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 Reduce associated costs

Benefits of stratified medicine

Non-small cell lung cancer

80-90% of casesAdenocarcinoma and squamous cell

carcinomaMajority are smoking relatedMajority present with incurable advanced

disease

Normal SquamousMetaplasia

Dysplasia in situ Cancer InvasiveCancer

Mutant EGFR

Pathways active

TKI against EGFR, blocks pathways

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

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

Iressa- 6 weeks & 4 months

Exon 21 L858R mutation case study (courtesy of Dr S Popat)

EGFR analysis

Oncologist

Path sample request

Sample received

EGFR analysis

Sample assessment

Report faxed to referrer

DNA extraction

Sampling

Sensitivity

Mutant signal is a % of the wild-type signal

WtEGFR

Wt EGFR

MtEGFR

WtEGFR

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 technologiesDNA sequencing, Pyrosequencing, Fragment-length analysis,

Real-time PCR (DxS kits), HRM, SNapShot, RFLPS

A few problems…..

And solutions

Reporting times (TaTs)

5.3 days5 days

Time of request

Sample receipt in molecular lab

Sample rejected as insufficient

tissue or tumour

Total analytical time

Insufficient information re.

requesting clinician

Manchester solution

Genetic lab

Onco/MDTs

PathPath

PathCentral M/Cr Trust

Onco/MDTs

Christie Trust

Other Trust

Other Trust

Path

Path

Request

Sample

Genetic lab

Onco/MDTs Path

Wythenshawe

Path

EGFR test request

CENTRAL REVIEW

Path

blocks

LOCAL REVIEW

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…)

UK NEQAS

UK Labs

non-UK Labs

2010 2011 run 1

23 labs24 labs

11 labs 16

labs

2010 2011 run 1

50% 50% 27%

73%

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)

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 possibleMolecular 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

TumourDNA, RNA, protein

IndividualDNA

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 ~4.5% adenocarcinoma patients Dramatic response to therapy Analysis by FISH, IHC or PCR Expected launch 2012-13

Future molecular markers: lung cancerEstablish 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