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, R. Brito1, J Tull1, R. Butler1
1All Wales Molecular Genetics Laboratory, Cardiff 2 Oxford Radcliffe Hospitals Trust
Overview
• Lung Cancer• Non-small Cell Lung Cancer (NSCLC)
• Tyrosine kinase inhibitors• Gefitinib/ Erlotinib
• Testing strategy• Sample types• EGFR & K-Ras analyses
• Problems to date & possible solutions
Non-small Cell Lung Cancer
• Lung cancer is the leading cause of cancer death worldwide– More than 38,000 patients diagnosed in the UK each year
• Types of Lung Cancer– Small Cell Lung Cancer (15%)– Non Small Cell Lung Cancer (85%)
• Often diagnosed at a late stage– 80% patients die within 1st year of diagnosis
• Current treatment– Early detection – Surgery and radiotherapy– Metastatic phase – Combined cytotoxic chemotherapy
• Median survival - ~8 months
EGFR Tyrosine kinase inhibitors– E.g. erlotinib, reversibly compete with
ATP binding to the EGFR TK domain• Reduced side effects• Median survival - ~24 months
– Effective in 10-20% of NSCLC patients• Women, ‘never smokers’, East Asians
(Japanese) and in patients with adenocarcinomas
• 88% of responders have acquired mutations within the EGFR TK domain
– Drugs alter the NSCLC molecular profile during the course of treatment
• Most responders eventually relapse– Acquisition of EGFR resistance
mutation, T790M and exon 20 insertions/ duplications
– Acquisition of K-Ras mutations
Patients with EGFR mutations do better with TKIsPatients without EGFR mutations do better with chemo
Testing strategy
• Sample types
• K-Ras & EGFR analyses
• Timeline
• Problems & possible solutions
Samples types
• Paraffin fixed biopsies– Histology assessment
• Extraction1. EZ1 DNA paraffin tissue kit
(Qiagen)• 190µl of G2 buffer - Incubate at 56oC
(10min)
• 10µl of proteinase K - Incubate at 56oC (Overnight)
• Extract DNA
– 50µl (1ng/µl – 60ng/µl)
2. Qiagen - DNA Blood Mini Kit • Uses xylene
• 48-72 hour incubation
• Concentration & volume of DNA dependent on tissue size + tests after
Samples types… … continued
• Bronchial brushings• Samples variably heterogeneous
– Extraction• PBS washes• EZ1 DNA tissue kit (Qiagen)
– 190µl of G2 buffer– 10µl of proteinase K
» Incubate at 56oC (at least 20min)– Extract DNA
• EDTA blood plasma– Separated within 4-6 hrs of collection– Extracted using EZ1 DNA virus kit (Qiagen)
K-Ras analysis• K-Ras analysis as exclusion test• 30-40% of NSCLC adenomas
– KRAS and EGFR mutations mutually exclusive
• Pyrosequencing– Interrogate codons 12, 13 and 61 of the
K-Ras gene– Sensitive to 5-10% mosaicism
c.34G>T (p.Gly12Cys)
Wildtype for codon 12
c.35G>A (p.Gly12Tyr)
K-Ras analysis … … continued
• DXS K-Ras Mutation Kit
– Scorpions real time PCR assay– Detects 7 Somatic mutations in the K-Ras
gene– Can detect less than 1% of mutant in a
background of wt genomic DNA
EGFR TK domain analysis• Bi-directional Sequencing
• Exons 18-21 (ATP cleft)
• 2 sets of primers per exon
– Paraffin fixed biopsies• Nested PCR
– Brushings• Straight forward
• Pros & Cons• Looks for all mutations within the TK-
domain
• BUT: Lowest degree of mutant alleles detectable by sequencing ranges from
15% - 50% (Rohlin et al., 2009)
Nested PCRExt_F
Ext_R
Int_F
Int_R
EGFR TK domain analysis• DXS Therascreen EGFR29
• Scorpions real time assay– Detects 29 of the most common
EGFR mutations (COSMIC) ≡ ~92%
• Pros & Cons• Detects <1% of mutant in a
background of wt genomic DNA
• As little as 3ng of DNA required
• Quick turnaround• Easy to automate• BUT: - Detects only 29
mutations
Results concordant with those obtained by sequencing – so far
Time line to results (KRAS & EGFR)
Bronchial brushingsBlood plasma - cfDNA
Paraffin fixed biopsies
Results so far
Problems & Possible Solutions
• Histopathology delays– Samples received from
various path laboratories
– Analysis time• No or insufficient tumour
• Insufficient sample
– Mistyping of lung tumour subtypes
• Education of Clinicians• Pre-analysed slides for
macro-dissection– Dedicated histopathology
department• Analysis time
• NSCLC subtypes
Problems & Possible Solutions … …continued
• Failure at DNA extraction– May not become evident until
PCR
– Insufficient sample
– Fixation method•Education of thoracic surgeons•Tailor extraction to fixation
Problems & Possible Solutions … …continued
• Only 40% of patients have a biopsy
– Alternatives to consider
• Cell free tumour DNA
• Circulating tumour cells (expensive)
Problems & Possible Solutions … …continued
• Sample heterogeneity (and/or assay sensitivity)– Brushings
• Risk of contaminating normal cells
– NSCLC show intratumoral heterogeneity
– Primary vs. metastases (discordant)
• Level of blood contamination from brushings dependent on experience of sampler
• Alternative tumour tissue
Problems & Possible Solutions … …continued
• K-Ras analysis– Is it necessary?
• EGFR & K-Ras mutually exclusive
– Shared pyrosequencing facilities
• Just screen for EGFR
• Try K-Ras DXS real time assay– Cost– Specificity
• Dedicated pyrosequencer
Time line to results – EGFR analysis alone
EGFR analysis aloneReport in 5 days
EGFR & K-Ras analysisReport in 9 days
Problems & Possible Solutions … …continued
• EGFR– Nested PCR – contamination
or assay optimisation– Sequencing – quality of
sequence data• Confirmation of mutations?
• Assay sensitivity/specificity– DXS – limited to specific
mutations– Sequencing limited to 15%
mosaicism
• Shorter overlapping fragments – eliminates nest
• Use of DXS real time negates sequencing shortfalls
• Use both DXS & sequencing• Alternative assays
– Digital PCR– COLD PCR prior to sequencing
Problems & Possible Solutions … …continued
• Obtaining blood to confirm novel mutations/ unknown variants
– Take a blood sample at consultation– National database of EGFR variants
• www.EGFR-info.com
Conclusions
• Service available with a 5-10 working day turn around time for biopsies, bronchial brushings and cfDNA
• Results using different methods and on different samples all concordant to date.
Future workLook to improve sensitivity of tests by looking at alternative mutation assays and/or sources of DNA