Angharad Williams All Wales Medical Genetics Service ACGS Summer Scientific Meeting
5th July 2016
Contents
• Background on Non-Small Cell Lung Cancer and EGFR testing
• Issues with diagnostic testing on biopsies
• How we have resolved these issues
• Cell free circulating DNA and it’s uses with cancer diagnostics
• Update on the new EGFR ctDNA service
Lung cancer is the leading cause of cancer death worldwide
Discovery Medicine; ISSN: 1539-6509; eISSN: 1944-7930. Discov Med 10(51):144-150, August 2010.
15%
Cell Nucleus
EGFR Tyrosine kinase domain (exons 18-21)
EGF
Permanently activated intracellular signaling
Cell proliferation Ref: Astra Zeneca
EGFR* as a Treatment Target in NSCLC *Epidermal Growth Factor Receptor
TKI agents compete with ATP for binding
tyrosine kinase domain
Cell Nucleus
Inhibition of cell signaling
Inhibition of EGFR-dependent
proliferation
EGF
Ref: Astra Zeneca
Permanently activated intracellular signaling
T790M resistance mutation confers resistance to TKIs
Osimertinib (3rd line TKI)
Cell proliferation processes
EGFR as a Treatment Target in NSCLC
FFPE tumour tissue
Macrodissection & DNA Extraction
Molecular Testing
Interpretation and Report
EGFR FFPE Diagnostic Service
FFPE slides Histopathology
What happens when FFPE fails...
1. 30-40% of NSCLC patients are not testable
2. Re-biopsy not feasible for testing of resistance on progression
...Our answer is cell free circulating tumour DNA or
ctDNA
ctDNA is tumour DNA that has been shed into the blood stream
Diaz and Bardelli, J Clin Oncol. 2014 ; 32(6): 579-586
ctDNA as a Biomarker in Cancer
Diaz and Bardelli, J Clin Oncol. 2014 ; 32(6): 579-586
Two Routes of EGFR ctDNA Service
Route 1 – Diagnostic Testing
• “Screen”
• FFPE analysis has failed
OR
• No biopsy taken
• Iressa™ (AstraZeneca)
Route 2 – On Progression
• Specific testing resistance
mutation
• On clinical progression
• TAGRISSO™ (Osimertinib
- AZD9291)
5 day turnaround time
ctDNA has a very short half life
Preservative tubes such as Streck or CellSave tubes
recommended
False negatives an issue
ctDNA Collection is Important
ctDNA Processing Workflow
Sample arrives in lab and spun to isolate the plasma
Plasma stored as 1ml
aliquots at -80ºc
ctDNA extracted from the plasma using the QIAamp Circulating Nucleic Acid on
the QIAVac system
Sample taken in Streck or CellSave tube
Droplet Digital PCR
EGFR ctDNA Diagnostic Testing on Blood
3 Droplet digital PCR assays of EGFR
Assay Allele frequency Company
L858R 40% BioRad
Exon 19 deletions 45% Life Tech
T790M (resistance)
50% BioRad
Why use ddPCR for Mutation Detection?
Diaz and Bardelli, 2014 Journal of Clincial Oncology 32
Technique Sensitivity Optimal Application
Sanger sequencing >10% Tumour tissue
Pyrosequencing 10% Tumour tissue
COLD-PCR and Pyro
2% Tumour tissue
Next-generation Sequencing 2% Tumour tissue
Q-PCR 1% Tumour tissue
ARMS 0.10% Tumour tissue
ddPCR, BEAMing 0.01% or lower ctDNA
Why use ddPCR for Mutation Detection?
Validated to 0.5% mutation:wild type at 10 ng of DNA
4% 1% 0.5% 0.1% 0.01%
Total 21 patients
16 reported
24% for screen
76% for T790M
6x T790M found (54%)
Range 1-5% in blood
2 Patients on Osimertinib
5 ongoing
Swansea, Cardiff, Carmarthen, N
Wales, Bath, Bristol
EGFR ctDNA Diagnostic Service So far ...
Example of 1% T790M Patient COLD-PCR and Pyrosequencing
ddPCR
Summary
• ctDNA is a useful non-invasive cancer biomarker
• However it’s short half life means it needs urgent processing
• We have introduced an additional EGFR testing service based on ctDNA to test patients who fail on FFPE biopsy or to track resistance
Angharad Williams (ctDNA Service) [email protected]
Acknowledgments Helen Roberts
Daniel Nelmes
Kirsty Hambridge
Michaela John
Rachel Butler
FFPE
Pros
• The presence of tumour is known
• Easily stored at room temp
Cons
• Histopathology and macrodissected
• Downstream problems with quality of DNA
• Invasive biopsy procedure
• No tumour sample available
• One fixed time point
ctDNA
Pros
• Extracted from blood in house
• No need for invasive biopsy
• Sampling longitudinally and heterogeneity
• Detection at low levels in the blood
Cons
• Short half life
• Uncertain how much tumour DNA is circulating – FALSE NEGATIVES
• Very low concentrations from extraction
ddPCR Protocol
WT
PCR
Amplification
As standard
Droplet Making
The PCR mix with
mutant/WT probes is
partitioned into ~20,000
oil droplets with
microfluidics
Fluorescence
Detection Fluorescence amplitude
is measured in the reader
as either WT (HEX),
Mutant (FAM), both or
empty (no fluorescence) TaqMan Assay
ddPCR Analysis
• A threshold is called between ‘positive’ droplets containing DNA and ‘negative’ droplets with no DNA
• Using open source website to predict thresholds
• definetherain.org.uk
threshold
ddPCR Analysis
Fractional abundance mutation:WT
WT
BOTH L858R
EMPTY
4%
1%
0.5%
0.1%
• The software then calls
droplets as either
mutant, WT, both or
empty based on
fluorescence signal
• We are taking >7
droplets as a pass
• Uses Poisson modeling to predict starting concentration of the target DNA
2D Amplification plot
Astra Zeneca Service Evaluation of EGFR Mutation Detection from ctDNA
Aims to evaluate the feasibility of delivering accurate blood-based EGFR testing in a timely manner across the UK