1. The Use of Circulating Tumour DNA as a Liquid Biopsy
Kirsty Hambridge
Genetic Technologist
Genetic Technologist Training Day
20th November 2014

2. Circulating Tumour DNA (ctDNA)
ctDNA is tumour DNA that has been shed into the bloodstream
ctDNA can be present in 0.01% - >90% of the total Cell Free DNA (cfDNA)
The amount of ctDNA is related to the tumour burden and varies between patients with different clinical presentations
Circulating tumour DNA is a component of cell free DNA, this is DNA present in the blood stream and this is the same sample as the free fetal DNA
The tumour DNA is released into the blood stream by secretion, apoptosis and necrosis, however most cfDNA fragments are between bp suggesting apoptosis produces the majority of cfDNA in circulation.
Diaz and Bardelli, 2014 Journal of Clincial Oncology 32

3. FFPE versus ctDNA FFPE Samples ctDNA Samples
Tumour DNA extracted from fixed biopsy samples or tumour resections
Problems with quality of DNA due to fixation
Mixture of normal and tumour DNA
Long time to process by histopathologists.
Macrodissected to enrich tumour content
Some patients have no tumour sample available
The sample represents the tumour at one fixed time point
ctDNA Samples
ctDNA shed directly from tumour
Extracted from the plasma component of whole blood
Large fragment sizes possible
Small quantities extracted ~ 30ng/ 5ml plasma
Separate out plasma within a few hours of receipt of blood sample.
Serial samples can be taken at various time points during the patient’s treatment
FFPE is an invasive procedure

4. ctDNA Collection
ctDNA has a very short half life ranging from 15 minutes to several hours
It is stable in plasma at ºc
Blood can be sampled in ETDA tubes but the plasma has to isolated and stored at -80ºc within one hour of collection
Preservative tubes can be used to stabilise the cfDNA in blood for up to 4 days at room temperature.

5. ctDNA Workflow Sample arrives in lab and spun to isolate the plasma
Plasma is stored at -80ºc
Blood sample taken in Cell Save preservative tubes
Sample is extracted on the same day as the downstream process set up due to ctDNA instability
Set up:
Pyrosequencing
Next-generation sequencing
Quantative PCR
BEAMing
Digital PCR
ctDNA is extracted from the plasma using the QIAamp Circulating Nucleic Acid on the QIAVac system

6. Problems with ctDNA
Due to the unstable nature of ctDNA the sample is has to be collected and processed correctly
Only get 30ng of cfDNA per 5ml plasma extraction
The amount of ctDNA is related to the tumour burden and varies between patients
Difficult to discriminate ctDNA from normal cfDNA
The technique used must be sensitive enough to pick up the low level variants
Diaz and Bardelli, 2014 Journal of Clincial Oncology 32

7. Current Projects: Patients are being stratified using an FFPE sample
Technique
Sensitivity
Pyro-sequencing 5%
Cold-PCR 2%
Enhanced ICE Cold-PCR
?0.1%
AZD5363 is a kinase inhibitor on the PIK pathway
Fulvestrant works in two ways. It’s an anti-oestrogen drug. It fits into the oestrogen receptor and blocks oestrogen from reaching the cancer cells. This means the cancer either grows more slowly or stops growing altogether. It also works by reducing the number of receptors on breast cancer cells.
Developed Cold-PCR that explotes the difference in denaturation temperatures in between WT and mutant allele
Currently devloping ICE Cold-PCR that uses a blocker (an oligo that is complimentary to the WT DNA ) and enriches the mutant
Digital PCR

8. Current Projects: Aristotle
3 colorectal cancer patients
FFPE sample and 4 serial ctDNA samples
NGS using Cancer Hotspot Panel v2 on the Ion Proton
Digital PCR
These are the somatic mutations found in the FFPE sample
Baseline Diagnosis
Completion of Long Course
Chemo/Radiotherapy
Progression of lung nodule, and new liver lesions consistent with metastatic disease
Start Long Course
Chemo/Radiotherapy

9. Acknowledgements All Wales Medical Genetics Service Rachel Butler
Cardiff University
Dr Daniel Nelmes
Velindra Hospital Cardiff
Dr Richard Adams
Dr Robert Jones