Liquid Biopsies COPY 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.
alternative to conventional biopsy methods.
What liquid biopsies are currently used?
- Urine
- Plasma/serum
- Saliva – important for detection for head and neck cancers
- CSF – detection of tumour DNA in primary brain tumours
What is amniotic fluid analysis an example of?
- An established liquid biopsy – but is invasive so has been substituted for non-invasive prenatal test that identifies foetal DNA.
- Foetal DNA can be detected in mothers blood.
- Can do paternal test, sex tests, and chromosomal abnormalities.
Why do we use blood as a liquid biopsy?
- Balance between cell renewal and cell death – cell death achieved by apoptosis or necrosis.
- Some materials are released to the bloodstream which gets removed by phagocytes. These materials remain in the blood for a certain amount of time before removal can be detected.
What are some of the materials we can detect in blood?
- E.g Circulating Endothelial Cells (CEC) – early detection of heart attack
- Circulating Tumour cells (CTCs) – cells broken off from tumours
- Disseminated tumour cells (DTCs)
- Cell free nucleotides – released by cells dying from apoptosis or necrosis, exhausted exercise, etc
- Extracellular micro-vesicles (exosomes) – contain proteins, RNA, bioactive lipids. These are messengers that communicate to other cells. Can detect early stages of metastatic breast cancer.
What information can you get from a liquid biopsy?
You can get an idea/multiple information on all the processes that are happening in the orgsanism at the moment of extraction such as:
- Germline and somatic information
- E.g if someone has a localised lung tumour, if WBC’s only analysed with germline info, won’t find info only occurred in lung cells. Need a solid biopsy from lung to check tumour cells.
- Circulating tumour cells detaching from a tumour.
What is the process of extracting a biopsy?
- 10mL blood collected by venipuncture (4-5mL plasma)
- If we are interested in circulating tumour DNA – isolate plasma
- If we are interested in extracting circulating tumour cells need to select another fraction of the blood sample.
What do we use if we are conducting liquid biopsies to detect biomarkers?
We need special tubes (EDTA) purple cap tubes as they have anticoagulant.
The selection of tubes has to prevent what?
- Blood clots
- Genomic DNA release (from white blood cells) – contaminates somatic information we are aiming to collect.
- Haemolysis
What are the types of tubes that can be used and their specifications?
What can be seen after centrifugation?
Depending on type of material we want to extract from a blood sample we will be isolating different layers.
What are Circulating Tumour Cells (CTCs)?
- 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.
What is the isolation and characterisation of CTCs?
- This is based on the biological properties and/or physical properties of tumour cells.
- Combined methods to isolate the cells – use cell surface techniques based on FACS or magnetic beads.
- Can isolate CTCs as they are CD45 negative, EpCAM positive, these cell surface markers are not found on any other cells so can prepare our experiment to isolate CTCs.
- Physical properties include size.
- After isolation, want to determine where the CTCs are coming from?
- Identified/characterised based on transcripts – PCR done on total RNA extracted from the cells.
What do we do after we isolate CTCs?
- Study the genomes, proteomes, transcriptome
- Carry out phenotypic studies
- Culture circulating tumour cells – can enucleate CTCs in mice to see if they form tumours elsewhere.
What do we use to study the genomes?
- NGS
- FISH
- Flow cytometry
- RT qPCR
- In vivo/in vitro culture
This analysis of CTCs gives information on the tumour they have detached from.
What is Circulating Tumour DNA (ctDNA)?
- Present in different fluids: plasma, serum, urine and others
- Low concentration (1-50mg 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 makeup (including irregularities/mutations) with 80-95% specificity and 60-85% sensitivity.
Where is ctDNA found?
cTDNA is found in the plasma portion of blood after centrifugation. Extract circulating free DNA first then ctDNA present in the circulating free DNA.
How do you isolate the circlulating free DNA?
- After centrifugation, transfer supernatant to a clean polypropylene tube and freeze it if needed.
- Isolation using magnetic bead, cellulose-based or silica-based systems
What information does ctDNA provide us with and what can we do with it?
As we are not interrogating cells we are limited and can only look at epigenome and genome.
Can analyse:
- Amplifications and deletions
- Point mutations
- Study of epigenetic modifications
- Translocations
What are the advantages of liquid biopsies?
- Lower invasiveness
- Higher patient compliance
- Higher cost/effectiveness
- Allow repeated access and multiple sampling – monitor stages of the disease
- No special training required for extraction
What are the disadvantages of liquid biopsies?
- Low amount of material
- Early diagnosis
- Data interpretation
Why are liquid biopsies used for cancer biomarkers?
- Cancer is a heterogeneous disease – tumour heterogeneity won’t provide a complete picture
- Molecular properties within a tumour differ and also between metastatic sites – intermetastatic heterogeneity.
- Can be multiple sites of metastasis in an organ, a solid biopsy may not collect all of the types of tumour cells present, liquid biopsies would detect info from all parts of the tissue and cells and all mutations present.
- Primary tumour information may not reflect the current disease condition
- Solid biopsies require identification of tumour site in advance.
- No need to identify the tumour site before taking a liquid biopsy and allow repeating sampling.
- Allow analysis of tissues difficult to access – important in cancer
What can liquid biopsies be used for?
- Any point during the disease – levels of progression
- Early diagnosis of the disease
- Determine molecular profile of tumour – determines treatment to be given
- During treatment, monitor response to therapy or surgery
What do promising biomarkers need to be?
Need to be clinically validated, not implemented as a diagnosis tool yet, but that provides highly specific and complementary information.
What is the detection of EGFR mutations in lung cancer?
- Blood samples look for mutations in epidermal growth factor receptor (EGFR) in patients with non small cell lung cancer.
- In non small cell lung cancer 10-20% have EGFR mutations – most common type of lung cancer
- Liquid biopsies are good for 25% of non small cell lung cancer patients who have insufficient tissue or unable to undergo tissue biopsy.
- Sensibiility of 75.7% and specificity of 99.8%
- Aim of test to identify patients with non small cell lung cancer who could be eligible for treatment with elotania (commercial name) that treats EGFR gene.
- Uses Cobas EGFR mutation Test v2. Results for T790M mutations – 4-8 hours to get results from blood plasma.
What is the pan-tumour liquid biopsy test for patients with advanced solid cancer?
- Liquid CDx
- Tests to identify patients who might benefit from treatment with complementary targeted therapy such as BARB inhibitors to treat patients with mutations in genes.
- Designed to identify mutations with solid cancer that can be used for targeted therapies.
What is the summary of liquid biopsies?
- CTCs and ctDNA are present in blood in low variable concentration and provide useful information about the individual genetic makeup.
- CTCs and ctDNA have potential clinical uses as cancer biomarkers:
- Screening
- Diagnosis
- Treatment
- Standardisation of techniques along with multicenter studies involving large cohorts of patients and controls are required to validate CTCs and ctDNA as clinical biomarkers.
