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
What type of cancer can liquid biopsies detect for?
- Breast cancer (main once)
- Colorectal
- Lung
- Prostate
State the reasoning of the usage of cancer biomarkers from liquid biopsies in modern times
- Promising biomarkers that need to be clinically validated
- Not implemented as diagnosis tool yet
- But that provides highly specific and complementary information
State 2 examples of FDA-approved diagnostic tests?
- Detection of EGFR mutations in lung cancer
a. EGFR = epidermal growth factor receptor
- Detection of EGFR mutations in lung cancer
- Pan-tumour liquid biopsy test for patients with advanced solid cancer
How can biomarkers be validated for diagnostic usage?
Standardization of techniques along with multicentre studies involving large cohorts of patients and controls are required to validate CTs and tDNA as clinical biomarkers.
