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

3. Benefits of stratified medicine

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

5. Mutant EGFR
Pathways active

6. TKI against EGFR, blocks pathways

7. Ref: T Mok IPASS trial. JCO 2009 27 (suppl):408s (abstract 8006)
RR better to both cytotoxic chemo and TKI in mutation positive patients
Mutation rate 50% of those tested (clinically enriched population though)
Even in this prospective trial, only about 35% had mutation status obtained
Ref: T Mok IPASS trial. JCO (suppl):408s (abstract 8006)

8. Ref: T Mok IPASS trial. JCO 2009 27 (suppl):408s (abstract 8006)
Survival better in EGFR mutation +ve patients treated with gefitinib
Essentially the same with chemo irrespective of EGFR status, ie not prognostic, despite better RR
Ref: T Mok IPASS trial. JCO (suppl):408s (abstract 8006)

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

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

11. Sampling

12. Sensitivity Mutant signal is a % of the wild-type signal Wt EGFR
Mt EGFR
Wt EGFR
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. Manchester solution Other Trust Path Path Path Path Path Path
LOCAL REVIEW
Request
Central M/Cr Trust
Other Trust
Path
EGFR test request
Path
Onco/MDTs
Path
Sample
Genetic lab
Path blocks
Path
Wythenshawe
Path
CENTRAL REVIEW
Christie Trust
Path
Genetic lab
Path
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. Histopathology Labs 27% 2010 2011 run 1 2010 2011 run 1 2010
Genetics Labs
UK Labs
non-UK Labs
run 1
50%
27%
73%
run 1
23 labs
24 labs
11 labs
16 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
Crizotinib and EML4- ALK fusions ~4.5% adenocarcinoma patients Dramatic response to therapy Analysis by FISH, IHC or PCR Expected launch

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