Liquid Biopsies Flashcards
What are the three types of medicine ?
One size fits all
Stratified medicine
Precision medicine
Describe stratification
Patients are grouped by : Disease Subtypes Demographics Clinical features Biomarkers
Describe personalisation
This is taking into account :
- Patient preferences
- Clinical features
- Medication history
- Environment
- Behaviours/habits
- Biomarkers
What is personalised medicine ?
A medical model which moves away from one size fits all to one which involves targeted therapies.
What are different technologies which are involved in personalised medicine ?
- Liquid biopsies
- Next gen sequencing
- CRISPR/Cas9
- 3D cultures - organoid technology
- Targeted therapies
What is a liquid biopsy?
Liquid biopsy = Sampling + analysis of non-solid biological tissue (e.g.blood)
- Minimally invasive, detects molecular biomarkers (associated with disease = disease biomarkers)
- Representative of the tissue from which it has spread.
Blood carries biomarkers very effectively.
What caused the start of liquid biopsies?
Discovery of free DNA/RNA in blood = Used in disease prognosis+diagnosis.
What is an example of a established liquid biopsy ?
Amniotic fluid analysis.
The problem with it - it’s highly invasive so it has been replaced by non-invasive pre-natal test which uses free circling foetal DNA in mothers blood.
Why do we use blood for liquid biopsies?
Blood contains dying cells (apoptosis/necrosis, constant cell renewal).
Cell material is released into bloodstream before phagocytes engulf
Detect by extracting a blood sample
What can we detect in the blood during biopsies ?
We can detect :
- Endothelial cells (early detection of heart attack)
- CTCs-Circulating tumour cells
- Cell-free DNA/RNA = increase in exercise/inflammation
- Tumour educated platelets
- Disseminated tumour cells
- Extracellular micro-vesicles (exosomes)
- Micro-RNA 105 (metastatic breast cancer )
What type of information are we interested in when processing liquid biopsies ?
Germline Information
Somatic Information
Why are we more interested in somatic information?
We can find germline hereditary information in any cell in our body.
Somatic info is only found at that specific tissue where changes have occurred.
-E.g. Localised lung tumour
If we analyse white blood cells/skin cells, we will not find the mutation that has occurred in the lung cells as it is not inherited. Need to conduct a solid biopsy from the lung to study the somatic information from the tumour cells. Blood sample detects DNA coming from tumour cells that are dying in the lung + detects circulating tumour cells that detach from lung tumour
What is somatic mutation?
Somatic mutation occurs in a single body cell and cannot be inherited (only tissues derived from the mutated cell are affected)
What is germline mutation
Germline mutation occurs in gametes and can be passed onto offspring (every cell in the entire organism will be affected)
How can we extract a liquid biopsy?
-Venipuncture procedure
(10mL of blood contains 4-5mL plasma)
-To study circulating tumour DNA = isolate the plasma.
Why do we need to use special extraction tubes ?
We need to use the EDTA (contains anti-coagulant) tube because :
- prevents blood clot
- prevents genomic DNA release (from WBCs)
- prevents haemolysis
What are the 2 types of tubes used for liquid biopsies?
- EDTA/Citrate
- Cell-free DNA tubes
Describe the properties of EDTA /Citrate tubes
EDTA /Citrate tubes contain anti-coagulants to prevent clotting.
Describe the properties of Cell-free DNA tubes
Paxgene-Qiagen,Streck
Contain a stabiliser to prevent release of gDNA (genomic DNA) from WBCs and haemolysis of RBCs
-after storing for 7 days at 4oC and then centrifuging, nice yellow plasma fraction due to no haemolysis
Describe the logistics and storage of EDTA tubes
-On site centrifugation within 6 hours of collection to isolate plasma and avoid white cell apoptosis.
If not possible , samples can be stored at 4oC for up to a week
-after storing for 7 days at 4oC and then centrifuging, red plasma fraction due to haemolysis
Describe the logistics and storage of Cell free DNA tubes
Samples can be stored for 6-14 days at 6oC-37oC
-transport, storage
What are the different fractions of blood following centrifugation?
Following 15mins centrifugation at 2,000 x g speed at 4oC
3 diff phases:
55% Plasma (yellow top layer)
Water,proteins,nutrients,hormones +cfDNA exosomes
(cell-free DNA )
<1% buffy coat (intermediate layer)
White blood cells, platelets
WBC and CTCs
45%Hematocrit (bottom red layer)
Red blood cells
-depending on what we want to extract from blood sample, interested in isolating top/intermediate layer
What different liquid biopsy samples are there?
- Saliva
- Cerebrospinal fluid
- Pleural liquid
- Urine (to detect metabolites)
- Serum plasma
What can we detect in a liquid biopsy?
- Circulating free DNA - e.g. Circulating tumour DNA
- CTC (circulating tumour cells )
- Small nucleic acids (miRNA)
e. g. miRNA 105 - Promotes breast cancer metastasis, detected in blood in early stages of breast cancer metastasis - Metabolites in urine
- Exosomes (extracellular vesicles) - lipid bilayer, proteins, RNAs, lipids, cell-cell communication
Which 2 important things are found in blood (liquid biopsy) that help us identify biomarkers?
- CTCs - Circulating Tumour Cells
- ctDNA - Circulating Tumour DNA
Describe the isolation and characterisation of CTCs
-Based on biological properties and physical properties
-Identified/characterised based on transcripts.
PCR done on total RNA extracted from cells
What are CTCs?
CTCs = Circulating Tumour Cells.
- These are cells that have detached from a tumour and travel through blood stream to other parts of the body
- Single cells/Clusters
- Marker for tumour growth/negative cancer prognosis, treatment response (CTCs = treatment is not being efficient)
- Extremely rare (low conc): 1-10 per 1ml of blood
- Found in a high background of normal cells in blood (erythrocytes, leukocytes, platelets, neutrophils) = need sensitive + specific methods to identify + isolate CTCs
Centrifuge blood sample for 15mins at 2000g speed at 4oC, what phase are CTCs found in?
Buffy coat intermediate layer (WBC layer)
How can we isolate tumour cells using markers?
Isolate CTCs using their Biological properties e.g:
- Cell surface markers techniques (FAX/magnetic)
- CTCs are CD45(blood cell marker ) negative.
- EpCAM ,CK(cytokeratins 8,18,19)positive = these cell surface markers not found in other types of blood cells
- Useful for detecting metastasis
Or isolate CTCs using their physical properties:
- Size - CTCs are larger than other blood cells
- Electric charge
- Density
What can we do once we isolate/extract circulating tumour cells?
Want to know CTC origin, so identify specific biomarkers on the isolated cells.
-e.g. suspect prostate cancer. identify prostate-specific biomarkers (PSA). extract CTC DNA/RNA - rtqPCR to identify PSA levels/PCR to amplify PSA then run on electrophoresis gel.
We can study :
- Transcriptome (set of RNA transcripts)
- Proteome (Entire set of proteins)
- Phenotypic studies (
- Genome (genetic material of organism)
- Morphology
- Use lots of different techniques - rtqPCR, culture CTCs in vitro, inoculate CTCs into mouse and see if tumour forms
What are some techniques we could use to study the CTC after extracting/isolating ?
- Next generation sequencing - study cell DNA
- FISH (Fluorescence in situ hybridisation) - study chromosome abnormalities
- Flow cytometry
- In vivo/vitro culture
- RTqPCR - detect biomarkers
these techniques provide info about the original tumour site (that CTCs detached from)
Describe cfDNA
- Present in fluids - plasma, serum, urine, CSF
- Low concentration (1-50ng DNA/1mL plasma)
- Amount is highly variable b/w diff. people + varies depending on health of person (Increased cfDNA in cancer, trauma, inflammation, exhaustive exercise)
- Presence of permanent background genomic DNA in plasma - cfDNA coming from cells dying by apoptosis/necrosis + being replaced
What are the properties of circulating tumour DNA ?
- ctDNA = highly fragmented w specific size range (500bp) (=don’t need to fragment in NGS)
- Provides information of current genetic makeup (irregularities/mutations) with 80-90% specificity and 60-85% sensitivity.
Why does free DNA increase during cancer ?
- Circulating free DNA (cfDNA) comes from cells that are continuously dying and being replaced.
- Cancerous tumour cells
What does the highly fragmented property of free DNA mean when it involves next generation sequencing ?
-To study cfDNA using NGS , don’t need to fragment cfDNA (an early step of process)
How can we extract the circulating free DNA ?
Following centrifugation, cfDNA is found within the top phase - Plasma
-cfDNA in top plasma layer, then identify ctDNA from total cfDNA
Describe the process of isolating cfDNA
Following centrifugation:
- Transfer the supernatant (plasma) to a clean polypropylene tube and freeze if need to store it (forever)
- Isolate cfDNA from plasma using magnetic bead/cellulose based/silica based systems. then store (forever) if required
What analysis can cfDNA provide us with ?
-amplifications/deletions
-point mutations
-translocations
-epigenetic modifications
Limited analysis - Not a cell so we analyse epigenome/genome using NGS, RTqPCR (to analyse diff. in cfDNA levels), Digital PCR.
What is RTqPCR?
This is reverse transcription polymerase chain reaction.
Combines reverse transcription of RNA and amplification
What are the advantages of liquid biopsies (compared to traditional solid biopsies?
- ↓ invasive - just extract blood by normal venipuncture = extract 4-5ml plasma
- ↑ patient compliance
- ↑ cost-effectiveness
- Allows repeated access and multiple sampling = can monitor disease progress
- No special training is required for extraction (not surgeon, any nurse can do it)
- Gives info about genetic characteristics + tumour characteristics w/o needing solid biopsy = difficult in tissues difficult to access - lung, prostate, kidney
What are the disadvantages liquid biopsies?
- Low amount of material extracted - CTCs, ctDNA (limitation) = requires sensitive techniques to isolate material + low CTCs/ctDNA in early tumour stages = hard to detect
- Early diagnosis (not many markers at early stages of disease)
- Data interpretation - lot of data from all tissues - we need to extract tumour cell info via biomarkers and separate from normal background cell info. + decide what to report back to patient
- Requires special tubes that prevent blood clotting, genomic DNA release, haemolysis
What are the steps in cancer treatment ?
- Cancer detection screening /early diagnosis
- Molecular profiling or prognostication
- Detection of residual disease
- Monitoring response
- Monitoring clonal evaluation
Cancer has biomarkers. Explain how liquid biopsies are suitable in diagnosis.
- Liquid biopsies can provide the genetic makeup of individuals
- Cancer is a heterogenous disease(between metastatic sites the molecular properties are different + intratumoural)
- Liquid biopsies help us identify different tumours as all parts of tissues. Solid biopsy = only pick up 1 part of tumour = some clones of tumour.
- Don’t need to know tumour site (do for solid biopsies). extracting liquid biopsy tells you tumour site
- Analyses tissues difficult to access - lung, kidney, prostate
- Extract info at any point of disease - early stages, throughout treatment
Discuss some further advantages of liquid biopsies in cancer
- We do not need to know the tumour’s location
- Allows repeat sampling
- Allow analysis of difficult to access tissues.
- Primary tumour information may not reflect current disease condition.
What does Genomic analysis involve?
- Mutation profiling
- Treatment selection
- Monitoring clonal evolution
What is liquid cdx
A lab test that detects specific gene mutations found in cell-free DNA (cfDNA) isolated from whole blood plasma specimens
Identifies mutations in patients with advanced solid cancer that can be treated using targeted therapies.
What are some way a liquid biopsy can be used ? (clinically)
- They can be taken at any stage of disease meaning they can be used to diagnose diseases.
- Cancer biomarkers can be detected
Liquid biopsy cancer biomarkers need to be ………..
clinically validated - not currently used as a diagnostic tool, but provides highly specific + complementary info to normal diagnostic tools (solid biopsy, x-ray)
-but exceptions - EGFR lung cancer,
Describe the EGFR mutation test
- Diagnostic test which uses blood samples to find mutations in the gene for epidermal growth factor receptor (EGFR)-lung cancer.
- EGFR mutations in 20% of non-small cell lung cancer patients (most common form lung cancer)
- 75.7% sensitivity, 99.8% specificity.
- Identifies patients who could be eligible for treatment by erlotinib.
What does quantitative analysis include?
Disease staging
Monitor response
Prognosis (predicting future events).
