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.
E.g blood (carries information from all over the body), urine, plasma serum, saliva and CSF (identification of DNA in tumours).
Give an example of a liquid biopsy?
Amniotic fluid - refer to diagram
How is blood a liquid biopsy?
- Cells are constantly renovating in our body, there is a balance between new and old cells
- Cell death is achieved by apoptosis or necrosis
- Debris within blood vessels is removed by phagocytes, they remain the blood for a short period of time. We can detect them if we take a blood sample.
- We can also detect endothelial cells e.g early detection of heart attacks. These include cells that have detached from tumours, cell free nucleotides from apoptosis or necrosis/ inflammation, extracellular vesicles known as exsosomes (vesicles that are made of a lipid bilayer, containing RNA and proteins, these are messengers that allow communication between cells), microRNA for the detecttion of metastatic breast cancer.
- A biopsy is like a screen shot, where we then identify things in the picture
- We are interested in the somatic information because we can find germline information in every cell in our body, however we can only isolate this somatic information from the tissue that it is found in.
- E.g if somebody has lung cancer, we need to carry out a lung biopsy from the lung to identify the tumoral cells and study the somatic information. Therefore, taking blood contains the tumoral cells and other circulating tumoral cells.
How do we carry out a biopsy?
What two types of tube do we use
- 10mL of blood collected by a venipuncture (4-5mL of plasma)
- These need to prevent blood clots, prevent genomic DNA release from white blood cells and prevent haemolysis. To prevent this from happening we use 2 main types of tubes:
What can we see when we centrifuge blood?
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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.
- They are found in the buffy coat:
What do we do once we have extracted our ctc cells?
- Studies that analyse the proteomics, transcriptomics, genomics and phenotypic studies
- Transfer to animals for studies of mechanism of cells
- PCR, NGS, flow cytometry ect
What is circulating tumour DNA? (ctDNA)?
- 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.).
- Presence of permanent genomic DNA background in plasma.
- 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.
Where do we find the ctDNA?
Plasma
How do we isolate the ctDNA?
- Transfer supernatant to a clean polypropylene tube and freeze it if needed
- Isolation using magnetic bead, cellulose-based or silica-based systems. We can extract DNA from the plasma and then store it
What and how can we study from ctDNA?
- 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): ctDNA presence quantification
What are the advantages of liquid biopsies and disadvantages of using solid 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 • Early diagnosis • Data interpretation
How can liquid biopsies be used as cancer markers?
- 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 tissues difficult to access.
What can liquid biopsies be used for?
- Early diagnosis of the disease
- Identification of location of tumour
- Monitor response to therapy
FDA approved test
• Detection of EGFR mutations in lung cancer
• This takes blood samples to look for mutations in genes coding for the epidermal growth factor receptor in patients with lung cancer.
What is a liquid cdx test?
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