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molecular basis of disease > liquid biopsies > Flashcards

liquid biopsies Flashcards

1
Q

What is personalised medicine?

A
  • model that moves from one-size fit all medicine to new approaches which helps better manage patients health, and targeted therapy for best outcomes.
  • liquid biposies are used for personalised medicine.
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2
Q

Define liquid bioposies.

A
  • sampling and analysing of non-solid biological tissue, primarily blood. It is a minimally invasive technology for detection of molecular biomarkers.
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3
Q

What are examples of liquid biopsies?

A
  • urine
  • bloods
  • saliva
  • CSF (useful for circulating tumour DNA)
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4
Q

What are advantages of liquid biopsies?

A
  • minimal invasive technology

- liquid biopsy sample is representative of tissue from which it has spread.

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5
Q

What is an example of well known liquid biopsy?

A
  • amniotic fluid analysis

- sex test, chromosomal abnormalities can be identified by analysing this liquid biopsy.

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6
Q

Why can we use blood as a liquid biopsy?

A
  • cells are continuously renovating , cell death leads to different turnover of material.
  • some material is continuously being released in the blood stream.
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7
Q

What is found in the blood biopsy sample?

A
  • circulating endothelial cells
  • circulating tumour cells (CDC)
  • disseminated tumour cells (DTCs)
  • tumour educated platelets (TEPs)
  • extracellular micro-vesicles (exosomes)
  • metabolites
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8
Q

How can you extract liquid biopsies?

A

-10mL blood collected by venipuncture (4-5mL plasma)

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9
Q

Why is important to use a special EDTA extraction tubes when collecting blood?

A

to prevent:

  • blood clots
  • genomic DNA release (from WBC)
  • haemolysis
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10
Q

What are properties and logistic and storage of EDTA, citrate?

A

-contain anticoagulant to prevent clotting.

Logistic and storage:
-onsite centrifugation within 6hrs of collection to isolate plasma and avoid WBC apoptosis.
if not possible, sample can be stored at 4 degree for upto a week.

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11
Q

What are properties and logistic and storage of cell-free DNA tubes(eg. paxgene-qiagen; streck)?

A

-contain stabilisers to prevent release of gDNA from white blood and haemolysis of RBC.

logistic and storage:

  • samples can be stored for 6-14 days at 6-37 degrees.
  • more convenient in terms of transport and storage.
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12
Q

What do you see after 15 mins of centrifugation at 2,000x g speed at 4 degree?

A
  1. plasma (55%):
    - ctDNA and exosomes
    - water, proteins, nutrients, hormones, etc.
  2. Buffy coat (<1%)
    - WBC, platelets , CTCs
  3. Hematocrit (45%)
    - RBC
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13
Q

What are 2 biomarkers in liquid biopsies?

A
  1. circulating tumour cells (CTC)

2. circulating tumour DNA(ctDNA)

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14
Q

What are characteristics of circulating tumour cells(CTCs)?

A
  • 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
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15
Q

What is isolation and characterisation of CTCs based on?

A
  • biological and physical properties (size, electrical charge)
  • identified/characterised based on transcripts - PCR done on total RNA extracted from the cells.
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16
Q

Give an example of identifying circulating tumour cells.

A
  • prostate cancer : Prostate specific antigen (PSA) circulating in blood.
  • extract circulating tumour cells, then isolate the DNA and RNA from these cells
  • culture them and amplify them using PCR to identify levels of PSA if any
  • run these samples on electrophoresis gel
17
Q

What are characteristics of circulating tumour DNA (ctDNA)?

A
  • present in different fluids: plasma, serum, urine and others
  • low concentration (1-50ng DNA/mL plasma)
  • Amount highly variable from person to person depending on health status in the same person (increase in cancer, trauma , etc)
  • presence of permanent genomic DNA background in plasma.
  • highly fragmented but with specific size range (<500bp)
  • provides info on current genetic makeup (including irregularities/mutations) with 80-95% specificity and 60-85% sensitivity.
18
Q

Where can we find circulating tumour DNA in centrifugation?

A

-found in plasma layer

19
Q

How do you isolate cell free DNA after centrifugation?

A
  • transfer supernatant to a clean polypropylene tube and freeze it if needed.
  • using magnetic beads or cellulose based or silica based system we can isolate that circulating tumour DNA and store it forever.
20
Q

What technologies are available to study the levels of circulating tumour DNA?

A
  • NGS
  • RTqPCR
  • Digital PCR
21
Q

What are advantages of liquid biposies?

A
  • lower invasiveness
  • higher patient compliance
  • higher cost/effectiveness
  • allow repeated access and multiple sampling
  • no special training required for extraction
22
Q

What are disadvantages of liquid biopsies?

A
  • low amount of material
  • early diagnosis
  • data interpretation
23
Q

Why is liquid biopsies better than solid biopsies?

A
  • with solid biopsies we can only pick up one part of tumour, but with liquid biopsies you can pick up mutations that are present in different metastatic site.
  • cancer is heterogenous disease.
  • molecular properties within a tumour differ and also between metastatic site.
  • no need to identify the tumour site before taking a biopsy and allow repeating sampling.
  • allow analysis of tissue difficult to access, ie lungs, heart.
24
Q

What can liquid biopsies be used for?

A
  • determine early stage of disease.
  • allows us to determine their molecular profile of disease helping in treatment decisions.
  • also to monitor response to therapy.
25
Q

What are clinical application of liquid biopsies?

A

-promising cancer biomarkers that need to be clinically validated, not implemented as diagnosis tool yet, but that provides highly specific and complementary information.

26
Q

Which gene is mutations in lung cancer found and what diagnostic tool is used to test this?

A
  • detection of EGFR gene mutations in lung cancer

- FDA -approved diagnostic test.

27
Q

What is the new FDA approved diagnostic test for patients with advanced solid cancer?

A

-liquid CDX

28
Q

What potential clinical use do CTCs and ctDNA have as cancer biomarkers?

A
  1. screening
  2. diagnosis
  3. treatment
29
Q

centrifugation is done for ….at speed …. at temp…

A

15 minutes at x2000xg speed at 4 degrees

30
Q

What differentiates circulating tumour cells from normal blood cells?

A

-circulating tumour cells are CD45 negative but become cytokines 18/19 positive, these surface markers are not found in other types of blood cells so we can identify them.

31
Q

what are non invasive prenatal test used for?

A

Fetal DNA is present in maternal plasma and is cleared after birth

  • Non invasive prenatal test for screening of Down syndrome, Edwards syndrome and Patau’s syndrome
  • replacing amniocentesis
32
Q

How can cell free DNA be used used to monitor health of solid organ transplants?

A

CfDNA acts as bio markers for monitoring the health of a solid organ transplant.
Endomyocardial biopsy is currently used to asses rejection risk but donor cfDNA increases in recipient blood and rejection risk increases

molecular basis of disease (29 decks)

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