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

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

Benefits of stratified medicine

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

Mutant EGFR Pathways active

TKI against EGFR, blocks pathways

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 2009 27 (suppl):408s (abstract 8006)

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 2009 27 (suppl):408s (abstract 8006)

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

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

Sampling

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

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)

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 %

Mutations analysed

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

A few problems….. And solutions

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

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

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

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…)

Histopathology Labs 27% 2010 2011 run 1 2010 2011 run 1 2010 Genetics Labs UK Labs non-UK Labs 2010 2011 run 1 50% 27% 73% 2010 2011 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)

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)

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

Molecular therapy: plenty of targets…

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)

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 2012-13

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

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)

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