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) Angharad.Williams8@wales.nhs.uk

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