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Molecular Basis of Disease(Sem 4) > 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 individual preferences
  • Clinical features
  • Medication history
  • Environment
  • Behaviours /habits
  • Biomarker
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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 involved 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

Sampling and analysis of non-solid biological tissue (e.g.-blood).Minimally invasive technology used to detect molecular biomarkers.
Representative of the tissue from which it has spread.

Blood can carry markers very effectively.

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7
Q

What caused the start of liquid biopsies?

A

The discovery of free DNA/RNA in blood. This can be used in prognosis and diagnosis of diseases.

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8
Q

What is an example of a established liquid biopsy ?

A

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The problem with it , is it is highly invasive so it has been replaced by non-invasive pre-natal test which uses free circling foetal DNA in mothers blood.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

There is constant cell renewal through cell death and renewing through necrosis /apoptosis.
Cell material is released into blood stream and it is cleared by phagocytes but may remain in the blood stream for a short time.

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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)
  • Tumour cells
  • Cell free nucleotides
  • 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 information in any cell in our body.
Somatic 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 as it is not inherited.

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13
Q

What is somatic information (mutation)?

A

occur in a single body cell and cannot be inherited (only tissues derived from mutated cell are affected)

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14
Q

What is germline information(mutation)

A

occur 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
In 10mL of blood can contain around 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 an anti-coagulant) one because :

  • prevents blood clot
  • prevents genomic DNA release
  • prevents haemolysis
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17
Q

What are the two 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

These 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 white blood and haemolysis of red blood cells

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

21
Q

Describe the logistics and storage of Cell free DNA tubes

A

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

22
Q

What are the different fractions of blood following centrifugation?

A

Following 15minutes at 2,000 x g speed at 4oC

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

<1% buffy coat
White blood cells, platelets
WBC and CTCS

45%Hematocrit
Red blood cells
RBC

23
Q

What are different liquid biopsy samples are there ?

A
  • Saliva
  • Cerebrospinal fluid
  • Pleural liquid
  • Urine
  • Serum plasma
24
Q

What can we detect in a liquid biopsy?

A
  • Circulating free DNA
  • Circulating tumour DNA
  • Small nucleic acids (miRNA)
    e. g. miRNA 104 - Breast cancer Metastasis
  • Metabolites in urine
  • CTC (circulating tumour cells )
  • Exosomes (extracellular vesicles)
25
Q

What are CTCs?

A
  • These are 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 or clusters
  • Marker for tumour growth/negative cancer prognosis, treatment response(means the treatment is not being efficient )
  • Extremely rare 1-10 per 1ml of blood
  • Found in a high background of normal cells meaning they are sensitive.
26
Q

Describe the isolation and characterisation of CTC

A

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

27
Q

How can we isolate tumour cells using markers ?

A
  • Biological properties for e.g:
  • Cell surface markers techniques (FAX)/magnetic
  • They are CD45(blood cell marker ) negative.
  • EpCAM ,CK( cytokeratin’s 8,18,19)positive
  • Useful for detecting metastasis
28
Q

What can we do once we extract circulating tumour cells?

A

We can study :

  • Transcriptome (set of RNA transcripts)
  • Proteome (Entire set of proteins)
  • Phenotypic studies (
  • Genome (genetic material of organism )
  • Use lots of different techniques
29
Q

What are some techniques we could use to study the CTC ?

A 
Next generation sequencing 
FISH (Fluorescence in situ hybridisation)
Flow cytometry
In vivo/vitro culture
RTqPCR
30
Q

Describe ctDNA

A

Present in different fluids ,plasma, serum, urine

  • Low concentration (1-50ng DNA/mL plasma )
  • Amount is highly variable for person to person and depends on health (Increase in cancer,trauma )
  • Presence of permanent genomic DNA in plasma
31
Q

Why does free DNA increase during cancer ?

A
  • Free DNA comes from cells which are continuously dying and being replaced.
  • Cancerous cells
32
Q

What are the properties of circulating tumour DNA ?

A
  • It is highly fragmented with a specific size range (,500bp)
  • Provides information of current genetic makeup(irregularities/mutations with 80-90% specificity and 60-85% sensitivity.
33
Q

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

A

-To study it using NGS , we will not need to fragment it first which is one of the first steps in the process.

34
Q

Where can we extract the circulating free DNA ?

A

It can be found following centrifugation within the top fraction -The Plasma

35
Q

Describe the process of isolating cfDNA

A

Following centrifugation:

  • Transfer the supernatant to a clean polypropylene tube and freeze if required
  • Isolation using magnetic bead, cellulose based/silica based systems
  • Isolation using magnetic bead, cellulose-based/silica based systems
36
Q

What analysis can cfDNA provide us with ?

A

-amplifications/deletions
-point mutation
-translocations
-epigenetic modifications
Not a cell so we analyse epigenome/genome using RTqPCR,NGS,Digital PCR

37
Q

What is RTqPCR?

A

This is reverse transcription polymerase chain reaction.

Combines reverse transcription of RNA and amplification

38
Q

What are the advantages of liquid biopsies ?

A
  • Lower invasiveness
  • Higher patient compliance
  • Higher cost/effectiveness
  • Allow repeated access and multiple sampling
  • No special training is required for extraction
39
Q

What are the disadvantages liquid biopsies?

A
  • Low amount of material (limitation)
  • Early diagnosis (not many markers at early stages of disease)
  • Data analysing
40
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 metastatic properties are different /intratumoural)
  • Liquid biopsies help us identify different tumours.
41
Q

Discuss some further advantages of liquid biopsies in cancer

A
  • We do not need to know the tumours location
  • Allows repeat sampling
  • Allow analysis of difficult to access tissues.
  • Primary tumour information may not reflect current disease condition.
42
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
43
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
44
Q

What does quantitative analysis include?

A

Disease staging
Response monitoring
Prognostication ( predicting future events).

45
Q

What does Genomic analysis involve?

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

Describe the EGFR mutation test

A
  • This is a FDA approved diagnostic test which uses blot samples to find mutations in the epidermal growth factor receptor (EGFR)-lung cancer.
  • 20% of non-small cell lung cancer patients (most common form )
  • 75.7% sensitivity, 99.8% specificity.
  • Identifies patients who could be eligible for treatment by erlotinib.
47
Q

What is liquid cdx

A

Identifies patients who can be treated by targeted therapy

Molecular Basis of Disease(Sem 4) (28 decks)

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