Liquid biopsies Flashcards
What is a liquid biopsy?
→ Sampling and analysis of non-solid biological tissue, primarily blood (also can include cerebrospinal fluid). It is a minimally invasive technology for detection of molecular biomarkers
→ Representative of the tissue/s from which it has spread
→ Amniotic fluid analysis is an example of an established liquid biopsy
What 2 types of information can we process from a liquid biopsy? Which one are we more interested in from a liquid biopsy?
We can process both germline and somatic information from a liquid biopsy - but mostly interested in somatic because germline info is more accessible
How do you collect a liquid biopsy?
→ So blood doesn’t clot
→ Also need the tube being filled with blood to prevent:
genomic DNA release (from white blood cells) (you don’t want white blood cells bursting in the blood sample as that will bring in germline info) haemolysis
What are the types of tubes that can therefore be used to collect liquid biopsies(blood) and what properties do they have/what are logistics they have?
→ Tube type 1: EDTA, Citrate
→ Properties of tube type 1: Contain anticoagulant to prevent clotting
→ Logistics and storage of tube type 1: 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
→ Tube type 2: Cell-free DNA tubes (e.g. Paxgene-Qiagen; Streck)
→ Properties of tube type 2: Contain a stabiliser to prevent release of gDNA from white blood and haemolysis of red blood cells
→ Logistics and Storage of tube type 2: Samples can be stored for 6-14 days at 6ºC-37ºC
After 15 minutes of centrifuging a blood sample tube at 2000g and temp 4 degrees (to avoid haemolysis), what 3 layers are visible?
→ Top 55% is plasma (containing water, proteins, nutrients, hormones etc)
→ Next 1% is a buffy coat (containing white blood cells, platelets)
→ Bottom 45% is red blood cells
Name 2 liquid biopsies biomarkers
→ Circulating Tumour cells (CTC)
→ Circulating Tumour DNA (ctDNA)
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
→ Identified/characterised based on transcripts- PCR done on total RNA extracted from the cells
What is circulating tumour DNA?
→ 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
→ 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
→ You find ctDNA in the top 55%/top part of the test tube of blood sample after centrifuged- in plasma section
How do you isolate circulating free DNA generally?
→ Transfer supernatant to a clean polypropylene tube and freeze it if needed
→ Isolate using magnetic bead, cellulose-based or silica-based systems.
We can use :
→ Next Generation Sequencing (NGS), Digital Droplet PCR (ddPCR), array CGH: to identify Amplifications and deletions, Translocations, Point mutations, Chromosomes abnormalities, epigenetic status (methylation)
→ Real Time Quantitative Polymerase Chain reaction (qPCR): can identify ctDNA presence quantification
What are advantages/disadvantages of Liquid biopsies (vs solid/traditional 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- detection of early tumour cells is difficult
→ Data interpretation
Why are liquid biopsies good for use in detection of cancer? Why biopsy generally?
(rather than solid)
→ 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
Summarise this lecture
→ Liquid biopsies represent a novel non-invasive and revolutionary alternative to conventional biopsy methods.
→ CTCs and ctDNA are present in blood in low variable concentration and provide useful information about the individual genetic make-up.
CTCs and ctDNA have potential clinical uses as cancer biomarkers:
1. Screening
2. Diagnosis
3. Treatment
→ Standardisation of techniques along with multi-centre studies involving large cohorts of patients and controls are required to validate CTCs and ctDNA as clinical biomarkers.
