyping and interpretation assessed
- 6 geno errors (22% of labs)
2011 – run1
- 47 labs -- deletion in exon 19 (tumour content 70%)
- No mutation (tumour content 50-60%)
- c.2579T>G; p.Leu858Arg (tumour content 40-50%)
- genotyping and interpretation assessed
- 3 geno errors (6% of labs)
- No errors by previously participating labs
2011 – run2 – 49 labs – 3 geno error (6% of labs)
50%
2011 run 1
Establishment of best-practice
Labs
 Accredited
 Successful EQA participation
Samples
 Best possible sample
 Assessment of tumour nuclei % recommended
 Macrodissection recommended but not essential
 All samples possible
Molecular analysis
 Minimum set of mutations (>95%)
 Molecular methodology is the choice of the lab
 Sensitivity of detection linked to sample assessment (<5-10%)
 Oversensitivity is problematic!
Reporting
 Patient and sample unequivocally identified
 % tumour, molecular technology and sensitivity of analysis should be stated
 The report should interpret the molecular finding and predict the patient’s
response (including information on UVs)
Future molecular markers

The study of the molecular features
of an individual in relation to a
pathological condition (DNA, RNA,
Proteins)
Cancer
Tumour
Individual
DNA, RNA, protein
DNA
Molecular therapy: plenty of targets…
Future molecular markers: lung
cancer
Establish robust NHS service model for future
molecular markers
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CRUK SMP
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EGFR, KRAS, BRAF, EML4-ALK
EML4-ALK gene fusions – Crizotinib
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FISH (IHC, PCR)
Crizotinib and EML4- ALK fusions
~4.5% adenocarcinoma patients
Dramatic response to therapy
Analysis by FISH, IHC or PCR
Expected launch 2012-13
Future molecular markers: lung
cancer
Establish robust service model for future molecular
markers
 EML4-ALK gene fusions – Crizotinib
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DDR2 mutations – Dasatinib sensitivity in squamous
[Hammerman et al, Cancer discovery 2011]
FGFR1 amplification – FGFR1 inhibitor in squamous
[Weiss et al, Sci Transl Med 2010]
Synthetic lethality
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2o mutations confer resistance [Choi et al, NEJM 2010]
Somatic BRCA or LKB1 loss
Response to EGFR TKIs due to genetic modifiers,
opportunities for companion drugs [Bivona, Nature
2011]
MET expression and inhibition
Cell free DNA (cfDNA)
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Shed directly from the tumour, detected in plasma / serum
Non-invasive, alternative source of tumour DNA
~30-40% of patients with NSCLC have no tumour sample
available
Mutation status detection – (e.g.) detect EGFR mutations
Recurrent disease monitoring – monitor acquired
resistance to TKI (e.g. T790M)
The future
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UK (EGFR) services are now established
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Need to determine appropriate no. of experienced
and qualified provider labs
Partnership with pathologists for seamless clinical
service
Predictive / prognostic utility
of future markers
Commissioning