liquid biopsies Flashcards
What is personalised medicine?
- model that moves from one-size fit all medicine to new approaches which helps better manage patients health, and targeted therapy for best outcomes.
- liquid biposies are used for personalised medicine.
Define liquid bioposies.
- sampling and analysing of non-solid biological tissue, primarily blood. It is a minimally invasive technology for detection of molecular biomarkers.
What are examples of liquid biopsies?
- urine
- bloods
- saliva
- CSF (useful for circulating tumour DNA)
What are advantages of liquid biopsies?
- minimal invasive technology
- liquid biopsy sample is representative of tissue from which it has spread.
What is an example of well known liquid biopsy?
- amniotic fluid analysis
- sex test, chromosomal abnormalities can be identified by analysing this liquid biopsy.
Why can we use blood as a liquid biopsy?
- cells are continuously renovating , cell death leads to different turnover of material.
- some material is continuously being released in the blood stream.
What is found in the blood biopsy sample?
- circulating endothelial cells
- circulating tumour cells (CDC)
- disseminated tumour cells (DTCs)
- tumour educated platelets (TEPs)
- extracellular micro-vesicles (exosomes)
- metabolites
How can you extract liquid biopsies?
-10mL blood collected by venipuncture (4-5mL plasma)
Why is important to use a special EDTA extraction tubes when collecting blood?
to prevent:
- blood clots
- genomic DNA release (from WBC)
- haemolysis
What are properties and logistic and storage of EDTA, citrate?
-contain anticoagulant to prevent clotting.
Logistic and storage:
-onsite centrifugation within 6hrs of collection to isolate plasma and avoid WBC apoptosis.
if not possible, sample can be stored at 4 degree for upto a week.
What are properties and logistic and storage of cell-free DNA tubes(eg. paxgene-qiagen; streck)?
-contain stabilisers to prevent release of gDNA from white blood and haemolysis of RBC.
logistic and storage:
- samples can be stored for 6-14 days at 6-37 degrees.
- more convenient in terms of transport and storage.
What do you see after 15 mins of centrifugation at 2,000x g speed at 4 degree?
- plasma (55%):
- ctDNA and exosomes
- water, proteins, nutrients, hormones, etc. - Buffy coat (<1%)
- WBC, platelets , CTCs - Hematocrit (45%)
- RBC
What are 2 biomarkers in liquid biopsies?
- circulating tumour cells (CTC)
2. circulating tumour DNA(ctDNA)
What are characteristics of 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 isolation and characterisation of CTCs based on?
- biological and physical properties (size, electrical charge)
- identified/characterised based on transcripts - PCR done on total RNA extracted from the cells.
Give an example of identifying circulating tumour cells.
- prostate cancer : Prostate specific antigen (PSA) circulating in blood.
- extract circulating tumour cells, then isolate the DNA and RNA from these cells
- culture them and amplify them using PCR to identify levels of PSA if any
- run these samples on electrophoresis gel
What are characteristics of circulating tumour DNA (ctDNA)?
- present in different fluids: plasma, serum, urine and others
- low concentration (1-50ng DNA/mL plasma)
- Amount highly variable from person to person 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 info on current genetic makeup (including irregularities/mutations) with 80-95% specificity and 60-85% sensitivity.
Where can we find circulating tumour DNA in centrifugation?
-found in plasma layer
How do you isolate cell free DNA after centrifugation?
- transfer supernatant to a clean polypropylene tube and freeze it if needed.
- using magnetic beads or cellulose based or silica based system we can isolate that circulating tumour DNA and store it forever.
What technologies are available to study the levels of circulating tumour DNA?
- NGS
- RTqPCR
- Digital PCR
What are advantages of liquid biposies?
- lower invasiveness
- higher patient compliance
- higher cost/effectiveness
- allow repeated access and multiple sampling
- no special training required for extraction
What are disadvantages of liquid biopsies?
- low amount of material
- early diagnosis
- data interpretation
Why is liquid biopsies better than solid biopsies?
- with solid biopsies we can only pick up one part of tumour, but with liquid biopsies you can pick up mutations that are present in different metastatic site.
- cancer is heterogenous disease.
- molecular properties within a tumour differ and also between metastatic site.
- no need to identify the tumour site before taking a biopsy and allow repeating sampling.
- allow analysis of tissue difficult to access, ie lungs, heart.
What can liquid biopsies be used for?
- determine early stage of disease.
- allows us to determine their molecular profile of disease helping in treatment decisions.
- also to monitor response to therapy.
What are clinical application of liquid biopsies?
-promising cancer biomarkers that need to be clinically validated, not implemented as diagnosis tool yet, but that provides highly specific and complementary information.
Which gene is mutations in lung cancer found and what diagnostic tool is used to test this?
- detection of EGFR gene mutations in lung cancer
- FDA -approved diagnostic test.
What is the new FDA approved diagnostic test for patients with advanced solid cancer?
-liquid CDX
What potential clinical use do CTCs and ctDNA have as cancer biomarkers?
- screening
- diagnosis
- treatment
centrifugation is done for ….at speed …. at temp…
15 minutes at x2000xg speed at 4 degrees
What differentiates circulating tumour cells from normal blood cells?
-circulating tumour cells are CD45 negative but become cytokines 18/19 positive, these surface markers are not found in other types of blood cells so we can identify them.
what are non invasive prenatal test used for?
Fetal DNA is present in maternal plasma and is cleared after birth
- Non invasive prenatal test for screening of Down syndrome, Edwards syndrome and Patau’s syndrome
- replacing amniocentesis
How can cell free DNA be used used to monitor health of solid organ transplants?
CfDNA acts as bio markers for monitoring the health of a solid organ transplant.
Endomyocardial biopsy is currently used to asses rejection risk but donor cfDNA increases in recipient blood and rejection risk increases
