Liquid Biopsies

Question 1

What is a liquid biopsy?

Answer 1

Sample and analysis of non solid biological tissue(minimally invasive) containing molecular biomarkers

Question 2

Give examples of liquid biopsy?

Answer 2

Normally blood

amniotic fluid

Question 3

Why isnt amniotic fluid tested and what is done instead?

Answer 3

Very invasive so pre-natal tests are done - test for circulating free DNA - foetus’ DNA is detected in mothers blood and can test for abnormalities e.g. downs syndrome

Question 4

Why can blood be used as a liquid biopsy?

Answer 4

Cells constantly dying, when dead, content released into bloodstream to be cleared by phagocytes. Before clearance, some levels are be detected.

Question 5

Which materials in circulating blood can be detected in biopsies

Answer 5

Extracellular micro-vesicles (exosomes)
Metabolites
Cell free nucleotides
Circulating tumour cells (CTCs)
Disseminated tumour cells (DTCs)
Tumour educated platelets (TEPs)
Circulating endothelial cells (CECs)

Question 6

What is detecting CECs useful for?

Answer 6

Circulating endothelial cells useful for early detection e.g. of heart attacks or secondary tumours)

Question 7

What is somatic and germline information? Which is more important to identify?

Answer 7

Mutations in DNA - germline in all cells so not useful. Somatic only in affected area e.g. metastasis - need solid biopsy of said area

Question 8

How are blood liquid biopsies collected and why?

Answer 8

Venipuncture

• prevents blood clots
• prevents germline dna release e.g. from white blood cells - cant contaminate genomic dna from WBCs into somatic info)
• prevent haemolysis

Question 9

How should a good liquid biopsy of blood look?

Answer 9

Yellowish plasma fraction on top

- if red indicates haemolysis - not good

Question 10

What are the types of tubes used to collect liquid biopsies?

Answer 10

EDTA / citrate

Cell free DNA tubes e.g. paxgene qiagen, streck

Question 11

What is the use of EDTA / citrate tube?

Question 12

What is the use of cell free dna tube?

Question 13

What are the layers of blood biopsy after centriguation from top to bottom? + what is the use of centrifugation

Answer 13

55% plasma - water, proteins, AND cfDNA including ctDNA, EXOSOMES
<1% Buffy coat - White blood cells and CTCs
45% haematocrit - red blood cells

• take the layer than is needed according to component needed within layer

Question 14

What are the 2 types of liquid biopsies biomarkers?

Answer 14

Circulating tumour cells (CTC)

Circulating tumour DNA (ctDNA)

Question 15

What are circulating tumour cells (CTC)?

Answer 15

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

Question 16

What is CTC a marker for?

Answer 16

Tumour growth and negative cancer prognosis (current treatment is inefficient) and treatment response

Question 17

Why is it difficult to extract CTC?

Answer 17

Extremely low levels and found in high background of normal cells so requires sensitive and specific methods - within the 55% plasma layer of centrifugation

Question 18

How are CTCs isolated?

Answer 18

Based on biological properties e.g. cell surface markers since cancer cells are CD45 negative cells and/or physical properties e.g. size or electrical charge

Question 19

How are CTCs identified?

Answer 19

Transcripts - PCR done on total RNA extracted from cell

Question 20

What downstream analysis can be done to CTCs?

Answer 20

NGS, FISH, flow cytometry, in vivo/in vitro culture, RTqPCR

Question 21

Where is ctDNA found?

Answer 21

Many fluids including plasma, serum , urine etc…

Question 22

What is different about ctDNA and cancer relations?

Answer 22

Unlike CTC, ctDNA does not mean there is cancer

There is a permanent baseline level of cfDNA and ctDNA(genomic DNA) in background plasma all the time

Question 23

What levels of ctDNA is present in blood?

Answer 23

• Low concentration but varies person to person (rises above baseline level based on health e.g. when patient develops cancer, trauma etc…)

Question 24

Why is ctDNA useful as it is? 2 reasons

Answer 24

Provides information on individual current genetic makeup including mutation + sensitive and specific

ctDNA is highly fragmented with specific size ranges (NGS requires fragmentation which is already done here)

Question 25

How is ctDNA isolated from supernatant and cfDNA?

Answer 25

Transfer supernatant to clean polypropylene tube and can freeze if needed ISOLATE JUST CTDNA USING MAGNETIC BEAD, CELLULOSE BASED OR SILICA BASED SYSTEMS

Question 26

What are the research uses of cfDNA?

Answer 26

NGS, ddPCR and array CGH to see amplification and deletions, translocation, point mutations, chromosome abnormalities and epigenetic status Real time quantitative polymerase chain reaction (qPCR) to quantify present of ctDNA

Question 27

Advantages of liquid biopsies

Answer 27

Lower invasiveness + not as risky as solid Higher patient compliance Higher cost/effectiveness Allow repeated access and multiple sampling (monitor disease and sample diff stages of disease) No special training required to extract Early DIAGNOSIS Determine molecular profile so decide TREATMENT plan

Question 28

Disadvantages of liquid biopsies

Answer 28

Low amount of material so need sensitive techniques Difficult for early diagnosis due to low levels Data interpretation

Question 29

Why are liquid biopsies useful specifically for cancer? why use cancer biomarkers?

Answer 29

Cancer is hetergeneous(diverse). Different molecular properties at diff metastatic sites. primary tumour information wont reflect curent condition. Do not need to identify tumour site before taking biopsy and can sample repeatedly for diff stages

Question 30

Are cancer biomarkers clinically validated? (FDA approved)

Answer 30

Most not yet 1) detection of EGFR mutations from ctDNA in lung cancer approved 2) pan tumour liquid biopsy for patients with advances solid cancer