L10: Liquid Biopsies Flashcards
What is a liquid biopsy?
→ Sampling and analysis of non-solid biological tissue, primarily blood
What other liquids are there for liquid biopsies?
→ urine,
→ plasma,
→ serum,
→ saliva
What are saliva biopsies useful for?
→ head and neck cancers
What is CSF biopsies used for?
→ circulating tumour DNA in brain tumours
What is an example of an established liquid biopsy?
→ amniotic fluid
What has amniotic fluid analysis been replaced with and why?
→ substituted with circulating foetal DNA in mothers blood
→ amniotic analysis is invasive
What are some markers for liquid biopsies?
→ cell free nucleotides → tumour educated platelets → circulating tumour cells → disseminated tumour cells → metabolites
Which cells are extracted for liquid biopsies?
Normally interested in somatic information because we can find germline information from any part of the body
→ germ line may not have tumour cells
Why are EDTAs used for venepuncture?
→ Preventing: blood clots
→ genomic DNA release (from white blood cells)
→ haemolysis
What does bursting of white blood cells release?
→ genomic DNA
What are the tubes used for liquid biopsises?
→ EDTA
→ citrate
→ cell free DNA
What are the properties that make EDTAs and citrates useful for LB?
→ contain anticoagulant to prevent clotting
What are the logistical and storage issues for EDTAs?
→ 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
What are the properties of cell-free DNA tubes that makes them suitable for LB?
→ Contain a stabiliser to prevent release of gDNA from white blood and haemolysis of red blood cells
How can cell-free DNA tubes be stored?
→ Samples can be stored for 6-14 days at 6ºC-37ºC
What makers can be found in the plasma?
→ cfDNA
→ exosomes
→ hormones
What markers are found in the buffy coat layer?
→ WBC and CTC
What is found in the haemotocrit?
→ RBCs
What are the actual LB biomarkers?
→ Circulating Tumour Cells (CTC)
→ Circulating Tumour DNA (ctDNA)
What are CTCs?
→ Cells that have detached from a tumour and travel through the bloodstream to other parts of the body- single cells or clusters
What is CTC a maker for?
→ tumour growth
→ negative cancer prognosis
What may it mean if CTCs are found when on treatment?
→ treatment may not be working
Why are specific methods needed to study CTCs?
→ Found in a high background of normal cells
How are CTCs isolated and characterised?
→ based on transcripts- PCR done on total RNA extracted from the cells.
→ use cell curface markers eg FACS or magnetic beads
What markers are found on CTC surfaces?
→ CD45 negative
→ EpCAM positive
How are CTCs differentiated?
→ cell surface markers
→ OR use their physical properties, size, charge, density
What is involved in CTC analysis?
→ study the omes with NGS, RTqPCR, flow cytometrry, FISH
What is ctDNA?
→ DNA that comes from cancerous cells and tumours
Where is ctDNA found?
→ plasma, serum, urine
→ Low concentration
What does the amount of ctDNA depend on?
→ health status in the same person (increase in cancer, trauma)
What is the size range of ctDNA?
→ Highly fragmented but with specific size range (<500bp)
Why is the first stage of NGS not needed for ctDNA?
→ highly fragmented
Where is the ctDNA supernatant transferred to?
→ clean polypropylene
What are the three ways ctDNA is isolated?
→ magnetic beads
→ cellulose-based
→ sillica based
What does use of NGS, and array CGH for ctDNA show?
→ Amplifications and deletions,
→ Translocations,
→ Point mutations,
→ Chromosomes abnormalities, epigenetic status (methylation)
Why is ctDNA sequencing limited?
→ not working with cells so can only interrogate genome and epigenome
What is ctDNA qPCR used for?
→ presence quantification
What are the advantages of liquid biopsies over solid biopsies?
→ Lower invasiveness even for tissues of limited access
→ Higher patient compliance
→ Higher cost/effectiveness
→ Allow repeated access and multiple sampling
→ No special training required for extraction
What are the advantages of disadvantages of liquid biopsies?
→ Low amount of material- need sensitive systems
→ Early diagnosis
→ Data interpretation
Why use liquid biopsies?
→ cancer is is a heterogenous disease
→ 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
What is one way lung cancer be detected?
→ Detection of EGFR mutations
What is LIQUID CDx?
→ Pan-tumour liquid biopsy test for patients with advanced solid cancer
Why are exosomes cancer biomakers?
→ participate in cancer progression and metastasis by transferring bioactive molecules between cancer and various cells
Why are miRNA markers for cancer?
→ plays a role is proliferative signalling
→ resisting cell death
