19- Liquid Biopsies

Question 1

what is a liquid biopsy?

Answer 1

sampling and analysis of non-solid biological tissue, mainly blood

blood, plasma serum, urine, CSF and saliva can be used

Question 2

what are the main types of biomarkers that can be detected via liquid biopsy?

Answer 2

circulating tumour DNA/ cells
disseminated tumour cells
tumour educated platelets
cell free nucleotides
circulating endothelial cells
exosomes
metabolites

Question 3

how do cell-free nucleotides serve as a biomarker?

Answer 3

they’re released by dying cells - levels can increase during situations like inflammation or tumour

serve as a biomarker for various conditions where there’s a higher rate of dying cells

Question 4

how do exosomes serve as a biomarker?

Answer 4

exosomes are extracellular micro-vesicles = nano-sized vesicles with a lipid bilayer containing proteins, RNA and bioactive lipids; act as cell-to-cell messengers

detection can indicate state of disease progression and intercellular communication

Question 5

how do circulating endothelial cells serve as a biomarker?

Answer 5

useful for the early detection of heart attacks

help identify cells that detach from tumours = cancer detection

Question 6

how do metabolites serve as a biomarker?

Answer 6

metabolites are released by cells, can indicate disease states and changes in cellular activity

Question 7

examples of tumour derived materials in blood that can serve as biomarkers - their significance?

Answer 7

circulating tumour DNA and/or cells - provides information on tumour genetics

disseminated tumour cells - indicates metastasis

tumour educated platelets - platelets influenced by tumour cells with an altered function, provides insight on tumour activity and progression

circulating free DNA - microRNAs can be isolated, different types indicative of metastasis and type of cancer

Question 8

explain whether liquid biopsies are better for collecting germline or somatic information

Answer 8

better for collecting SOMATIC information

germline information can be found in any cell, whereas somatic information is different across different regions of the body, and isolating information is harder
- germline mutations associated/ offering predisposition to specific conditions can be detected via WBC analysis = obtain from blood sample

as blood circulates around the body, it can collect tissue-specific events (e.g. apoptosis, localised inflammation, infection) and released biomarkers from specific tissues = e.g. blood sample can detect DNA coming from the lung tumour cells
- isolating somatic information is more important = isolate where the information is coming from (e.g. disseminated lung tumour cells from lung cancer)

Question 9

describe collecting and analysing a blood sample for specific biomarkers

Answer 9

collection:
- 10mL blood collected by venepuncture – sample will contain 4-5mL plasma
- special cell-free DNA extraction tubes used to prevent blood clots, genomic release from WBCs contaminating somatic sample, and haemolysis

analysis:
following 15 mins centrifugation at 2,000 x g speed at 4ºC - three layers will be visible
- plasma layer will contain circulating tumour DNA and exosomes
- buffy coat will contain WBCs, platelets, circulating tumour cells
- haematocrit will contain RBCs

Question 10

compare EDTA vs cell-free DNA tubes

Answer 10

EDTA tubes
- contain anticoagulant to prevent clotting
- store sample at 4 degrees for up to a week
- requires on site centrifugation within 6 hours for 15 mins centrifugation at 2,000 x g speed at 4ºC to prevent haemolysis, isolate plasma and avoid WBC apoptosis
- less convenient storage, transport to processing lab needs to be quick, limited use

cell-free DNA tubes
- tubes contain a stabiliser to prevent WBC genomic DNA release and haemolysis
- can be stored for 2 weeks at 6-37 degrees
- show a good plasma fraction
- convenient storage and transport

Question 11

what are circulating tumour cells/ CTCs?

Answer 11

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

Question 12

describe CTCs/ circulating tumour cells as biomarkers - what are they? what do they indicate? how are they found?

Answer 12

CTCs = cells that have detached from a tumour and travel through the bloodstream to other parts of the body as single cells or clusters

presence is a marker for tumour growth, negative cancer prognosis and ineffective treatment response

following 15 mins centrifugation at 2,000 x g speed at 4ºC of blood sample - CTCs found in the buffy coat
- need to be isolated as they’re present in low concentrations at 1-10 cells per 1ml
- present within a high background of cells

Question 13

how are CTCs isolated?

Answer 13

working with either the buffy coat or entire blood sample = CTCs isolated based on biochemical/ physical or biological properties

• relying on cell surface markers
  = e.g. tumour cells are often CD45-ve and EpCAM +ve
  = don’t share certain cell surface markers with other cells
  = use magnetic extraction for isolation based on cell surface marker differences
• physical properties = tumour cells are often bigger, have a different electric charge

Question 14

how can CTCs be further characterised?

Answer 14

specific cell surface markers are associated with specific types of cancer
- e.g. PSA for prostate cancer
- can determine cancer origin, it it’s a primary tumour or metastasis

further characterisation through:
- PCR based assays
- targeting specific transcripts
identifying tumour-associated markers
- gene expression profiles

allows us to quantify expression levels of specific genes/ markers associated with tumour cells, confirm the presence of specific tumour-associated transcripts

Question 15

what can be studied from isolated CTCs?

Answer 15

as we’re working with whole cells - transcriptomes, genomes, proteasomes, phenotypes

Question 16

what techniques can be used to study CTCs?

Answer 16

cultured cells in vitro/vivo to study their behaviour

NGS sequencing of CTCs DNA

FISH studies to detect chromosomal abnormalities

RT-qPCR to detect different markers

flow cytometry

Question 17

what is ctDNA/ circulating tumour DNA?

Answer 17

ctDNA is DNA coming from tumour cells

Question 18

what is cell-free DNA?

Answer 18

cell-free DNA is DNA coming from all the cells in the body

Question 19

describe ctDNA as a biomarker - what is it? what does it indicate? how is it found?

Answer 19

ctDNA = DNA coming from tumour cells

presence is highly variable between people, depending on their health status - ctDNA levels are higher:
- after exercise
- during cancer, trauma or inflammation
- from normal cell turnover and apoptosis
presence provides information of current genetic make-up (including irregularities/mutations)

found following 15 mins centrifugation at 2,000 x g speed at 4ºC of blood sample - in the top plasma layer
- cell-free DNA is extracted first = ctDNA extracted from this
- ctDNA is often highly fragmented

Question 20

sensitivity and specificity of ctDNA?

Answer 20

80-95% specificity and 60-85% sensitivity

Question 21

how is ctDNA isolated?

Answer 21

following centrifugation of blood sample - cell-free DNA is extracted from the top plasma layer

ctDNA extracted from cell-free DNA

isolation of ctDNA using magnetic bead, cellulose-based or silica-based systems = all three methods depend on binding affinities/ interactions between system and DNA

Question 22

different methods for analysis on ctDNA? what can be studied?

Answer 22

methods:
= NGS - no need for fragmenting ctDNA beforehand as it’s already so fragmented
= digital droplet PCR
= array CGH
= real-time quantitative/qPCR

studying genetic/ DNA mutations, alterations and epigenetic status - e.g. point mutations, amplifications, methylation
- studies are more limited = we can only look at the epigenome

Question 23

advantages of liquid biopsies over solid/traditional biopsies

Answer 23

• lower invasiveness
• higher patient compliance
• higher cost effectiveness
• allows repeated access and multiple samplings - can monitor disease progression through extracting samples at different disease stages
• no special training needed for sample extraction
• important for tissues that are hard to access (e.g. kidneys, lungs) - blood will contain the information from these tissues without difficulty

Question 24

disadvantages of liquid biopsies over solid/traditional biopsies

Answer 24

low amount of material = CTCs and ctDNA present in low concs, need sensitive techniques to isolate material

difficult for early diagnosis = low levels of ctDNA and CTCs will be present, harder to detect especially when catching it early offers patient a better prognosis

data interpretation = lots of background noise/info, need to isolate the information we want, hard to distinguish everything separately

Question 25

explain the importance of liquid biopsies for the detection of cancer biomarkers

Answer 25

by analysing CTCs and ctDNA found within a blood sample - liquid biopsies are used for:

• early diagnosis of a disease/ cancer = challenging with low concs of CTCs and ctDNA
• monitor response to therapies during treatment
• pick up heterogeneity within a tumour and between metastatic sites = cancer is a heterogenous disease, liquid biopsies pick up information from all cells within a tumour/ metastasis
• identify differing genetic make-up, mutations and molecular profiles between different tumour cells of a cancer = detect specific causative mutations
• no need to identify the location of a tumour beforehand = tissue-specific biomarkers isolated form liquid biopsies
• allows analysis of hard to access tumours - e.g. kidney, lungs
• can predict metastasis and prognosis of different cancers

Question 26

describe liquid biopsies as a diagnostic tool for detecting EGFR mutations in lung cancer - significance of EGFR mutation? affect on patient care/ treatment?

Answer 26

EGFR mutations = epidermal growth factor receptor mutated , contributes to development of non-small lung cancer

blood samples analysed, mutations in EGFR gene identified with non-small lung cancer patients = makes them eligible for a targeted therapy called EGFR mutations in lung cancer

Question 27

describe pan-tumour liquid biopsies as a diagnostic tool for advanced solid cancer

Answer 27

detects various types of cancer/ cancer-related biomarkers from a blood sample = e.g. common genetic mutations, chromosomal alterations

used as a diagnostic test for early-stage cancers, monitoring treatment therapies, measuring eligibility for FDA-approved targeted therapies like BARB inhibitors