Novel technologies applied to personalised medicine

Question 1

What is a liquid biopsy?

Answer 1

Sampling and analysis of non-solid biological tissue, primarily blood. It is a minimally invasive technology for detection of molecular biomarkers. Representative of the tissue/s from which it has spread.

Question 2

What is an example of an established liquid biopsy?

Answer 2

Amniotic fluid analysis

Question 3

What factors can be detected when using blood as a liquid biopsy?

Answer 3

Extracellular micro-vesicles (exosomes)

Metabolites

Cell free nucleotides

Tumour educated platelets (TEPs)

Circulating tumour cells (CTCs)

Disseminated tumour cells (DTCs)

Circulating Endothelial Cells (CEC)

Question 4

In liquid biopsies, specific techniques must be used to prevent what?

Answer 4

Blood clots

genomic DNA release (from white blood cells)

Haemolysis

Question 5

What type of liquid biopsy tubes can be used?

Answer 5

EDTA, Citrate

Cell-free DNA tubes (e.g. Paxgene-Qiagen; Streck)

Question 6

What are the properties of each of the liquid biopsy types?

Answer 6

EDTA, Citrate: Contain anticoagulant to prevent clotting

Cell-free DNA tubes (e.g. Paxgene-Qiagen; Streck): Contain a stabiliser to prevent release of gDNA from white blood and haemolysis of red blood cells

Question 7

What are the logistic and storage of each of the liquid biopsy tubes?

Answer 7

EDTA, Citrate: On-site centrifugation within 6hrs of collection to isolate plasma and avoid white cells apoptosis. If not possible, sample can be stored at 4ºC for a up to a week

Cell-free DNA tubes (e.g. Paxgene-Qiagen; Streck): Samples can be stored for 6-14 days at 6ºC-37ºC

Question 8

After 15 mins centrifugation at 2,000 x g speed at 4ºC, what does the liquid biopsy contain?

Answer 8

55% Plasma:
Water, proteins, nutrients, hormones etc

cfDNA, exosomes

<1% Buffy coat:
White blood cells, platelets, circulating tumour cells

45% Hematocrit:
Red blood cells

Question 9

What are circulating tumour cells?

They are a marker for what?

Answer 9

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

Tumour growth and negative cancer prognosis and treatment response.

Question 10

Circulating tumour cells are found in a high background of…

Answer 10

Normal cells! - sensitive and specific methods are needed to study them.

Question 11

Circulating tumour cells are extremely common.

True or false

Answer 11

False

Extremely rare: 1-10 per 1ml of blood.

Question 12

How are circulating tumour cells isolated and characterised ?

Answer 12

• Isolation and characterisation from buffy coat
• Biological properties and /or physical properties E.G circulating tumour cells are Epcam positive, CD45 negative whereas leucocytes are typically CD45 positive. Thus to isolate CTDs from leucocytes is by detecting such markers isolating using different antibodies that are attached to magnetic particles
• Flow cytometry also common method
• Identified/characterised based on transcripts- PCR done on total RNA extracted from the cells.

Question 13

What is circulating tumour DNA present in?

Answer 13

Different fluids: plasma, serum, urine and others.

Question 14

Circulating tumour DNA have a low concentration (1-50ng DNA/mL plasma).

True or false

Answer 14

True

Question 15

The amount of circulating tumour DNA is highly variable for person to person depending on what?

Answer 15

Health status in the same person (increase in cancer, trauma, etc.).

Question 16

The presence of permanent genomic DNA background in plasma requires what?

Answer 16

Methods to separate ctDNA and normal DNA.

Question 17

What is highly fragmented but with specific size range (<500bp) and thereby does not need to be fragmented as part of the first step of NGS ?

Answer 17

Circulating tumour DNA

Question 18

What provides information of current genetic make-up (including irregularities/mutations) with 80-95% specificity and 60-85% sensitivity?

Answer 18

Circulating tumour DNA

Question 19

How is Circulating tumour DNA isolated?

Answer 19

• Isolate plasma layer
• Transfer supernatant to a clean polypropylene tube and freeze it if needed
• Isolation using magnetic bead, cellulose-based or silica-based systems.

Question 20

What three technologies can be used to analyse circulating tumour DNA?

Answer 20

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

Question 21

What are the advantages of using liquid biopsies as apposed to solid biopsies/traditional biopsies?

Answer 21

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

Question 22

What are the disadvantages of using liquid biopsies as apposed to solid biopsies/traditional biopsies?

Answer 22

Low amount of material
Early diagnosis
Data interpretation

Question 23

Why are liquid biopsies used?

Answer 23

• Cancer is a heterogeneous disease
• Molecular properties within a tumour differ and also between metastatic sites.
• Primary tumour information may not reflect the current disease condition.
• No need to identify the tumour site before taking a biopsy and allow repeating sampling.
• Allow analysis tissues difficult to access

Question 24

Liquid biopsies can be used to investigate localised and metastatic disease

True or false

Answer 24

True

Question 25

What cancers have been detected using circulating tumour DNA

Answer 25

Breast cancer
Lung cancer
Colorectal cancer
Prostate cancer

Question 26

What are promising biomarkers that need to be clinically validated, not implemented as diagnosis tool yet*, but that provides highly specific and complementary information?

What is the exception to this?

Answer 26

Detection of liquid biopsies/ctDNA and CTCs/

Detection of EGFR mutations in lung cancer and Pan-tumour liquid biopsy test for patients with advanced solid cancer as diagnostic tests

Question 27

What is CRISPR/Cas9?

Answer 27

A genome editing technology which belongs to the Second Generation Gene Engineering tool

Question 28

Why is CRISPR used in cancer?

Answer 28

• 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.

Question 29

What are two components of CRISPR/Cas9?

Answer 29

• Cas9 (CRISPR associated protein 9): RNA-guided DNA endonuclease enzyme. Induces double strand breaks in DNA.
• gRNA: RNA that contains a 20nt sequence complementary to the DNA target sequence.

Question 30

How does CRISPR/Cas9 work?

Answer 30

• The 20 nt sequence present in the crRNA of the sgRNA guides the Cas9 endonuclease to the genomic DNA target sequence located immediately upstream of a NGG ‘‘protospacer adjacent motif’’ (PAM). Cas9 protein induces a double strand break on the target sequence. Strands are repaired by the cell DNA repair system which randomly introduces errors into the sequence giving rise to non-functional proteins (KO). Changes are inherited by daughter cells.

Question 31

What are the different CRISPR/Cas9 pathways used to generate a knockout?

Answer 31

Non-homology end joining (NHEJ) repair (Undirected knockout): Joins free DNA ends and this system can sometimes introduce errors when joining DNA end. Thus can cause a stop codon and generate a knockout.

Homology-directed repair (HDR):

If exploit HDR system, need an oligonucleotide repair template. Mutation introduced in repair DNA strand. Using a single-stranded template ensures that this template is not randomly inserted elsewhere in the genome or is targeted by the Cas9 enzyme.

Question 32

What is a limitation of Crispr?

Answer 32

Off-targets: Sites in the genome partially homologous with the gRNA targeting sequence that can be undesirably modified e.g can introduce double strand break in desired and undesired targeting sequence due to the similarity between both of them

Question 33

What are the applications of crispr?

Answer 33

• Gene disruption (KO)
• Gene insertion
• Gene deletion
• Gene correction
• Gene addition
• Gene activation (CRISPRa)
• Gene repression (CRISPRi

Question 34

How is Crispr used to develop human therapeutics in cancer?

Answer 34

1. PD-1 KO- treatment of melanoma: First Crispr related trial approved. T cells edited
2. CAR-T cell therapy- treatment of haematological cancers

Chimeric antigen receptor T cells genetically engineered cells that produce an artificial T-cell receptor for use in immunotherapy.

By using Crispr insert gene for CAR into t cell so that t cells express car receptor and thus expand and infused into patient to recognize and kill cells by activating immune response.

3. Correction of pathogenic mutations
   CRISPR used to repair mutants in EGFR. This gene is most commonly mutated in non small celllung cancer(NSCLC). By inducing HDR repair system which utilises DNA template in cells that carry pathogenic mutation, sequence can be corrected

Question 35

How is Crispr used to develop drugs?

Answer 35

• Identifying genes and mutations that can make cells resistant to drugs

E.g using cell line to test new drug, cell line transfected with Cas9 nuclease, mix cells with lentiviral genome wide CRISPR sgRNA library. Each sgRNA will introduce a different mutant into a different cell and can divide this population of cells into two groups. Administer drug to one cell group and utilise an untreated group not treated with drug (control). Can then use NGS to discover which cells have survived and what mutations they may harbour.

Question 36

How is CRISPR/Cas9 used for gene drives?

Answer 36

Functional characterisation of novel genes associated with a disease.

e.g Using in vitro cancer cell line and transfecting with Cas9 and gRNA knocking out gene of interest
and normal cell line (with gene). This identifies
Proliferation, Viability, Apoptosis, RNAseq Etc.