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Novel Technologies 1 Flashcards

1
Q

What are liquid biopsies and what are they representative of

A

Sampling and analysis of non-solid biological tissue e.g. blood, urine, plasma and serum, saliva, CSF for detection of molecular biomarkers

Representative of the tissue/s from which it has spread (blood has information from all organs)

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2
Q

What is an example of an established liquid biopsy

A

Amniocentesis

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3
Q

What factor makes blood a good liquid biopsy

A

Cells continuously renovate themselves (cell turnover) via cell renewal and death

Waste material is then continuously released into the bloodstream to be removed by phagocytes

Blood samples will have these waste materials

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4
Q

What materials circulate in blood

A

Circulating endothelial cells - useful for early detection of heart attacks

Circulating tumour cells

Cell free nucleotides released by dying cells - can increase after exercise/inflammation

Exosomes - micro-vesicle messengers that can communicate between cells

microRNA - e.g. microRNA105 has been described to promote breast cancer metastasis

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5
Q

What can circulating endothelial cells detect for

A

Heart attacks

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6
Q

Why may cell free nucleotides increase

A

Increased cell death

Exercise

Inflammation

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7
Q

How are miRNA’s involved in breast cancer

A

microRNA105 has been described to promote breast cancer metastasis

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8
Q

Can liquid biopsies find germline or somatic information

A

You can find germline and somatic information

Normally interested in somatic information - as this normally can only be found in tissue cells in which this mutation has occurred which the blood sample may be carrying!

E.g. circulating tumour cells detached from a tumour

Germline information can be found in any cell in the body

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9
Q

Where is the best cell to analyse for germline genome analysis

A

White blood cells which may carry particles and information from other bodily cells

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10
Q

What must you consider and prevent when extracting blood

A

Blood clots
Genomic DNA release (from white blood cells)
Haemolysis

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11
Q

What tubes are used for liquid biopsies

A

EDTA,citrate

Cell-free DNA tubes

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12
Q

What are the components of blood and what can you find in each component

A

55% plasma
Water, proteins, nutrients, hormones, etc.
cfDNA, exomes

<1% buffy coat
White blood cells, platelets, circulating tumour cells

Haematocrit
Red blood cells

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13
Q

What are circulating tumour cells

A

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

Marker for tumour growth and negative cancer prognosis and treatment response

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14
Q

Where in the blood are circulating tumour cells found

A

Buffy coat, alongside WBC’s

Rare 1-10 per ml of blood

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15
Q

How can you isolate circulating tumour cells

A

Isolation - based on biological and/or physical properties

E.g. based on cell surface markers, such as FACS or magnetic beads
Tumour cells are CD45 negative, EpCAM positive etc.

E.g. size (bigger), electric charge, density etc.

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16
Q

How can you characterise circulating tumour cells

A

Identified/characterised based on transcripts from PCR done on total RNA extracted from the cells

Found where they are from, e.g. looking for markers specific for a location

PSA (prostate specific antigen) seen on cell = prostate cancer tumour

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17
Q

In what type of studies can circulating tumour cells be used in

A

Transcriptome, genome, proteome studies

Phenotype studies

Culture in vivo or in vitro inoculation in a mouse to see if they form tumours

Use techniques such as NGS to sequence DNA, FISH to study chromosome abnormalities, rt/qPCR to find different markers

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18
Q

Where can you find ctDNA

A

Present in different fluids: plasma, serum, urine and others

In blood, it is found in the plasma layer alongside all other circulating free DNA

Low concentration (1-50ng DNA/mL plasma)

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19
Q

What might increase the amount of ctDNA

A

Increased in cancer, trauma, inflammation, exhaustive exercise etc.

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20
Q

Does ctDNA need fragmenting for NGS

A

No, as it is already highly fragmented with a specific size range (~500 bp)

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21
Q

What is the problem of using ctDNA

A

Presence of permanent genomic DNA background in plasma

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22
Q

How can you isolate circulating free DNA

A

Transfer plasma supernatant to a clean polypropylene tube and freeze it if needed

Isolation using magnetic bead, cellulose-based or silica-based systems

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23
Q

How can you study ctDNA/ what methods can be used

A

Next Generation Sequencing (NGS), Digital Droplet PCR (ddPCR), array CGH

Amplifications and deletions, Translocations, Point mutations, Chromosomes abnormalities, epigenetic status (methylation)

Real Time Quantitative Polymerase Chain reaction (qPCR) = ctDNA presence quantification

24
Q

What are the advantages of using ctDNA

A

Less invasive, higher patient compliance, higher cost/effectiveness

Allow repeated access and multiple sampling, no special training required for extraction

25
What are the disadvantages of using ctDNA
Low amount of material, it is not good for detecting early diagnosis Data interpretation - there is a lot of information that needs to be filtered
26
What are the different types of tumour heterogeneity
Intratumoural heterogeneity - within a primary tumour Intermetastatic heterogeneity - between two mestatses Intrametastatic heterogeneity - within metastatic leisons Interpatient heterogeneity
27
What problems does tumour heterogeneity bring
• Biopsy from primary tumour information may not reflect the current disease condition
28
How do liquid biopsies overcome issues faced by regular biopsies
Less invasive and allows repeat sampling Blood is representative of a range of tumour clones You do not need to identify the tumour site You can analyse tissues difficult to access
29
How are liquid biopsies used/What are they used for
They can be used in early diagnosis in disease - though it is a challenge due to low concentrations Determine molecular profile of a tumour Monitor response to treatment and surgery, by measuring minimal residual disease Identify resistance mechanisms Monitoring of clonal dynamics
30
How is ctDNA used
ctDNA can be used to predict metastasis and prognosis of breast cancer, and detection of specific mutations in colorectal or lung cancer
31
What are the problems with identifying novel biomarkers with ctDNA
Promising biomarkers need to be clinically validated, and have not been implemented as diagnosis tool yet*, but it provides highly specific and complementary information *Except EGFR in NSCLC
32
What biomarker has been used as a diagnostic tool
Detection of EGFR mutations have been implemented for non-small cell lung cancer to identifies those eligible for treatment with erlotinib (a targeted therapy)
33
Why do 25% of NSCLC patients have to undergo liquid biopsies
Due to insufficient tissue or otherwise unable to undergo tissue biopsy Looks for 42 mutations including T790M Low sensitivity but high specificity
34
What is sensitivity and specificity
ANS
35
What is liquid CDx
It is a pan tumour liquid biopsy test for patients with advanced solid cancer - identifying patients who may benefit from approved targeted therapies
36
How can liquid CDx help pharma companies
Enables companion diagnostic development for biopharma companies developing precision therapeutics
37
What is genome editing
Genome editing: group of technologies that give scientists the ability to change an organism’s DNA
38
What is CRISPR-Cas9
Second Generation Gene Engineering tool Facilitates knockout or knock-in of DNA sequences at a desired site in the genome Dramatically increases the efficiency of mutagenesis Can be used in vitro and in vivo
39
What are the origins of CRISPR
Defence mechanism of the adaptive immune response system of the bacterium Streptococcus pyogenes that confers resistance to exogenous nucleic acids (e.g., viral DNA) by integrating foreign DNA into a CRISPR loci to transcribe crRNA's to cleave the foreign DNA using CAS proteins
40
What is Cas9
RNA-guided DNA endonuclease which induced ds breaks in DNA
41
What are the components of CRISPR-Cas9
``` Cas9 gRNA - guide RNA with two components - variable crRNA (with target sequence) permanent tracrRNA (allows Cas9 to recognise guide RNA) ```
42
What are the two components of guide RNA
The variable crRNA which is complementary to the target sequence, allowing it to find the target Permanent tracrRNA which allows Cas9 to recognise guide RNA and follow along
43
What must the target sequence have in order for CRISPR to work
A PAM sequence - NGG
44
What happens after Cas9 cuts the DNA
DNA repair occurs - either via NHEJ leading to errors/frameshift/gene KO Or via homology directed repair, which requires the additional use of a template oligonucleotide DNA template - knock in or directed KO (insertion of stop codon)
45
What is NHEJ
Non homologous end joining - ligation of the ds-breaks
46
What is HDR
Homology directed repair Uses template DNA to correct the fault or insert directed faults e.g. stop codon
47
What are the potential methods of transfection
Using viral vectors e.g. lentivirus, lipofection, electroporation, nucleofection etc. As a vector it can be inserted as a DNA format mRNA and direct protein insertion increases the efficiency by eliminating transcription and translation
48
What are the two main limitations of CRISPR-Cas9
Efficiency and off-target effects Sites in the genome partially homologous with the gRNA targeting sequence that can be undesirably modified
49
How can you make CRISPR-Cas9 more efficient
Modified cas9 proteins or homologous proteins of cas9 might be used Modifying transfection methods as above (using protein rather than RNA)
50
How can you reduce CRISPR-Cas9 off-target effects
Modifying PAM can increase specificity of gRNA Prevents targeting of similar sequences If similar sequence in in intron then it is not a big problem
51
What are the applications of CRISPR-Cas9 in terms of genetic modifications
Disruption (KO) Indels Correction Gene activation/repression
52
How is CRISPR-Cas9 used in cancer therapeutics
PD1KO CAR-T cell Correct of mutations e.g. EGFR
53
Describe CRISPR KO of PD1
Treating melanoma, sarcoma and myeloma Aim - use CRISPR-Cas9 to perform 3 genetic modification on T-cells with the above cancers Insertion of NY-ESO-1 receptor which is related to the cancer/testis antigen expressed by various tumours Directing T-cells to cancer cells as it binds proteins expressed in cancer cells Deletion of TCR (primary T cell receptor) and PD1 genes
54
Why KO PD1
PD1 is a negative regulator of T cell function, thus KO increases activation of the immune system against cancer cells
55
What is CAR-T cell therapy used for
Treats acute lymphoblastic leukaemia in children and various lymphomas in adults
56
How can CRISPR-Cas9 be used to find mutations that confer drug resistance
Transfect cells of interest in vitro with Cas9 protein, then add a library of different guide RNA's which target different genes Each vector with gRNA will carry a label to identify which gRNA was transfected Compare transfected with non-transfected cell resistance to drugs Sequencing allows you to look at resistant cell genomes and identify the mutation that may confer resistance
57
Describe how CRISPR-Cas9 can be used in functional genomics
Functional characterisation of novel genes associated with disease Setting up of cell lines + transfection of CRISPR-Cas9 to target a gene of interest to KO - compare KO between cancer and normal cell lines This allows you to find out the consequences if that cell function is lost You can work out if it is a oncogene or a tumour suppressor gene KO of TSG = increased cell division KO of oncogene = reduced proliferation

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