Technology / Other

Question 1

What is polymerase chain reaction (PCR)?

Answer 1

A fast and reliable lab technique used to amplify DNA sequences

Question 2

What is the main steps of a PCR reaction?

Answer 2

1. Denaturation - Sample is heated to 94-96oC to separate the DNA into two single strand pieces of DNA
2. Annealing - Sample is cooled to 68oC to allow the primers (F/R) to anneal to the single stranded DNA
3. Extension - Sample is heated to 72oC to allow DNA extension by DNA polymerase (Taq Polymerase)
4. Cycle for the required quantity of DNA (Usually 30-40 cycles)

Question 3

For Reverse Transcriptase PCR, what must occur prior to PCR amplification?

Answer 3

Conversion of the sample RNA to cDNA with the enzyme reverse transcriptase

Question 4

What are the necessary components of a PCR reaction?

Answer 4

• DNA template. The sample DNA that contains the target sequence
• DNA polymerase. e.g. Taq Polymerase. to synthesise the complementary DNA strand
• Primers - Short pieces of DNA complementary to the target DNA sequence from which the new strand of DNA can be synthesised.
• Nucelotides. Deoxynucleotide triphosphatases (dNTPs) - Single units of the bases ‘A’, ‘T’, ‘G’, and ‘C’ which act as building blocks for the new strand.

Question 5

What causes a PCR reaction to plateau?

Answer 5

• Inhibitors of the polymerase found in the sample
• Reagent limitation (running out)
• Accumulation of pyrophosphate molecules
• Self annealing of the products

Question 6

What is the main limitation of PCR due and what is it caused by?

Answer 6

End point quantification of the PCR products is unreliable due to the plateau effect. This is why quantitative RT-PCR is necessary.

Question 7

What is fluorescence in situ hybridisation (FISH)?

Answer 7

FISH uses fluorophore-couples DNA or RNA sequences (probes) to detect the presence or absence of or to locate the position of, complementary sequences in fixed cells or tissues using fluorescent microscopy.

Question 8

What are the major advantages of FISH testing?

Answer 8

• High sensitivity and specificity for recognising targeted DNA or RNA
• Direct application to both metaphase chromosomes and interphase nuclei
• Visualisation of hybridisation at the single level
• Relatively fast (can get a result within 24 yrs)

Question 9

What is multiplex FISH?

Answer 9

Multiplex FISH enables the analysis of multiple targets and visualisation of co-localised signals in a single specimen. Specturally different fluorophores are uses for each hybridisation probe.

Question 10

What is the basic principle that underpins how FISH works?

Answer 10

FISH is based on the denaturation of probe (DNA sequence) and target (interphase or metaphase cells on a slide) and hybridisation of the probe to complementary sequence (target) on the slide.

Question 11

What are locus specific FISH probes used for?

Answer 11

They bind to a specific region of a chromosome to determine which chromosome the locus/gene is located or how many copies of a locus/gene exists within the genomes

Question 12

What do centromeric FISH probes target and what are they used for?

Answer 12

These probes target the repetitive sequences found in the middle of each chromosome (centromere). These probes are used to determine whether an individual has the correct number of chromos0mes. They can be used in combination with locus-specific probes to determine whether an individual is missing genetic material from a particular chromosome.

Question 13

What are enumeration FISH probes and what are they used for?

Answer 13

Enumeration FISH probes are used for the detection of deletions, duplications and ploidy. Usually one of the colours in the probe set is used as a ploidy cotnrol to confirm the copy number (centromeric probe). A normal signal would be two copies of each colour indicating two normal copies of the locus. A missing signal/colour indicates loss of chromosomal material and extra signals indicate a gain.

Question 14

What are breakapart FISH probes used for?

Answer 14

For the detection of translocations when the second gene/partner is unknown.
A probe consists of two fluorophores (red/green) on either side of a known breakpoint. A normal signal pattern would look like two fusions. Abnormal signals way take a variety of forms depending on whether there has been copy number changes and whether of of the fusions results in a deletion

Question 15

What are dual colour/dual fusion FISH probes used for?

Answer 15

For the detection of translocations when the second partner gene is known. Each probe (usually red/green)( span a breakpoint and are effectively cleaved during a translocation then fused with the other cleaved probe to make a fusion signal

Question 16

What are the two key concepts in tumour immunology?

Answer 16

Immunosurveillance and Immunoediting

Immunosurveillance - recognition of tumour neoantigens on the surface of tumour cells by the immune system.

Immuniediting occurs in three phases
Phase 1- Elimination phase where tumour cells are neutralised by natural killer (NK), CD4+ and CD8+ cells.
Phase 2 - Equilibrium between immune cells and tumour cells.
Phase 3 - Escape phase where the immune system is unable to destroy the tumour cell - corresponding to the appearance of a clinically detectable tumour

Question 17

What is tumour mutational burden (TMB)?

Answer 17

TMB refers to the number of somatic gene mutations present in a tumour. It is defined as the number of mutations per megabase of DNA analysed. This varies across tumour types

Question 18

What tumour features can impact TMB?

Answer 18

• Variations across different cancer types in levels of TMB
• Tumour heterogeneity
• Primary/Metastatic - Primary tumours have higher heterogeneity compared to metastatic (higher TMB)

Question 19

How can TMB influence the effect of the immune system in recognising tumour cells?

Answer 19

Cancers with low TMB have fewer mutations and therefore decreases the chance that one will activate the immune system

Cancer cells with high TMB have more mutations, increasing the chance that one will activate the immune system

Question 20

What treatment type has TMB been considered as a proposed biomarker for and how do they work?

Answer 20

Immune Checkpoint Inhibitors (ICIs) such as CTLA-4 inhibitors, PD1 inhibitors and PDL1 inhibitors.

One mechanism that tumours use to survive is to increase immune checkpoint molecules that can help to suppress antitumor immune responses. ICIs reinvigorate antitumor immune responses by interrupting co-inhibitory signaling pathways and promote immune-mediated elimination of tumor cells.

Question 21

Why is TMB a marker for ICI response?

Answer 21

Tumours with high TMB had a higher number of neoantigens and therefore can identify patients who would in turn benefit from ICI therapy.

Question 22

What are the high/low thresholds or TMB and what response is seen in patients treated with ICIs?

Answer 22

TMB-High - >20mutations/Mb, response 58%

TMB-Low - <20mutations/Mb, response 20%

Question 23

What methods can be used to detect TMB?

Answer 23

Calculation of TMB is generally carried out using WGS, WES or panel-based approachess. Research has favoured WGS/WES whereas clinical applications have more often utilised panels due to a lower sequencing cost, lower DNA inpur requirement and a shorter turnaround time

Question 24

What are the primary factors which influence how TMB is calculated?

Answer 24

1) Tumour cell content & sequencing coverage
Quality of TMB data is dependant on greater tumour content and sequencing coverage. Targeted panels allow for sequencing to a greater depth than WGS/WES enabling a higher sensitivity when the tumour content is low (<10%) and can detect variants down to lower frequencies.

2) Pre analytical/processing factors
Tissue is normally fixed in formaldehyde (FFPE) which can lead to significant noise in NGS and impact TMB calculations

3) Sequencing strategy and size of assay
As the size of a panel becomes smaller. the uncertainty associated with TMB becomes greater with the coefficient of variation rapidly increasing when the panel size is less than 1Mb

4) Bioinformatics pipelines
Pipeline filtering can differ between analysis methodologies. Most will filter out synonymous and germline variants as they are unlikely to be involved in creating neoantigens however this is not standardised. Lack of matched tumour-germline analysis in standard clinical practice means germline variants cannot be filtered out. Choice of variant caller (and its sensitivity) can impact how TMB calculated

5)TMB thresholds
Not yet standardised. Different studies have utilised different cut offs for high TMB

Question 25

What are the main uses for minimal residual disease (MRD) monitoring?

Answer 25

• High resolution determination of efficacy of therapy
• Allow target driven titration of dose and duration of treatment
• Relapse risk stratification after induction to allow triage to optimal consolidation therapy
• To determine prognosis after completion of standard treatment
• To spare toxicity and cost of stem cell transplant in those with a low risk of relapse
• Assignment of maintenance therapy after completion of standard treatment

Question 26

What are the main technologies which can be used for MRD monitoring?

Answer 26

FISH

• Cytogenetically defined rearrangements
• Low sensitivity

Quantitative RT-PCR (RNA)

• Monitors expression of leukaemic transcripts
• Most commonly monitors expression of translocation products (e.g. BCR-ABL1)
• Molecular breakpoints should be defined at diagnosis to allow monitoring
• Uses a standard curve derived from the amplification of serial dilutions of a cell line or plasmid DNA
• Allele-specific oligonucleotide (ASO)-primers required for MRD monitoring.
• Amplification conditions and sensitivity testing for each ASO primer is established by serially diluting the diagnostic material in normal mononuclear cells.
• Patient specific and relatively inexpensive

Immunological (Flow)

• Utilises specific immunological profile of the proteins on the surface of white blood cells either by fluorescently labelled antibodies or flow cytometry.
• Flow cytometry Identifies aberrant cell surface marker expression that is not seen in normal bone marrow or blood. An immunophenotype can be identified at presentation, however some relapsed disease may present with a different immunophenotype due to evolution.

QF-PCR
- Highly polymorphic markers are used to detect chimaerism in which no other markers are available.

NGS

• High sensitivity
• Specific primers for quantification not necessary
• Expensive
• Strong emphasis on bioinformatics expertise

ddPCR

• Allows for quantification without a standard curve
• More specific than RT-PCR
• No guidelines for its use in clinical setting

Question 27

What can RNA-Seq (NGS) be used to analyse?

Answer 27

• Alternative gene spliced transcripts
• Post-transcriptional modifications
• Gene fusions
• Mutations/SNPs
• Changes in gene expression over time, or differences in gene expression in different groups or treatments

Question 28

Through what mechanisms do ctDNA enter circulation?

Answer 28

Cell death through apoptosis and necrosis and ctDNA is also thought to arise due to active release from tumours

• DNA integrity can help to distinguish the mechanism of cfDNA release:
o Apoptosis results in smaller DNA fragments which can vary in size but have been stated to be typically around size of ~160-200 bp which is linked to the size of DNA fragment around a nucleosome and on the linker histone4,15-18.
o Necrosis results in much larger DNA fragments up to thousands of bp in length4,15-18
o Active release from tumours can result in variable sizes of DNA, between the size of those from apoptosis or necrosis.

Question 29

What are the pre analytical steps which need to be carried out to capture ctDNA?

Answer 29

• cfDNA is isolated from the plasma portion of EDTA whole blood samples which have been separated by centrifugation. It can also be obtained from serum although plasma is preferred.
• There is no formal accepted method for processing of plasma samples; however, due to high clearance rates and the haemolysis of blood cells, separation needs to occur in a timely fashion of typically less than 6 hours1,14,19. This applies to blood collected in EDTA vacutainers.
• The processing of whole blood to plasma is a critical step as the ctDNA can become diluted by the breakdown of normal cells and release of wild type cfDNA or it can be degraded and broken down1,14.
• In situations, where it would not be possible to process an EDTA blood in such a timeframe there are preservative tubes such as the Streck Cell Free BCT tubes which maintain stability of the blood and integrity of the ctDNA19.

Question 30

What are the main benefits of using ctDNA?

Answer 30

• Minimally invasive
• Relatively cheap
• Easy to access and repeatable
• Could be used for monitoring and early detection of resistance
• High specificity test but lower sensitivity and risk of false negative
• Reflects whole tumour

Question 31

What methods can be used for detecting variants in ctDNA and the pros and cons?

Answer 31

Targeted PCR based approaches include qualitative methods such as allele specific PCR, PCR using peptide nucleic acid clamps and quantitative approaches such as and ddPCR.
o Advantageous as they require less input DNA, have a high sensitivity and allow a quick turnaround time for key variants.
o Only able to look at a small set of variants

• Next generation sequencing (NGS) based approaches are becoming more appealing due to the increase in the number of potential targets genes / variants which can influence patient care. NGS approaches would allow for multiple regions of interest to be analyses as opposed to fewer targeted variants. This would perhaps offer a clear picture of the ‘clonal differences in tumour cell populations’1.
o Differences between methodologies – amplification versus hybridisation based – would need to be considered.
o Sensitivity of NGS methodologies for ctDNA have improved due to the introduction of unique molecular identifiers (UMIs)14,18. These label each fragment with a UMI during the first PCR cycle during the library preparation stage; this means that any PCR products which have derived from this fragment can be combined into a consensus sequence. This minimises PCR artefacts and errors