EGFR gene mutation testing in NSCLC

1. EGFR gene mutation testing in NSCLC Rachel Butler All Wales Molecular Genetics Laboratory

2. Benefits of stratified medicine • Reduce adverse reactions • Reduce morbidity and patient distress • Reduce associated costs • Improve patient response through correct dose or effective therapy • Reduce associated costs

3. Benefits of stratified medicine

4. Squamous Metaplasia Invasive Cancer Normal Dysplasia in situ Cancer Non-small cell lung cancer • 80-90% of cases • Adenocarcinoma and squamous cell carcinoma • Majority are smoking related • Majority present with incurable advanced disease

5. Mutant EGFR Pathways active

6. TKI against EGFR, blocks pathways

7. Ref: T Mok IPASS trial. JCO 2009 27 (suppl):408s (abstract 8006)

8. Ref: T Mok IPASS trial. JCO 2009 27 (suppl):408s (abstract 8006)

9. Iressa- 6 weeks & 4 months Exon 21 L858R mutation case study (courtesy of Dr S Popat)

10. EGFR analysis Report faxed to referrer Oncologist EGFR analysis Path sample request DNA extraction Sample received Sample assessment

11. Sampling

12. Sensitivity Wt EGFR WtEGFR MtEGFR WtEGFR Mutant signal is a % of the wild-type signal

13. Sample enrichment: Macrodissection Essential that Pathologist assesses specimen for tumour quantity and quality: • Selects appropriate tissue block • Representative block containing a high number of tumour cells • Selects and marks tumour cells • Area should contain >30% tumour cells • DNA extracted from tumour cells only, to enrich for EGFR mutations (if present)

14. Sample enrichment: COLD-PCR • Modification of PCR to directly amplify mutation:WT heteroduplexes • Can improve sensitivity to ~1-3% • Used for samples with low tumour %

15. Mutations analysed

16. Molecular technologies DNA sequencing, Pyrosequencing, Fragment-length analysis, Real-time PCR (DxS kits), HRM, SNapShot, RFLPS

18. A few problems….. And solutions

19. Reporting times (TaTs) Total analytical time 5 days 5.3 days Time of request Sample receipt in molecular lab Sample rejected as insufficient tissue or tumour Insufficient information re. requesting clinician

20. Path Path Path Path Path Manchester solution Other Trust LOCAL REVIEW Other Trust Central M/Cr Trust Request Path EGFR test request Onco/MDTs Sample Genetic lab Path blocks Wythenshawe Path CENTRAL REVIEW Christie Trust Genetic lab Onco/MDTs Onco/MDTs

21. EGFR sequence variants • Detected by screening technologies (sequencing, pyrosequencing etc) • EGFR variants not previously described or without clinical data • Benign, sensitising or resistant?

22. External quality assurance • Format: • 3 validated FFPE samples distributed • Labs to analyse and report by usual processes (1 month) • Assessed by 2 independent assessors for • Correct genotype (result), Interpretation, Clerical accuracy (i.e. name, dob…)

23. 27% 11 labs 23 labs 73% 50% 50% 16 labs 24 labs 2010 2011 run 1 2010 2011 run 1 UK Labs non-UK Labs Histopathology Labs Genetics Labs • 2010 • - 27 labs - - deletion in exon 19 (tumour content 60-70%) • - No mutation (tumour content 40-50%) • - c.2582A>T; p.Leu861Gln (tumour content 40-50%) • - genotyping and interpretation assessed • - 6 geno errors (22% of labs) • 2011 – run1 • - 47 labs -- deletion in exon 19 (tumour content 70%) • - No mutation (tumour content 50-60%) • - c.2579T>G; p.Leu858Arg (tumour content 40-50%) • - genotyping and interpretation assessed • - 3 geno errors (6% of labs) • - No errors by previously participating labs 2011 – run2– 49 labs – 3 geno error (6% of labs)

24. Establishment of best-practice Labs • Accredited • Successful EQA participation Samples • Best possible sample • Assessment of tumour nuclei % recommended • Macrodissection recommended but not essential • All samples possible Molecular analysis • Minimum set of mutations (>95%) • Molecular methodology is the choice of the lab • Sensitivity of detection linked to sample assessment (<5-10%) • Oversensitivity is problematic! Reporting • Patient and sample unequivocally identified • % tumour, molecular technology and sensitivity of analysis should be stated • The report should interpret the molecular finding and predict the patient’s response (including information on UVs)

25. Future molecular markers • The study of the molecular features of an individual in relation to a pathological condition (DNA, RNA, Proteins) Cancer Tumour DNA, RNA, protein Individual DNA

26. Molecular therapy: plenty of targets…

27. Future molecular markers: lung cancer Establish robust NHS service model for future molecular markers • CRUK SMP • EGFR, KRAS, BRAF, EML4-ALK • EML4-ALK gene fusions – Crizotinib • FISH (IHC, PCR)

28. Crizotinib and EML4- ALK fusions ~4.5% adenocarcinoma patients Dramatic response to therapy Analysis by FISH, IHC or PCR Expected launch 2012-13

29. Future molecular markers: lung cancer Establish robust service model for future molecular markers • EML4-ALK gene fusions – Crizotinib • 2o mutations confer resistance [Choi et al, NEJM 2010] • DDR2 mutations – Dasatinib sensitivity in squamous [Hammerman et al, Cancer discovery 2011] • FGFR1 amplification – FGFR1 inhibitor in squamous [Weiss et al, Sci Transl Med 2010] • Synthetic lethality • Somatic BRCA or LKB1 loss • Response to EGFR TKIs due to genetic modifiers, opportunities for companion drugs [Bivona, Nature 2011] • MET expression and inhibition

30. Cell free DNA (cfDNA) • Shed directly from the tumour, detected in plasma / serum • Non-invasive, alternative source of tumour DNA • ~30-40% of patients with NSCLC have no tumour sample available • Mutation status detection – (e.g.) detect EGFR mutations • Recurrent disease monitoring – monitor acquired resistance to TKI (e.g. T790M)

31. The future • UK (EGFR) services are now established • Need to determine appropriate no. of experienced and qualified provider labs • Partnership with pathologists for seamless clinical service • Predictive / prognostic utility of future markers • Commissioning