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