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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The problem with it , is it is highly invasive so it has been replaced by non-invasive pre-natal test which uses free circling foetal DNA in mothers blood.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
What are CTCs?
- 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
Describe the isolation and characterisation of CTC
-Based on biological properties and physical properties -Identified/characterised based on transcripts. PCR done on total RNA extracted from cells
27
How can we isolate tumour cells using markers ?
- 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
What can we do once we extract circulating tumour cells?
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
What are some techniques we could use to study the CTC ?
``` Next generation sequencing FISH (Fluorescence in situ hybridisation) Flow cytometry In vivo/vitro culture RTqPCR ```
30
Describe ctDNA
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
Why does free DNA increase during cancer ?
- Free DNA comes from cells which are continuously dying and being replaced. - Cancerous cells
32
What are the properties of circulating tumour DNA ?
- 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
What does the highly fragmented property of free DNA mean when it involves next generation sequencing ?
-To study it using NGS , we will not need to fragment it first which is one of the first steps in the process.
34
Where can we extract the circulating free DNA ?
It can be found following centrifugation within the top fraction -The Plasma
35
Describe the process of isolating cfDNA
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
What analysis can cfDNA provide us with ?
-amplifications/deletions -point mutation -translocations -epigenetic modifications Not a cell so we analyse epigenome/genome using RTqPCR,NGS,Digital PCR
37
What is RTqPCR?
This is reverse transcription polymerase chain reaction. | Combines reverse transcription of RNA and amplification
38
What are the advantages of liquid biopsies ?
- Lower invasiveness - Higher patient compliance - Higher cost/effectiveness - Allow repeated access and multiple sampling - No special training is required for extraction
39
What are the disadvantages liquid biopsies?
- Low amount of material (limitation) - Early diagnosis (not many markers at early stages of disease) - Data analysing
40
Cancer has biomarkers. Explain how liquid biopsies are suitable in diagnosis.
- 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
Discuss some further advantages of liquid biopsies in cancer
- 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
What are some way a liquid biopsy can be used ? (clinically)
- They can be taken at any stage of disease meaning they can be used to diagnose diseases. - Cancer biomarkers can be detected
43
What are the steps in cancer treatment ?
- Cancer detection screening /early diagnosis - Molecular profiling or prognostication - Detection of residual disease - Monitoring response - Monitoring clonal evaluation
44
What does quantitative analysis include?
Disease staging Response monitoring Prognostication ( predicting future events).
45
What does Genomic analysis involve?
- Mutation profiling - Treatment selection - Monitoring clonal evolution
46
Describe the EGFR mutation test
- 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
What is liquid cdx
Identifies patients who can be treated by targeted therapy

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

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