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[MBD] NoNa > Liquid Biopsies > Flashcards

Liquid Biopsies Flashcards

1
Q

What are the three types of medicine ?

A

One size fits all
Stratified medicine
Precision medicine

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2
Q

Describe stratification

A 
Patients are grouped by :
Disease 
Subtypes 
Demographics 
Clinical features
Biomarkers
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3
Q

Describe personalisation

A

This is taking into account :

  • Patient preferences
  • Clinical features
  • Medication history
  • Environment
  • Behaviours/habits
  • Biomarkers
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4
Q

What is personalised medicine ?

A

A medical model which moves away from one size fits all to one which involves targeted therapies.

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5
Q

What are different technologies which are involved in personalised medicine ?

A
  • Liquid biopsies
  • Next gen sequencing
  • CRISPR/Cas9
  • 3D cultures - organoid technology
  • Targeted therapies
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6
Q

What is a liquid biopsy?

A

Liquid biopsy = Sampling + analysis of non-solid biological tissue (e.g.blood)

  • Minimally invasive, detects molecular biomarkers (associated with disease = disease biomarkers)
  • Representative of the tissue from which it has spread.

Blood carries biomarkers very effectively.

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7
Q

What caused the start of liquid biopsies?

A

Discovery of free DNA/RNA in blood = Used in disease prognosis+diagnosis.

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8
Q

What is an example of a established liquid biopsy ?

A

Amniotic fluid analysis.
The problem with it - it’s highly invasive so it has been replaced by non-invasive pre-natal test which uses free circling foetal DNA in mothers blood.

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9
Q

Why do we use blood for liquid biopsies?

A

Blood contains dying cells (apoptosis/necrosis, constant cell renewal).

Cell material is released into bloodstream before phagocytes engulf

Detect by extracting a blood sample

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10
Q

What can we detect in the blood during biopsies ?

A

We can detect :

  • Endothelial cells (early detection of heart attack)
  • CTCs-Circulating tumour cells
  • Cell-free DNA/RNA = increase in exercise/inflammation
  • Tumour educated platelets
  • Disseminated tumour cells
  • Extracellular micro-vesicles (exosomes)
  • Micro-RNA 105 (metastatic breast cancer )
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11
Q

What type of information are we interested in when processing liquid biopsies ?

A

Germline Information

Somatic Information

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12
Q

Why are we more interested in somatic information?

A

We can find germline hereditary information in any cell in our body.
Somatic info is only found at that specific tissue where changes have occurred.

-E.g. Localised lung tumour
If we analyse white blood cells/skin cells, we will not find the mutation that has occurred in the lung cells as it is not inherited. Need to conduct a solid biopsy from the lung to study the somatic information from the tumour cells. Blood sample detects DNA coming from tumour cells that are dying in the lung + detects circulating tumour cells that detach from lung tumour

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13
Q

What is somatic mutation?

A

Somatic mutation occurs in a single body cell and cannot be inherited (only tissues derived from the mutated cell are affected)

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14
Q

What is germline mutation

A

Germline mutation occurs in gametes and can be passed onto offspring (every cell in the entire organism will be affected)

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15
Q

How can we extract a liquid biopsy?

A

-Venipuncture procedure
(10mL of blood contains 4-5mL plasma)
-To study circulating tumour DNA = isolate the plasma.

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16
Q

Why do we need to use special extraction tubes ?

A

We need to use the EDTA (contains anti-coagulant) tube because :

  • prevents blood clot
  • prevents genomic DNA release (from WBCs)
  • prevents haemolysis
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17
Q

What are the 2 types of tubes used for liquid biopsies?

A
  • EDTA/Citrate

- Cell-free DNA tubes

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18
Q

Describe the properties of EDTA /Citrate tubes

A

EDTA /Citrate tubes contain anti-coagulants to prevent clotting.

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19
Q

Describe the properties of Cell-free DNA tubes

Paxgene-Qiagen,Streck

A

Contain a stabiliser to prevent release of gDNA (genomic DNA) from WBCs and haemolysis of RBCs
-after storing for 7 days at 4oC and then centrifuging, nice yellow plasma fraction due to no haemolysis

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20
Q

Describe the logistics and storage of EDTA tubes

A

-On site centrifugation within 6 hours of collection to isolate plasma and avoid white cell apoptosis.

If not possible , samples can be stored at 4oC for up to a week
-after storing for 7 days at 4oC and then centrifuging, red plasma fraction due to haemolysis

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21
Q

Describe the logistics and storage of Cell free DNA tubes

A

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

-transport, storage

22
Q

What are the different fractions of blood following centrifugation?

A

Following 15mins centrifugation at 2,000 x g speed at 4oC

3 diff phases:

55% Plasma (yellow top layer)
Water,proteins,nutrients,hormones +cfDNA exosomes
(cell-free DNA )

<1% buffy coat (intermediate layer)
White blood cells, platelets
WBC and CTCs

45%Hematocrit (bottom red layer)
Red blood cells

-depending on what we want to extract from blood sample, interested in isolating top/intermediate layer

23
Q

What different liquid biopsy samples are there?

A
  • Saliva
  • Cerebrospinal fluid
  • Pleural liquid
  • Urine (to detect metabolites)
  • Serum plasma
24
Q

What can we detect in a liquid biopsy?

A
  • Circulating free DNA - e.g. Circulating tumour DNA
  • CTC (circulating tumour cells )
  • Small nucleic acids (miRNA)
    e. g. miRNA 105 - Promotes breast cancer metastasis, detected in blood in early stages of breast cancer metastasis
  • Metabolites in urine
  • Exosomes (extracellular vesicles) - lipid bilayer, proteins, RNAs, lipids, cell-cell communication
25
Q

Which 2 important things are found in blood (liquid biopsy) that help us identify biomarkers?

A
  • CTCs - Circulating Tumour Cells

- ctDNA - Circulating Tumour DNA

26
Q

Describe the isolation and characterisation of CTCs

A

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

27
Q

What are CTCs?

A

CTCs = Circulating Tumour Cells.

  • These are cells that have detached from a tumour and travel through blood stream to other parts of the body
  • Single cells/Clusters
  • Marker for tumour growth/negative cancer prognosis, treatment response (CTCs = treatment is not being efficient)
  • Extremely rare (low conc): 1-10 per 1ml of blood
  • Found in a high background of normal cells in blood (erythrocytes, leukocytes, platelets, neutrophils) = need sensitive + specific methods to identify + isolate CTCs
28
Q

Centrifuge blood sample for 15mins at 2000g speed at 4oC, what phase are CTCs found in?

A

Buffy coat intermediate layer (WBC layer)

29
Q

How can we isolate tumour cells using markers?

A

Isolate CTCs using their Biological properties e.g:

  • Cell surface markers techniques (FAX/magnetic)
  • CTCs are CD45(blood cell marker ) negative.
  • EpCAM ,CK(cytokeratins 8,18,19)positive = these cell surface markers not found in other types of blood cells
  • Useful for detecting metastasis

Or isolate CTCs using their physical properties:

  • Size - CTCs are larger than other blood cells
  • Electric charge
  • Density
30
Q

What can we do once we isolate/extract circulating tumour cells?

A

Want to know CTC origin, so identify specific biomarkers on the isolated cells.
-e.g. suspect prostate cancer. identify prostate-specific biomarkers (PSA). extract CTC DNA/RNA - rtqPCR to identify PSA levels/PCR to amplify PSA then run on electrophoresis gel.

We can study :

  • Transcriptome (set of RNA transcripts)
  • Proteome (Entire set of proteins)
  • Phenotypic studies (
  • Genome (genetic material of organism)
  • Morphology
  • Use lots of different techniques - rtqPCR, culture CTCs in vitro, inoculate CTCs into mouse and see if tumour forms
31
Q

What are some techniques we could use to study the CTC after extracting/isolating ?

A
  • Next generation sequencing - study cell DNA
  • FISH (Fluorescence in situ hybridisation) - study chromosome abnormalities
  • Flow cytometry
  • In vivo/vitro culture
  • RTqPCR - detect biomarkers

these techniques provide info about the original tumour site (that CTCs detached from)

32
Q

Describe cfDNA

A
  • Present in fluids - plasma, serum, urine, CSF
  • Low concentration (1-50ng DNA/1mL plasma)
  • Amount is highly variable b/w diff. people + varies depending on health of person (Increased cfDNA in cancer, trauma, inflammation, exhaustive exercise)
  • Presence of permanent background genomic DNA in plasma - cfDNA coming from cells dying by apoptosis/necrosis + being replaced
33
Q

What are the properties of circulating tumour DNA ?

A
  • ctDNA = highly fragmented w specific size range (500bp) (=don’t need to fragment in NGS)
  • Provides information of current genetic makeup (irregularities/mutations) with 80-90% specificity and 60-85% sensitivity.
34
Q

Why does free DNA increase during cancer ?

A
  • Circulating free DNA (cfDNA) comes from cells that are continuously dying and being replaced.
  • Cancerous tumour cells
35
Q

What does the highly fragmented property of free DNA mean when it involves next generation sequencing ?

A

-To study cfDNA using NGS , don’t need to fragment cfDNA (an early step of process)

36
Q

How can we extract the circulating free DNA ?

A

Following centrifugation, cfDNA is found within the top phase - Plasma
-cfDNA in top plasma layer, then identify ctDNA from total cfDNA

37
Q

Describe the process of isolating cfDNA

A

Following centrifugation:

  • Transfer the supernatant (plasma) to a clean polypropylene tube and freeze if need to store it (forever)
  • Isolate cfDNA from plasma using magnetic bead/cellulose based/silica based systems. then store (forever) if required
38
Q

What analysis can cfDNA provide us with ?

A

-amplifications/deletions
-point mutations
-translocations
-epigenetic modifications
Limited analysis - Not a cell so we analyse epigenome/genome using NGS, RTqPCR (to analyse diff. in cfDNA levels), Digital PCR.

39
Q

What is RTqPCR?

A

This is reverse transcription polymerase chain reaction.

Combines reverse transcription of RNA and amplification

40
Q

What are the advantages of liquid biopsies (compared to traditional solid biopsies?

A
  • ↓ invasive - just extract blood by normal venipuncture = extract 4-5ml plasma
  • ↑ patient compliance
  • ↑ cost-effectiveness
  • Allows repeated access and multiple sampling = can monitor disease progress
  • No special training is required for extraction (not surgeon, any nurse can do it)
  • Gives info about genetic characteristics + tumour characteristics w/o needing solid biopsy = difficult in tissues difficult to access - lung, prostate, kidney
41
Q

What are the disadvantages liquid biopsies?

A
  • Low amount of material extracted - CTCs, ctDNA (limitation) = requires sensitive techniques to isolate material + low CTCs/ctDNA in early tumour stages = hard to detect
  • Early diagnosis (not many markers at early stages of disease)
  • Data interpretation - lot of data from all tissues - we need to extract tumour cell info via biomarkers and separate from normal background cell info. + decide what to report back to patient
  • Requires special tubes that prevent blood clotting, genomic DNA release, haemolysis
42
Q

What are the steps in cancer treatment ?

A
  • Cancer detection screening /early diagnosis
  • Molecular profiling or prognostication
  • Detection of residual disease
  • Monitoring response
  • Monitoring clonal evaluation
43
Q

Cancer has biomarkers. Explain how liquid biopsies are suitable in diagnosis.

A
  • Liquid biopsies can provide the genetic makeup of individuals
  • Cancer is a heterogenous disease(between metastatic sites the molecular properties are different + intratumoural)
  • Liquid biopsies help us identify different tumours as all parts of tissues. Solid biopsy = only pick up 1 part of tumour = some clones of tumour.
  • Don’t need to know tumour site (do for solid biopsies). extracting liquid biopsy tells you tumour site
  • Analyses tissues difficult to access - lung, kidney, prostate
  • Extract info at any point of disease - early stages, throughout treatment
44
Q

Discuss some further advantages of liquid biopsies in cancer

A
  • We do not need to know the tumour’s location
  • Allows repeat sampling
  • Allow analysis of difficult to access tissues.
  • Primary tumour information may not reflect current disease condition.
45
Q

What does Genomic analysis involve?

A
  • Mutation profiling
  • Treatment selection
  • Monitoring clonal evolution
46
Q

What is liquid cdx

A

A lab test that detects specific gene mutations found in cell-free DNA (cfDNA) isolated from whole blood plasma specimens

Identifies mutations in patients with advanced solid cancer that can be treated using targeted therapies.

47
Q

What are some way a liquid biopsy can be used ? (clinically)

A
  • They can be taken at any stage of disease meaning they can be used to diagnose diseases.
  • Cancer biomarkers can be detected
48
Q

Liquid biopsy cancer biomarkers need to be ………..

A

clinically validated - not currently used as a diagnostic tool, but provides highly specific + complementary info to normal diagnostic tools (solid biopsy, x-ray)
-but exceptions - EGFR lung cancer,

49
Q

Describe the EGFR mutation test

A
  • Diagnostic test which uses blood samples to find mutations in the gene for epidermal growth factor receptor (EGFR)-lung cancer.
  • EGFR mutations in 20% of non-small cell lung cancer patients (most common form lung cancer)
  • 75.7% sensitivity, 99.8% specificity.
  • Identifies patients who could be eligible for treatment by erlotinib.
50
Q

What does quantitative analysis include?

A

Disease staging
Monitor response
Prognosis (predicting future events).

[MBD] NoNa (28 decks)

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