LB

Question 1

What is a liquid biopsy?

Answer 1

→ Sampling and analysis of non-solid biological tissue, primarily blood

Question 2

What other liquids are there for liquid biopsies?

Answer 2

→ urine,
→ plasma,
→ serum,
→ saliva

Question 3

What are saliva biopsies useful for?

Answer 3

→ head and neck cancers

Question 4

What is CSF biopsies used for?

Answer 4

→ circulating tumour DNA in brain tumours

Question 5

What is an example of an established liquid biopsy?

Answer 5

→ amniotic fluid

Question 6

What has amniotic fluid analysis been replaced with and why?

Answer 6

→ substituted with circulating foetal DNA in mothers blood

→ amniotic analysis is invasive

Question 7

What are some markers for liquid biopsies?

Answer 7

→ cell free nucleotides
→ tumour educated platelets
→ circulating tumour cells
→ disseminated tumour cells
→ metabolites

Question 8

Which cells are extracted for liquid biopsies?

Answer 8

Normally interested in somatic information because we can find germline information from any part of the body
→ germ line may not have tumour cells

Question 9

Why are EDTAs used for venepuncture?

Answer 9

→ Preventing: blood clots
→ genomic DNA release (from white blood cells)
→ haemolysis

Question 10

What does bursting of white blood cells release?

Answer 10

→ genomic DNA

Question 11

What are the tubes used for liquid biopsises?

Answer 11

→ EDTA
→ citrate
→ cell free DNA

Question 12

What are the properties that make EDTAs and citrates useful for LB?

Answer 12

→ contain anticoagulant to prevent clotting

Question 13

What are the logistical and storage issues for EDTAs?

Answer 13

→ 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

Question 14

What are the properties of cell-free DNA tubes that makes them suitable for LB?

Answer 14

→ Contain a stabiliser to prevent release of gDNA from white blood and haemolysis of red blood cells

Question 15

How can cell-free DNA tubes be stored?

Answer 15

→ Samples can be stored for 6-14 days at 6ºC-37ºC

Question 16

What makers can be found in the plasma?

Answer 16

→ cfDNA
→ exosomes
→ hormones

Question 17

What markers are found in the buffy coat layer?

Answer 17

→ WBC and CTC

Question 18

What is found in the haemotocrit?

Answer 18

→ RBCs

Question 19

What are the actual LB biomarkers?

Answer 19

→ Circulating Tumour Cells (CTC)

→ Circulating Tumour DNA (ctDNA)

Question 20

What are CTCs?

Answer 20

→ Cells that have detached from a tumour and travel through the bloodstream to other parts of the body- single cells or clusters

Question 21

What is CTC a maker for?

Answer 21

→ tumour growth

→ negative cancer prognosis

Question 22

What may it mean if CTCs are found when on treatment?

Answer 22

→ treatment may not be working

Question 23

Why are specific methods needed to study CTCs?

Answer 23

→ Found in a high background of normal cells

Question 24

How are CTCs isolated and characterised?

Answer 24

→ based on transcripts- PCR done on total RNA extracted from the cells.
→ use cell curface markers eg FACS or magnetic beads

Question 25

What markers are found on CTC surfaces?

Answer 25

→ CD45 negative | → EpCAM positive

Question 26

How are CTCs differentiated?

Answer 26

→ cell surface markers | → OR use their physical properties, size, charge, density

Question 27

What is involved in CTC analysis?

Answer 27

→ study the omes with NGS, RTqPCR, flow cytometrry, FISH

Question 28

What is ctDNA?

Answer 28

→ DNA that comes from cancerous cells and tumours

Question 29

Where is ctDNA found?

Answer 29

→ plasma, serum, urine | → Low concentration

Question 30

What does the amount of ctDNA depend on?

Answer 30

→ health status in the same person (increase in cancer, trauma)

Question 31

What is the size range of ctDNA?

Answer 31

→ Highly fragmented but with specific size range (<500bp)

Question 32

Why is the first stage of NGS not needed for ctDNA?

Answer 32

→ highly fragmented

Question 33

Where is the ctDNA supernatant transferred to?

Answer 33

→ clean polypropylene

Question 34

What are the three ways ctDNA is isolated?

Answer 34

→ magnetic beads → cellulose-based → sillica based

Question 35

What does use of NGS, and array CGH for ctDNA show?

Answer 35

→ Amplifications and deletions, → Translocations, → Point mutations, → Chromosomes abnormalities, epigenetic status (methylation)

Question 36

Why is ctDNA sequencing limited?

Answer 36

→ not working with cells so can only interrogate genome and epigenome

Question 37

What is ctDNA qPCR used for?

Answer 37

→ presence quantification

Question 38

What are the advantages of liquid biopsies over solid biopsies?

Answer 38

→ 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

Question 39

What are the advantages of disadvantages of liquid biopsies?

Answer 39

→ Low amount of material- need sensitive systems → Early diagnosis → Data interpretation

Question 40

Why use liquid biopsies?

Answer 40

→ 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

Question 41

What is one way lung cancer be detected?

Answer 41

→ Detection of EGFR mutations

Question 42

What is LIQUID CDx?

Answer 42

→ Pan-tumour liquid biopsy test for patients with advanced solid cancer

Question 43

Why are exosomes cancer biomakers?

Answer 43

→ participate in cancer progression and metastasis by transferring bioactive molecules between cancer and various cells

Question 44

Why are miRNA markers for cancer?

Answer 44

→ plays a role is proliferative signalling | → resisting cell death