H. Mugalaasi 1 , J. Davies 2 , L Medley 2 , R. Brito 1 , J Tull 1 , R. Butler 1

1. 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

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 1st 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 • 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) • 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. Wildtype for codon 12 c.34G>T (p.Gly12Cys) c.35G>A (p.Gly12Tyr) 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

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. Nested PCR Ext_F Ext_R Int_F Int_R 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)

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. 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

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

17. 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

18. 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 Problems & Possible Solutions … …continued

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

20. 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

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