Liquid Biopsies

Question 1

What is a liquid biopsy

Answer 1

Sampling and analysis of non-solid biological tissue, primarily blood. It is a minimally invasive technology for the detection of molecular biomarkers. Representative of the tissue/s from which it has spread.

Question 2

What type of biopsy do we have

Answer 2

• Solid biopsy
• liquid biopsy

Question 3

What can we use as samples for liquid biopsies

Answer 3

• Saliva
• CSF
• Serum
• Urine

Question 4

How can blood be used a a liquid biopsy

Answer 4

Cells in blood undergo apoptosis or necrosis and release their genetic material into the blood.

Cells can actively secrete genetic material Ito the blood while alive

Question 5

What do we detect in blood

Answer 5

• Extracellular micro-vesicles (exosomes)
• Metabolites
• Cell-free nucleotides
• Tumour educated platelets
• Circulating endothelial cells
• Disseminated tumour cells
• Circulating tumour cells

Question 6

What type of info do we get from liquid biopsies

Answer 6

• We get both germline and somatic information
• More of a snapshot of what was happening at the time of drawing the blood sample

Question 7

How is a blood sample collected

Answer 7

10ml collected by venipuncture and stored in tubes that prevent clotting and haemolytic

Question 8

How is blood samples stored

Answer 8

• EDTA, citrate tubes: contains anticoagulants and used for onsite centrifugation within 6hrs
• Cell free DNA tubes: contains stabilisers to prevent DNA release, can be stored for up to 2 weeks at 6-37 degrees C

Question 9

After centrifuging what does the blood separate into

Answer 9

55% plasma:

• Water
• Nutrients
• Hormones
• ctDNA
  -Exosomes

1% Buffy coat:

• WBC
• Platelets
• Circulating tumour cells
  45% Hematocrit:
• RBC

Question 10

What can we detect in liquid biopsies

Answer 10

• Circulating Tumour DNA
• Circulating Tumour cells
• MicroRNAs
• Exosomes
• Metabolites

Question 11

What are circulating tumour cells

Answer 11

• 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.

Question 12

How can we isolate and characterise CTCS

Answer 12

• Based on their biological and physical properties
• Identified/characterised based on transcripts- PCR done on total RNA extracted from the cells

Question 13

What are circulating tumour DNA

Answer 13

• Present in different fluids: plasma, serum, urine and others.
• Low concentration (1-50ng DNA/mL plasma).
• Amount highly variable for person to person and depending on health status in the same person (increase in cancer, trauma, etc.).
• Presence of permanent genomic DNA background in plasma.

Question 14

How can we test for ctDNA

Answer 14

• Highly fragmented but with specific size range (<500bp)
• Provides information of current genetic make-up (including irregularities/mutations) with 80-95% specificity and 60-85% sensitivity.

Question 15

How can we isolate ctDNA

Answer 15

• Transfer supernatant to a clean polypropylene tube and freeze it if needed
• Isolation using magnetic bead, cellulose-based or silica-based systems.

Question 16

What procedure can we do with ctDNA

Answer 16

• Next Generation Sequencing (NGS), Digital Droplet PCR (ddPCR),
  array CGH: Amplifications and deletions, Translocations, Point
  mutations, Chromosomes abnormalities, epigenetic status
  (methylation)
• Real Time Quantitative Polymerase Chain reaction (qPCR): ctDNA
  presence quantification

Question 17

List some advantages of liquid biopsies

Answer 17

• Lower invasiveness
• Higher patient compliance
• Higher cost/effectiveness
• Allow repeated access and multiple
  sampling
• No special training required for extraction

Question 18

List some disadvantages of liquid biopsies

Answer 18

• Low amount of material
• Early diagnosis
• Data interpretation

Question 19

Why would we use liquid biopsies in detecting cancer

Answer 19

• Cancer is a heterogeneous disease.
• Molecular 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 20

What are cancer biomarkers

Answer 20

• Promising biomarkers that need to be clinically validated, not implemented as diagnosis tool yet*, but that provides highly specific and complementary information