Novel Technologies 1 Flashcards
What are liquid biopsies and what are they representative of
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)
What is an example of an established liquid biopsy
Amniocentesis
What factor makes blood a good liquid biopsy
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
What materials circulate in blood
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
What can circulating endothelial cells detect for
Heart attacks
Why may cell free nucleotides increase
Increased cell death
Exercise
Inflammation
How are miRNA’s involved in breast cancer
microRNA105 has been described to promote breast cancer metastasis
Can liquid biopsies find germline or somatic information
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
Where is the best cell to analyse for germline genome analysis
White blood cells which may carry particles and information from other bodily cells
What must you consider and prevent when extracting blood
Blood clots
Genomic DNA release (from white blood cells)
Haemolysis
What tubes are used for liquid biopsies
EDTA,citrate
Cell-free DNA tubes
What are the components of blood and what can you find in each component
55% plasma
Water, proteins, nutrients, hormones, etc.
cfDNA, exomes
<1% buffy coat
White blood cells, platelets, circulating tumour cells
Haematocrit
Red blood cells
What are circulating tumour cells
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
Where in the blood are circulating tumour cells found
Buffy coat, alongside WBC’s
Rare 1-10 per ml of blood
How can you isolate circulating tumour cells
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.
How can you characterise circulating tumour cells
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
In what type of studies can circulating tumour cells be used in
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
Where can you find ctDNA
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)
What might increase the amount of ctDNA
Increased in cancer, trauma, inflammation, exhaustive exercise etc.
Does ctDNA need fragmenting for NGS
No, as it is already highly fragmented with a specific size range (~500 bp)
What is the problem of using ctDNA
Presence of permanent genomic DNA background in plasma
How can you isolate circulating free DNA
Transfer plasma supernatant to a clean polypropylene tube and freeze it if needed
Isolation using magnetic bead, cellulose-based or silica-based systems
How can you study ctDNA/ what methods can be used
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
What are the advantages of using ctDNA
Less invasive, higher patient compliance, higher cost/effectiveness
Allow repeated access and multiple sampling, no special training required for extraction
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
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
What problems does tumour heterogeneity bring
• Biopsy from primary tumour information may not reflect the current disease condition
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
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
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
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
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)
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
What is sensitivity and specificity
ANS
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
How can liquid CDx help pharma companies
Enables companion diagnostic development for biopharma companies developing precision therapeutics
What is genome editing
Genome editing: group of technologies that give scientists the ability to change an organism’s DNA
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
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
What is Cas9
RNA-guided DNA endonuclease which induced ds breaks in DNA
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)
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
What must the target sequence have in order for CRISPR to work
A PAM sequence - NGG
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)
What is NHEJ
Non homologous end joining - ligation of the ds-breaks
What is HDR
Homology directed repair
Uses template DNA to correct the fault or insert directed faults e.g. stop codon
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
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
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)
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
What are the applications of CRISPR-Cas9 in terms of genetic modifications
Disruption (KO)
Indels
Correction
Gene activation/repression
How is CRISPR-Cas9 used in cancer therapeutics
PD1KO
CAR-T cell
Correct of mutations e.g. EGFR
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
Why KO PD1
PD1 is a negative regulator of T cell function, thus KO increases activation of the immune system against cancer cells
What is CAR-T cell therapy used for
Treats acute lymphoblastic leukaemia in children and various lymphomas in adults
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
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
