1. Analysis of the Epidermal Growth Factor Receptor and K-Ras genes in patients with Non-small Cell Lung Cancer H. Mugalaasi 1, J. Davies 2, L Medley 2, R. Brito 1, J Tull 1, R. Butler 1 1 All Wales Molecular Genetics Laboratory, Cardiff 2 Oxford Radcliffe Hospitals Trust

2. 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

3. 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 1 st year of diagnosis Current treatment –Early detection – Surgery and radiotherapy –Metastatic phase – Combined cytotoxic chemotherapy Median survival - ~8 months

4. 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 TKIs Patients without EGFR mutations do better with chemo

5. Testing strategy Sample types K-Ras & EGFR analyses Timeline Problems & possible solutions

6. Samples types Paraffin fixed biopsies –Histology assessment Extraction 1.EZ1 DNA paraffin tissue kit (Qiagen) 190µl of G2 buffer - Incubate at 56 o C (10min) 10µl of proteinase K - Incubate at 56 o C (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

7. 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)

8. 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)

9. 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

10. 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 PCR Ext_F Ext_R Int_F Int_R

11. 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

12. Time line to results (KRAS & EGFR) Bronchial brushings Blood plasma - cfDNA Paraffin fixed biopsies

13. Results so far

14. 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

15. 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

16. Problems & Possible Solutions … …continued Only 40% of patients have a biopsy –Alternatives to consider Cell free tumour DNA Circulating tumour cells (expensive)

17. 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

18. 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

19. Time line to results – EGFR analysis alone EGFR analysis alone Report in 5 days EGFR & K-Ras analysis Report in 9 days

20. 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

21. 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

22. 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 work Look to improve sensitivity of tests by looking at alternative mutation assays and/or sources of DNA