Liquid Biopsies Flashcards
Describe liquid biopsies
Liquid biopsies is one of the factors used for personalised medicine with its role primarily acting as a biomarkers
What is a liquid biopsie?
- Sampling and analysis of non-solid biological tissue, primarily blood.
- Blood is used as it recieves inputs from all organs of the body including any tumours
- Other examples include saliva, pleural liquid, urine, CSF liquid
- It is a minimally invasive technology for detection of molecular biomarkers
- Solid biopsie is a very invasive technique
- Representative of the tissues from which it has spread.
State an example of an established liquid biopsie?
- Amniotic fluid analysis
- Invasive
- Non invasive, pre natal tests are being used (circulating free DNA)
What are CTCs and what is their biomarker role for in liquid biopsies?
- CTC = Circulating tumour cells
- Rare 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.
- Found in a high background of normal cells! - sensitive and specific methods are needed to study them
What is the isolation, characterisation and analysis of CTs based on?
- Isolation and charaterisation: Biological and/or physical properties, Transcripts via PCR performed on total RNA from cells
- Downstream analysis: Techniques like NGS, flow cytometry, FISH etc. , Used to study genomics, phenotype, protemics etc.
Describe the sources of ctDNA and featurs about it (3)?
- ctDNA = Circulating tumour DNA - cancer > ^ ctDNA
- 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.).
- Presence of permanent genomic DNA background in plasma (from cell turnover)
Describe the physical features of cDNA
Highly fragmented (many pieces with different sizes) but with specific size range (<500bp)
ctDNA, What technique can be used to analyse and where is it found in centrifugation of liquid biopsie?
- NGS used primarily
- Provides information of current genetic make-up (including irregularities/mutations) with 80-95% specificity and 60-85% sensitivity.
- Found in plasma in cDNA (circulating free DNA)
- Extract ctDNA and isolate tDNA from there
How is ctDNA isolated after the centrifugation of liquid biopsie?
- After centrifugation, transfer supernatant to a clean polypropylene tube and freeze it if needed
- Isolation of cDNA using magnetic bead, cellulose-based or silica-based systems.
What techniques are used for the analysis of ctDNA and state what they can detect?
- Next Generation Sequencing (NGS), Digital Droplet PC (ddPCR), array CGH:
- Amplifications and deletions
- Translocations
- Point mutations
- Chromosomes abnormalities
- Epigenetic status (methylation)
- Real Time Quantitative Polymerase Chain reaction (qPCR):
- cDNA presence quantification
State the advantages (5) of using liquid biopsies over solid biopsies?
- Advantages:
- Lower invasiveness
- Higher patient compliance
- Higher cost effectiveness
- Allow repeated access and multiple sampling
- No special training required for extraction
State the disadvantages (3) of using liquid biopsies over solid biopsies?
- Disadvantages:
- Low amount of material
- Early diagnosis is difficult
- Data interpretation
Describe features of cancer as reasons as to why liquid bipsoies are a good method for cancer biomarkers?
Key features of cancer:
- Cancer is a heterogeneous disease, therefore..
a. Heteogenous = condition with several root causes - Molecular properties within a tumour differ arise and also between metastatic sites. Therefore..
a. Metastatic sites = secondary malignant growths - Primary tumour information may not reflect the current disease condition.
Describe features of cancer as reasons as to why liquid biopsies are a good method for cancer biomarkers?
- Why liquid biopsies are used:
- No need to identify the tumour site before taking a biopsy and allow repeating sampling.
- Allows for identification of tissue specific markers and the different tumour sites
- Allow analysis of tissues difficult to access.
What can the cancer biomarkers of liquid biopsies be used for?
- Early detection, screening and testing
- Individual genetic makeup
- Identification of resistance mechanisms
