Mutation Detection

Question 1

What does locus specific FISH identify?

Answer 1

Bind to a particular region of a chromosome to determine on which chromosome the locus/gene is located or the number of copies that exists

Question 2

What does Alphoid/Centromeric FISH identify?

Answer 2

These map to the repetitive centromeric loci found in the middle of each chromosome. They can be used to identify the number of chromosomes. Can be used with locus-specific to determine whether material missing from a chromosome.

Question 3

What does enumeration FISH identify?

Answer 3

For the detection of deletions, duplications and chromosome ploidy. Usually one set of colours is used as a ploidy control to confirm copy number (i.e. centromeric probe). A normal signal is two copies of each colour.

Question 4

What FISH probes can be used to identity a fusion?

Answer 4

Breakapart Probes - For detection of translocations when the partner gene is unknown. Probe consists of two fluorophores (Red/Green) on either side of a known breakpoint. A variety of abnormal signal patterns can be seen depending on whether there are copy changes and whether the resulting fusion has deletions.

Dual colour/Dual fusion Probes - Detection of fusions with known partner gene. Each probe spans the breakpoint and is effectively cleaved during a translocation and fused together to produce a fusion signal.

Question 5

What is third generation sequencing?

Answer 5

Third generation sequencing, also known as long-read sequencing, is a class of DNA sequencing which works by reading sequences at the single molecule level.

This is in contrast to second generation (NGS) sequencing which requires breaking long strands of DNA into small segments then infering nucleotides by amplification and synthesis.

Question 6

What are the main benefits of third generation sequencing over second generation?

Answer 6

• Lack of PCR means less bias and aretefacts generated from PCR
• Allows for a more homogenous coverage of the genome through overcoming mismapping seen in short reads due to repetitive sequences
• Fast
• Phasing of haplotypes possible
• Analysis of long repeat expansions possible

Question 7

Why has third generation sequencing not yet been routinely implemented in clinical practice?

Answer 7

It still currently has a high error rate making it unsuitable. However advancements are being made year upon year and the error rate for this method is decreasing.

Question 8

What are the main steps of tissue processing?

Answer 8

1) Fixation (Formalin)
2) Tissue processing (Dehydration, Clearing Infiltration)
3) Tissue Embedding (Paraffin)
4) Sectioning with microtome
5) Tissue staining
6) Cover slippin & Mounting

Question 9

What factors can impact the quality of a tissue sample?

Answer 9

• Fixation
• Extraction Method
• Fragmentation
• Deamination on artefacts
• Tumour Heterogeneity
• Presence of Normal Tissue
• Chemsitry of assay used for testing

Question 10

What kind of quality issues can fragmentation cause?

Answer 10

Low amounts of amplifiable DNA

Associated with lower coverage (NGS)

Question 11

What is a deamination artefact and what change is associated with it?

Answer 11

Hydrolytic deamination of cytosine to form uracil (or thymine if the cytosine is methylated). This results in non-reproducible C>T/G>A sequencing artefacts that are observed after PCR amplification when using FFPE.

Question 12

What are the non-fixation variables which may affect the quality of a tissue sample?

Answer 12

• The duration of transport from patient to laboratory.
• Size of tumour specimens.
• Environmental factors such as exposure to heat, light and the concentration and age of formalin used for fixation.
• The age of blocks examined is another consideration as long-term storage in suboptimal environments can cause significant DNA damage.
• Tumour heterogeneity, and the presence of normal tissue.
• Chemistry and design strategy used for NGS (hybridisation vs amplicon).

Question 13

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

Answer 13

• 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 14

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

Answer 14

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 15

What can RNA-Seq be used to analyse?

Answer 15

• 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 16

What are the core ‘ingredients’ of a polymerase chain reaction (PCR)?

Answer 16

Ingrediants

• DNA template to be copied
• Primers (short stretches of DNA that initiate the PCR reaction, designed to bind either side to the DNA you want to amplify)
• DNA nucleotide bases (dNTPs). DNA bases (A, C, G and T) are the building blocks of DNA and are needed to construct the new strand of DNA.
• Taq polymerase enzyme to add in the new DNA bases
• Buffer to ensure the right conditions for the reaction.

Question 17

What are the steps of a polymerase chain reaction (PCR)?

Answer 17

1) Denaturing (94-96 degrees)– when the double-stranded template DNA is heated to separate it into two single strands.
2) Annealing (50-56 degrees) – when the temperature is lowered to enable the DNA primers to attach to the template DNA.
3) Extending (72 degrees) – when the temperature is raised and the new strand of DNA is made by the Taq polymerase enzyme. The bacteria’s DNA polymerase is very stable at high temperatures, which means it can withstand the temperatures needed to break the strands of DNA apart in the denaturing stage of PCR. DNA polymerase from most other organisms would not be able to withstand these high temperatures, for example, human polymerase works ideally at 37˚C (body temperature).

These three stages are repeated 20-40 times, doubling the number of DNA copies each time.

The new fragments of DNA that are made during PCR also serve as templates to which the DNA polymerase enzyme can attach and start making DNA.

Question 18

What are the three main steps in Sanger sequencing?

Answer 18

1. PCR with fluorescent chain terminating dNTPs
   • The DNA sequence is used as a template for “chain termination PCR”.
   • Same mixture as normal PCR but with addition of modified nucleotides (dNTPs) called dideooxynucleotides (ddNTPs)
   • The ddNTPs lack the 3’OH group, therefore extension ceases when ddNTP is incorporated.
2. Size separation by Gel electrophoresis
   • Separation of DNA sequences by length.
   • Shorter fragments move faster than longer fragments.
3. Gel Analysis and determination of sequence
   • Reading the gel to determine the DNA sequence from smallest to largest.
   • DNA polymerase only synthesises 5’ to 3’, each terminal ddNTP will correspond to a specific nucleotide in the original sequence.

Question 19

What is immunohistochemistry (IHC)?

Answer 19

Immunohistochemistry (IHC) is a powerful microscopy-based technique for visualizing cellular components, for instance proteins or other macromolecules in tissue samples. The strength of IHC is the intuitive visual output that reveals the existence and localization of the target-protein in the context of different cell types, biological states, and/or subcellular localization within complex tissues: for these reasons IHC is widely used in many research and clinical laboratories.

Question 20

What is flow cytometry?

Answer 20

Flow cytometry is a popular cell biology technique that utilizes laser-based technology to count, sort, and profile cells in a heterogeneous fluid mixture. Using a flow cytometer machine, cells or other particles suspended in a liquid stream are passed through a laser light beam in single file fashion, and interaction with the light is measured by an electronic detection apparatus as light scatter and fluorescence intensity. If a fluorescent label, or fluorochrome, is specifically and stoichiometrically bound to a cellular component, the fluorescence intensity will ideally represent the amount of that particular cell component. Flow cytometry is a powerful tool because it allows simultaneous multiparametric analysis of the physical and chemical characteristics of up to thousands of particles per second. This makes it a rapid and quantitative method for analysis and purification of cells in suspension. Using flow, it is possible to determine the phenotype and function and even sort live cells.

Question 21

What techniques can be used for the detection of methylation?

Answer 21

Methylation-Specific MLPA (MS-MLPA)
- Used to detect methylation status alongside copy number alterations (procedure for CN alterations same as routine MLPA

Methylation-specific PCR (MS-PCR)

• Simple single tube PCR – PCR primers designed specifically for methylated and un-methylated alleles following bisulphite modification.
• By designing primers that will amplify products of different sizes for the methylated and un-methylated products this makes it a simple fragment analysis assay.

Pyrosequencing

• Bisulfite conversion followed by PCR
• Distinct from sanger sequencing, in which labelled dideoxynucleotides are incorporated randomly in the reaction terminating extension of strands representative of each nucleotide position;
• Uses a sequencing-by-synthesis system in which nucleotides are dispensed one at a time, incorporated into the extending strand and degraded prior to the next nucleotide dispensation. Light is given off proportionate to the amount of nucleotide added to the elongating strand and recorded by an inbuilt CCD camera. Excess nucleotide is degraded by apyrase, after which the next nucleotide is dispensed.
• Comparing the peak light emission of incorporation of C or T at a CpG site within the amplicon gives a precise measure of the amount of methylation at that position within the sample.

Methylation-Sensitive High-Resolution Melting (MS-HRM)
- Methylated and unmethylated PCR products show different melting profiles with thermal denaturation.

Question 22

What is bisulphite modification/conversion?

Answer 22

During DNA replication (cell division) methyl groups (DNA methylation) are resynthesized on the newly replicated strand by one of the enzymes called DNA Methyl Transferases (DNMT). Those enzymes are not present in standard PCR reaction. Therefore, a PCR product obtained from amplification of a specific locus does not contain information about methylation status of the cytosines and the methylation information is lost. The DNA template has to be chemically modified with the use of sodium bisulfite to preserve methylation information before PCR amplification. Sodium bisulfite deaminates non-methylated cytosines to uracil and leaves methylated cytosines untouched (in other words methylated cytosines are resistant to modification induced by sodium bisulfite), thus allowing the alleles to be distinguished.
Bisulphite modification is not always 100% efficient. Bisulphite modified DNA can revert.

Question 23

How does Quantitative Real Time PCR (qPCR/qrt-PCR) work? (not RT-PCR = reverse transcriptase PCR)

Answer 23

• Ability to monitor the amplification the target sequence.
• Reaction monitored in “real time” (rather than looking at bands on a gel or further processing it with capillary electrophoresis).
• Can be used quantitatively to measure/monitor disease.
• The reaction is placed into a Real Time machine that “watches” the reaction occur with a camera or fluorescence detector.
• There are different techniques used to allow the progress of the reaction to be monitored but they all link the amplification of DNA to the generation of fluorescence which can simply be detected with a camera during each PCR cycle.

Question 24

What are the steps of Quantitative Real Time PCR?

Answer 24

1) Denaturation

2) Annealing
• The TaqMan probe has a gene specific sequence and binds downstream of the primer.
• At the 5’ end it has a reporter dye and at the 3’ end a quencher.
• When the probe is intact, the dye and quencher are in proximity and fluorescence resonance energy transfer (FRET) occurs, preventing fluorescence emission as the quencher absorbs the fluorescenct dye from the reporter.

3) Extension
The primers are extended by DNA polymerase.

4) Cleavage
At the target site the probe will bind downstream of a primer for the region of interest - as the primer extends, the 5’ nuclease activity of Taq DNA polymerase cleaves off the reporter dye.

5)Emission
Once the reporter is separated from the quencher the signal will increase.

More reporter dye is released at each cycle and fluorescence therefore increases with the number of cycles. This reaches an exponential (log) phase then plateaus..

Question 25

How does digital droplet PCR (ddPCR) work?

Answer 25

• Sample partitioning
• Traditional PCR, a single sample offers only a single measurement, but in ddPCR, the sample is partitioned into 20,000 nanoliter-sized droplets.
• Partitioning enables the measurement of thousands of independent amplification events within a single sample.
• Involves generating droplets by mixing the aqueous sample with oil which causes the dispersion into single droplets (as opposed to Chip dPCR which involves partitioning the reaction mix over a chip).
• Each partition should contain a single template, which will either be positive or negative (fluorescent or not fluorescent).

Question 26

Through what mechanisms do ctDNA enter circulation?

Answer 26

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 27

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

Answer 27

• 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 28

What are the main benefits of using ctDNA?

Answer 28

• 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 29

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

Answer 29

• 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 errors18

Question 30

What is a single nucleotide polymorphism (SNP)?

Answer 30

DNA sequence variation occurring commonly within a population (e.g. >1%) in which there is a single nucleotide change e.g. C to T. Occur at a frequency of approximately 1 every 1000 base pairs, and therefore there are roughly 4-5 million SNPs in a person’s genome.
More than 100 million SNPs have been found in the human genome from populations around the world.

The genomic distribution of SNPs is not homogenous; SNPs occur in non-coding regions more frequently than in coding regions

SNPs in the coding region are of two types:
· Synonymous SNPs, which do not affect the protein sequence, but can still affect its function.
· Nonsynonymous SNPs which do change the amino acid sequence of protein. The nonsynonymous SNPs are of two types: missense and nonsense.

Question 31

What types of variants can SNP arrays detect?

Answer 31

• SNP arrays allow for analysis of DNA copy number data and the ability to detect gains, losses, loss of heterozygosity (LOH), and mosaicism on a single array.
• The have the additional advantage of being able to detect copy-neutral LOH (also called uniparental disomy). Copy-neutral LOH is a form of allelic imbalance. In copy-neutral LOH, one allele or whole chromosome from a parent is missing. This problem leads to duplication of the other parental allele. Copy-neutral LOH may be pathological.

Question 32

What are the three main components of a SNP array analysis?

Answer 32

1. An array containing immobilized allele-specific oligonucleotide (ASO) probes.
2. Fragmented nucleic acid sequences of target, labelled with fluorescent dyes.
3. A detection system that records and interprets the hybridization signal.

Question 33

What can impact the signal intensity in a SNP array?

Answer 33

· The amount of target DNA in the sample (to detect copy number), and
· The affinity between target DNA and probe (binding will occur more efficiently between sequences with 100% of matched bases than those with 90% matched bases due to a SNP mismatch).

Question 34

What are the benefits and limitations of SNP array analysis?

Answer 34

Benefits of SNP array
• No need for metaphases, utilises DNA
• potential for blood to be analysed instead of marrow
• samples could be obtained from patients too frail for bone marrow aspirate
• potential to reduce failure rates (no dividing cells required)

Drawbacks and limitations of SNP arrays:
· Unable to detect balanced rearrangements/gene fusions and whole genome ploidy changes.
· Mosaicism is not always reliable under 20-30% (depending on size and direction) i.e. so not good for Minimal residual disease (MRD) detection, or specific nucleotide mutations.