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The Use of Circulating Tumour DNA as a Liquid Biopsy

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Presentation on theme: "The Use of Circulating Tumour DNA as a Liquid Biopsy"— Presentation transcript:

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

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