Liquid Biopsies Flashcards
What is a liquid biopsy?
Sampling and analysis of non-solid biological tissue, primarily blood. It is a minimally invasive technology for detection of molecular biomarkers. Representative of the tissue/s from which it has spread.
How can we use a liquid biopsy to detect cell death?
As a result of cell turnover, from regular apoptosis or necrosis, there is cell material that is being continuously released into the bloodstream
These materials are cleared by specialised cells called phagocytes
However they remain in the bloodstream for a short period of time where we can detect it in the blood
What can we detect in blood?
In blood we can detect:
- Circulating endothelial cells- useful for early detection of heart attacks
- Cells that have detached from tumours- called circulating tumour cells (CTCs)
- Cell free nucleotides that are being released from cells dying from apoptosis and necrosis
(In certain conditions such after inflammation or a tumour the levels of those cell free nucleotides can increase as there is a higher rate of dying cells)
- We can also detect extracellular micro-vesicles (exosomes)
Exosomes are nano-sized vesicles that are made up of the lipid bilayer containing proteins, RNA and bioactive lipids
They are though to be messengers between cells
What are the types of liquid biopsy tubes?
EDTA, Citrate:
- Contain anticoagulant to prevent clotting
- (On-site centrifugation within 6hrs of collection to isolate plasma and avoid white cells apoptosis. If not possible, sample can be stored at 4ºC for a up to a week)
Cell-free DNA tubes:
- Contain a stabiliser to prevent release of gDNA from white blood and haemolysis of red blood cells
- (Samples can be stored for 6-14 days at 6ºC-37ºC)
What happens when you centrifuge blood?
After 15 mins centrifugation at 2,000 x g speed at 4ºC…
55% is plasma with water, proteins, nutrients and cfDNA, exosomes etc.
<1% Buffy coat WBC and CTCs
45% haematocrit of RBCs
What are circulating tumour cells?
- Cells that have detached from a tumour and travel through the bloodstream to other parts of the body- single cells or clusters.
- Marker for tumour growth and negative cancer prognosis and treatment response.
- Extremely rare: 1-10 per 1ml of blood.
- Found in a high background of normal cells! - sensitive and specific methods are needed to study them.
- So some researchers prefer to isolate the buffy layer
What are the ways we can isolate cells based on characteristics? (Give examples)
Biological properties and /or physical properties
Through identified/characterised based on transcripts
For example to isolate them we can use their biological properties such as isolation based on cell surface markers such as FOX and magnetic nanoparticles
So we can isolate CTCs because they are CD45 negative unlike leukocytes and positive or EpCAM and CK
Or we can use other their physical properties such as size and density
Then we identify markers specific e.g. for prostate cells after isolating the DNA from the CTC and amplifying it using PCR and run those samples on electrophoresis
Where is Circulating Tumour DNA (ctDNA) found?
Present in different fluids: plasma, serum, urine and others.
Low concentration (1-50ng DNA/mL plasma).
Amount highly variable for person to person and depending on health status in the same person (increase in cancer, trauma, etc.).
There is a presence of permanent genomic DNA background in plasma as part of the normal cell turnover process.
How can we measure ctDNA?
In this case we do not need to carry out the first fragmentation step as part of the next gen seq. process
- Highly fragmented but with specific size range (<500bp)
- Provides information of current genetic make-up (including irregularities/mutations) with 80-95% specificity and 60-85% sensitivity.
How do you isolate ctDNA?
Transfer supernatant to a clean polypropylene tube and freeze it if needed
We can store it forever theoretically
Isolation of DNA using magnetic bead, cellulose-based or silica-based systems and store it forever
What type of information can ctDNA provide?
Next Generation Sequencing (NGS), Digital Droplet PCR (ddPCR), array CGH:
- Amplifications and deletions, Translocations, Point mutations, Chromosomes abnormalities, epigenetic status (methylation)
Real Time Quantitative Polymerase Chain reaction (qPCR):
- nctDNA presence quantification
What are the advantages and disadvantages of liquid biopsies?
Advantages:
- Lower invasiveness
- Higher patient compliance
- Higher cost/effectiveness
- Allow repeated access and multiple sampling
- No special training required for extraction
Disadvantages:
- Low amount of material e.g. low CTC levels and ctDNA
- When it comes to early diagnosis it is less likely to detect a cancer at an early non-metastatic stage
- Data interpretation in regards to isolating the cells of interest from the rest
Why would you use liquid biopsies for cancer detection?
Cancer is a heterogeneous disease.
Molecular properties within a tumour differ and also between metastatic sites.
Primary tumour information may not reflect the current disease condition.
No need to identify the tumour site before taking a biopsy and allow repeating sampling.
Allow analysis of tissues difficult to access such as lungs and prostate.
