Development of an EGFR/KRAS testing service for Non-Small Cell Lung Cancer (NSCLC)

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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
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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)
• Adenocarcinoma (ADC)
• Squamous cell carcinoma (SCC)
• Large cell carcinoma (LCC)

Percentage of NSCLC subtypes in UK
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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

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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)

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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
• 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

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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!!!
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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

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KRAS mutations

• KRAS mutations occur in 30 of NSCLC tumours (8)
• Codon 12 most common mutation site

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Project Aims

• Develop and set-up methods for EGFR and KRAS
  analysis
• Determine best methods for analysis of EGFR and
  KRAS mutations
• Develop and validate appropriate methodologies
  for testing

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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)

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Challenges with NSCLC testing using FFPE samples

• Frequently low sample quantity Low DNA yield
• Variable tumour content within sample (lt5 to
  100)
• Poor DNA quality - Degradation (lt300bp), PCR
  inhibitors
• Genetic heterogeneity inter- and intra-tumour
  variation
• Pathology departments involvement at this stage
  important to maximise tumour macro-dissection

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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 Direct Sequencing WAVE HS dHPLC fragment
collection WAVE Surveyor Ex19 Fragment Length
Analysis Ex21 Pyrosequencing
KRAS Direct Sequencing WAVE Surveyor
Pyrosequencing
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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
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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
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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 (lt10)

• 33 (16/48) of patient samples positive for KRAS
  mutations tested by both pyrosequencing and
  direct sequencing

Percentage of KRAS mutations identified (by codon)
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EGFR Results
Sample EGFR ve Control Mutation Exon 19 Del
(c.2240-2254del p.L747 T751del)
ve control
Sequencing
WT
267
298
257
174
Uncleaved product
WAVE Surveyor
102
80
ve control WT
WAVE HS dHPLC
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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)
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Additional methods
Ex 19 Fragment length analysis
L858R mutant
Ex 21 Pyro-sequencing
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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

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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

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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 macro-dissection
• 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

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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 Ex18-21 PCR
KRAS codons 12, 13 and 61 PCR
Direct Sequencing /Pyrosequencing
Direct Sequencing/WAVE Surveyor/Fragment Length
Analysis
Report
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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

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References

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• 3 DAddario 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
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• 7 Sequist L.V. Lynch T.J. EGFR Tyrosine Kinase
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• 8 Do, H. et al. High resolution melting analysis
  for rapid and sensitive EGFR and KRAS detection
  in formalin fixed paraffin embedded biopsies. BMC
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