Robert Beer

Transcription

1 Robert Beer All Wales Medical Genetics Service Genetic Technologist Training Day 22 nd November 2017

2 Contents Stratified Medicine NHS EGFR Diagnostic Testing Services Cell free circulating DNA (ctdna) Why use ddpcr and ddpcr Validation ddpcr Setup and Analysis

3 What is Stratified Medicine? + ~ - + Image from

4 Non Small Cell Lung Cancer Discovery Medicine; ISSN: ; eissn: Discov Med 10(51): , August Li T, Kung H-J, Mack PC, et al. J Clin Oncol. 2013;31(8):

5 EGFR* as a Treatment Target in NSCLC *Epidermal Growth Factor Receptor EGFR Erlotinib, Gefitinib (Activating mutations) Osimertinib (Resistance mutations) KRAS EML4- Alk Tumorigenesis RAF Crizotinib

6 EGFR and ALK NHS Diagnostic Services FFPE tumour tissue FFPE slides Macrodissection & DNA Extraction Molecular Testing Interpretation and Report Histopathology EGFR Molecular service FFPE Blood ALK Cytogenetic service

7 What happens when FFPE fails Approx % 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

8 ctdna is tumour DNA that has been shed into the blood stream Diaz and Bardelli, J Clin Oncol ; 32(6):

9 ctdna as a Biomarker in Cancer

10 ctdna Processing Workflow Sample taken in Streck or CellSave tube Must be received <96 hrs Do no refrigerate! Sample arrives in lab and spun to isolate the plasma Plasma stored as 1ml aliquots at -80ºc Droplet Digital PCR ctdna extracted from the plasma using the QIAamp Circulating Nucleic Acid on the QIAVac system

11 What is ddpcr? Droplet Digital PCR Thousands of droplets are made, each containing a few molecules of DNA PCR carried out on each droplet Massive partitioning is the key to ddpcr

12 Why use ddpcr for Mutation Detection? Technique Sensitivity Optimal Application Sanger sequencing >10% Tumour tissue Pyrosequencing 10% Tumour tissue COLD-PCR and Pyro ~5% Tumour tissue Next-generation Sequencing 5% Tumour tissue Q-PCR 1% Tumour tissue ARMS 0.10% Tumour tissue ddpcr, BEAMing 0.01% or lower ctdna Diaz and Bardelli, 2014 Journal of Clincial Oncology 32

13 Two Routes of EGFR ctdna Service Route 1 Diagnostic Testing Testing of L858R, Exon 19 Deletions and T790M FFPE analysis has failed OR No biopsy taken Iressa (AstraZeneca) Route 2 On Progression Specific testing of T790M resistance mutation On clinical progression TAGRISSO (Osimertinib - AZD9291) 5 working day turnaround time

14 EGFR ctdna Diagnostic Testing 3 Droplet digital PCR assays of EGFR Assay Allele frequency Source Company L858R 40% BioRad Exon 19 deletions 45% Life Tech T790M (resistance) 50% BioRad Mutations are detected using a mix of flourecenent FAM and HEX probes

15 ddpcr Limit of Detection Validation Validated to 0.5% mutation:wild type at 10ng of DNA 4% 1% 0.5% 0.1% 0.01%

16 Preparing a ddpcr Run We follow the Bio-Rad SOP for ddpcr setup There are 4 setup stages: 1. Prepare PCR-ready samples - add supermix, FAM/HEX probes, controls and DNA 2. Generate droplets - up to 20, PCR amplification of droplets 4. Droplet reading using the QX200 droplet reader

17 Preparing a ddpcr Run 3 controls (at least) on each run: 1. Positive control for calling thresholds and QC 2. Normal control check for background amplification 3. No template control (NTC) Check for contamination Important to add enough DNA We found 20ng (for ctdna) works well but this might mean adding whole sample!

18 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

19 ddpcr Analysis and QC 1. Thresholds called by Define the Rain and fractional abundance of positive control checked against previous run data 2. >10,000 droplets to pass a sample 3. Check quality of all control samples before patient samples 4. We are calling >5 droplets as a mutation positive result (+ clear NTC and normal control) Patient +VE Control Normal NTC

20 Summary Without the sensitivity of ddpcr we would not be able to detect low level ctdna mutations Detection of these low level mutations allows us to provide more treatment opportunity for patients than was available previously We are gradually finding more and more uses for ultra sensitive approaches such as ddpcr

21 Thank You Robert Beer Acknowledgments Rachel Butler Angharad Williams Aislinn Cooper Justyna Tull Laura Ferguson