Mutation Detection Flashcards

Question

On what basis were genes included in the ACMG secondary findings list?

Answer 1

Based on clinical features, likelihood of early diagnosis, molecular characteristics, clinical testing options, and medical actionability.

Answer 2

For clinically significant findings that have potential health implications, these include: - the timing of the impact upon health, when this will come about, now or in the future - its scope, who it affects, the individual, their offspring or other family members - its scale, whether its impact upon health is significant or trivial - The probability of impact, whether the variant is completely penetrant or only marginally so.

Answer 3

The boundary between clinical and research activities is becoming increasingly blurred, particularly in the subspecialty of clinical genetics and is growing with the use of genomic technology. This arises because in a resource scarce environment, some genetic tests (e.g for rare diseases) can only be accessed through research protocols because there are no relevant genetic tests validated for clinical use within the NHS

Answer 4

Beneficence - it has the potential for reducing the risk of, or preventing, disease Non-maleficence - to do no harm Justice respect for autonomy

Answer 5

Discussed at GTAB: - The seriousness of the presenting problem and the nature of the other findings - Whether the finding represents a known clinical entity or risk factor or a finding that requires further investigation (e.g. variants of uncertain clinical significance, VUCS) - Whether the finding has been validated to an acceptable standard - The availability of any treatment/prophylaxis, and its likely success - Whether the finding is a risk factor for disease or represents a disease process - The age of the patient and co-existing morbidities and conditions - Prior knowledge of the patient’s wishes

Answer 6

The intentional search for additional pathogenic variants during genomic sequencing.

Answer 7

A pipeline is a term in computer science for chaining or connecting software tools/programs/scripts creating a stepwise workflow to execute the analytical steps necessary for a complete bioinformatic analysis. Each step in the pipeline is typically designed to take input from the previous step and generate output that is used as input for the next step.

Answer 8

Contains raw sequencing

Answer 9

Text file containing demultiplexed sequence reads Often used as the first input for bioinformatics pipelines to generate quality and alignment metrics

Answer 10

Generated from FASTQs. Reads are mapped against the reference genomes to allow for aligned reads to be viewed. Also provides some quality information e.g. depth of sequencing at different locations

Answer 11

Compressed BAM files to help with space saving

Answer 12

File contains information about a position in the genome, usually variants. May also include annotations This is the output of variant calling section of BI pipeline

Answer 13

Stores genomic regoins and is usually used to define the regions fo interest for an assay e.g. variants will only be called within the regions defined by the BED file

Answer 14

The input- FASTQ Quality control- filter out poor quality reads and removing artefacts Sequence alignment- map to reference genome and produce BAMs Variant calling- Identify variations between sequence and reference Variant filtering: filter out false positives or poor quality variants (e.g. due to poor mapping, strand bias) Variant annotation: characterize variant with location, HGVS, VAF, databases Variant prioritisation: some pipelines may only show variants that are thought to be clinically relevant, filter out polymorphisms- must be fully validated or could miss important variant

Answer 15

Full control - if commercial, an organisation must put their trust into the claims made by a commercial operator - allows modification of filtering criteria, annotations and databases Data security - all in house so less cocersn than with a commercial operator Cost - often cheaper than regularly paying for commercial BI

Answer 16

Cost -Can be costly to set up at the beginning Expertise - Requires a high amount of expertise and validation to ensure pipelines are effective and that variants of interest are not filtered out Time - likely to take a considerable amount of time to work through bugs and test quality of outputs.

Answer 17

Low VAF- close to LOD Low TC samples Identify areas of poor quality/drop out Large deletions/insertions Horizontally complex variant: 2 or more sequence alterations on same read in close proximity, so that they may resemble single variant. Vertically complex variant: three or more alleles are represented by different sequence reads

Answer 18

GC rich regions Homopolymeric tracts Strand bias Artefacts due to poor quality input DNA (e.g. deamination from FFPE) Insufficient template DNA

Answer 19

Variant present which affects alignment Read maps to multiple locations in the reference genome (e.g. pseudogene) Reference genome is incomplete so sequence is missing (e.g. centromeric regions) Errors introduced during sequencing

Answer 20

segmental duplications or pseudogenes can result in the same sequence being present in 2 or more locations in the genome.

Answer 21

NGS sequence reads that map to these duplicated regions e.g. psuedogenes will not have unique mapping and therefore may be removed from downstream analyses. If clinically relevant genes have a pseudogene it may be difficult to get sufficient coverage of the gene for variant calling Alternatively, called variants may be in the pseudogene and not the gene itself

Answer 22

PMS2 and Lynch Syndrome

Answer 23

Paired-end sequencing- sequence both ends of the DNA fragment producing forward and reverse reads Paired-end sequencing can be useful for detecting structural variants (deletions, insertions or inversions)

Answer 24

FISH (Fluorescence in situ hybridization) is a technique using DNA probes with fluorophore-coupled nucleotides to detect complementary sequences in fixed cells or tissues. Its advantages include high sensitivity, direct application to both metaphase chromosomes and interphase nuclei, and single-cell level visualization

Answer 25

FISH involves denaturation of probe. Treated with formadimide which reduces the melting point of the DNA and helps in faster hybridization, where the probe binds to complementary sequences on the slide. The procedure includes slide preparation, codenaturation, hybridization, stringent washing, counterstaining, and visualization using a fluorescent microscope

Answer 26

The three main types of FISH probes are locus-specific probes, alphoid or centromeric repeat probes, and whole chromosome probes. They are used for detecting specific genetic regions, determining chromosome number, and examining complex chromosomal abnormalities, respectively.

Answer 27

FISH is widely used in oncology for diagnosing and prognosticating various cancers such as sarcoma, CML, CLL, ALL, AML, NHL, and MCL. It helps in defining treatment options, disease monitoring, and treatment response assessment.

Answer 28

The advantages of FISH include high resolution, applicability to various cell types (including non-living, fixed, and paraffin-embedded cells), relatively fast results (usually within 24 hours), and the ability to count many cells.

Answer 29

The disadvantages of FISH include its limited scope (it will not detect abnormalities in regions not probed), lack of high throughput, and lower resolution compared to PCR techniques.

Answer 30

Locus-specific probes bind to particular regions of a chromosome and are used to determine chromosome location or copy number of specific genes. They can be breakapart probes for detecting translocations, dual-color dual-fusion probes for known translocations, or enumeration probes for detecting deletions, duplications, and chromosome ploidy.

Answer 31

G banding FISH QF-PCR RT-qPCR MLPA SNP arrays NGS

Answer 32

Low resolution (>5Mb), labor-intensive, slow turnaround time, unable to detect UPD, requires dividing cells and manipulation of the cell cycle, risk of cultural artefacts, and some abnormalities not detected in cultured cells.

Answer 33

Hybridisation of probes to DNA, ligation of probes, PCR amplification of ligated probes, separation by capillary electrophoresis, and analysis and quantification.

Answer 34

It allows for the generation of different sized products for electrophoretic resolution.

Answer 35

1. Denaturation of genomic DNA 2. Hybridisation of probes to the sample 3. Ligation of probes 4. PCR amplification of ligated probes using universal primers 5. Separation of amplified products by capillary electrophoresis 6. Analysis and quantification

Answer 36

Allows copy number detection and methylation profiling

Answer 37

By using probes with a methylation-sensitive restriction site and restriction enzume Hha1; methylated DNA remains undigested and generates a signal during PCR.

Answer 38

It requires reverse transcriptase to create cDNA from RNA before proceeding with the MLPA reaction.

Answer 39

mRNA expression profiling.

Answer 40

Split Read (SR) Read Pair (RP) Assembly-based (AS) Read Depth (RD)

Answer 41

Because no single method is comprehensive enough to detect the full range of DNA variations on its own.

Answer 42

Limited by the length of reads and less reliable in regions with duplications.

Answer 43

It is reliable for detecting deletions and duplications and can count the number of CNVs, though it has poor breakpoint resolution.

Answer 44

Single nucleotide polymorphism (SNP) - a DNA sequence variation occurring commonly within a population (e.g. >1%) in which there is a single nucleotide change e.g. C to T. On average, SNPs occur every 1000bp meaning that there are roughly 4 to 5 million SNPs per person’s genome.

Answer 45

Synonymous SNPs (sSNP) do not cause a change in amino acid. Non-synonymous (nsSNP) when an amino acid is altered and are nonsense or missense

Answer 46

Patients DNA is fragmented and amplified Bead cheap containing thousands of probes containing common SNPs Patients DNA hybridized to probe and single base extension with dNTPs. These are fluorescently labelled. Beads are scanned and the detection system interprets hybridization signal and coverts intensity into genotype DNA binds if the patient contains the SNPs. This leads to the some heterozygous calls

Answer 47

Illumina - 850K SNPs - 3262 target regions - Uses single base extension with a fluorescently labelled dNTP (green if one base and red if another) Affymetrix array - 1.8 million markers. 946,000 for CNV detection, 906,600 SNPs - All targets bind all probes and the presence of SNPs reduce the affinity of a signal and weaker fluorescence

Answer 48

By chance, people will be homozygous for some SNPs and heterozygous for other SNPs. The SNPs selected in the bead-chip type arrays are carefully chosen for variability within a population. In the B-Allele Chart, BB homozygotes have a data value of 1.0, AA homozygotes have a data value of 0.0 and AB heterozygotes have a data value of 0.5. This results in three clusters on the BAF plot

Answer 49

AB genotype Normally: B/A- 0.5 BAF Gain - Duplication- 0.33 or 0.66 BAF Deletion - BAF 0 or 1 Can also be used to determine clone level- based on how separated the BAF is

Answer 50

Both copies of the chromosome are from the same parent (either inherited or acquired in cancer), there will be two copies of each SNP, but because both regions are identical, every SNP will be homozygous. This will look the same as a deletion in the B-Allele frequency chart, but no copy number change will be visible in the LogR ratio chart.

Answer 51

LOH as a cancer mechanism Uniparental disomy (inherited or acquired) Consanguinity

Answer 52

Can detect CN-LOH Higher resolution than G banding More accurate clone level compared to G-banding, FISH but limited below 20% Less subjective analysis High throughput

Answer 53

Can't detect balanced translocations 5% limit of detections Can't detect SNVs High level of artefacts Quite laborious Clonality limited to 20%ish so can't be used for MRD

Answer 54

MDS ALL MM CNS (methylation array)

Answer 55

LOH CNVs Methylation

Answer 56

Array CGH: It focuses on detecting variations in DNA copy number by comparing the fluorescence intensity of a test sample against a reference sample. SNP Array: In addition to detecting copy number changes, SNP arrays assess the presence of single nucleotide polymorphisms SNPs and uses in silico tools for CNV calling.

Answer 57

Patient and control DNA are labeled with fluorescent dyes and applied to bead chip with thousands of probes for regions fo interest Patient and control DNA compete to hybridize which is then scanned my microarray If gain then patient DNA will be more present than control and if loss then control DNA more present

Answer 58

Can detect genomic imbalances as small as 100bp, depending on coverage More cost effective better reproducibility and less batch-to-batch variation Multiple consecutive probes indicating the same copy-number change are required to determine a gain or loss. This enhances the accuracy of the interpretation.

Answer 59

Low detection frequency of mosaicism (<30% of abnormal cells) False positives Can't detect LOH or UPD

Answer 60

Type of CGH BAC clones are propagated in vectors in bacteria, purified, amplified and then spotted onto a glass slide using ultra fine needles. Multiple copies of each BAC are spotted onto the array and distributed across the array Due to the large size, BACs are very stable and hybridisation is specific

Answer 61

Less CNVs of uncertain significance detected High signal to noise ratio accurate copy number information

Answer 62

Abnormalities may be missed as unable to distinguish gains/losses <85kb or those which fall in 600kb gaps, LOH/UPD not detected less sub arrays per slide therefore expensive Not really used in routine practice

Answer 63

Expression arrays allow the simultaneous investigation of the expression of thousands of genes and typically involve comparing two or more highly related cellular or tissue sources that differ in an informative way. Used to investigate expression profiling in tumours and expression profiling of MicroRNAs.

Answer 64

Bilsulphite modification followed by microarray hybridisation: CpG islands in promoters of specific genes are hypermethylated in cancer genomes. Sodium bisulphite converts cytosine to uracil but leaves 5-methylcytosine (methylated cytosine) unchanged. Oligonucleotide probes on the microarray hybridise specifically to either the converted or unconverted sequence. ChIP-on-chip, which locates protein binding sites that may help identify functional elements in the genome. For example, in the case of a transcription factor as a protein of interest, one can determine its transcription factor binding sites throughout the genome. DamID (DNA adenine methyltransferase identification): an alternative to ChIP-chip, transcription factor or chromatin-binding proteins of interest are fused to DNA adenine methyltransferase.

Answer 65

Microarrays are now widely applied to the study of human cancer, for delineating molecular subtypes, and for assessing disease progression and treatment response Breast - 70-gene prognostic signature (Mammaprint) developed on Agilent platform found to be strong predictor for metastasis-free survival - not yet used in diagnostic setting

Answer 66

RNA interference (RNAi) is a post-transcriptional method of gene silencing. The technique involves printing RNAi reagents onto a standard glass microarray slide, which is then placed in tissue culture dishes and cultured cells in medium are added to the arrays. Cells that adhere to the spots internalise the printed material and become transfected, leading to silencing of a specific gene, and the remaining cells form a non-transfected lawn between spots. Microarrays are then fixed and prepared for immunofluorescence, staining for DNA and F-actin, in situ hybridisation, apoptosis detection or other assays.

Answer 67

A copy number variant (CNV) is a loss (deletion) or gain (duplications and triplications) of genomic material relative to the reference genome. CNVs can be intragenic, typically deletions/duplications larger than 50bp, or intergenic, involving multiple genes.

Answer 68

Intergenic CNVs: The ACMG technical standards for the interpretation and reporting of constitutional copy-number variants- these guidelines are used for both rare disease and inherited cancer but not for acquired neoplasia Intragenic CNVs: ACGS Best Practice Guidelines for Variant Classification in Rare Disease 2023 and CanVIG-UK Consensus Specification for Cancer Susceptibility genes (CSGs) of ACGS Best Practice Guidelines for Variant Classification 2023. Acquired CNVs: The 2020 ACMG technical standards do not apply to acquired CNVs in neoplasia and there are currently no consensus guidelines adopted in the UK.

Answer 69

Population frequency: presence in unaffected individuals supports benign classification. E.g. Database of Genomic Variants (DGV), gnomAD, In-house laboratory databases Genomic Content of CNV: Includes known pathogenic or cancer associated genes. DECIPHER database, COSMIC, In-house databases, ClinVar. Professional guidelines including NICE, NCCN, WHO Size of CNVs- how many coding genes are involved. Haploinsufficiency vs triplosensitivity: Consider whether one allele sufficient for normal function or if an additional copy would change the function. Literature evidence: has this CNV been reported previously in an affected individual.

Answer 70

Haploinsufficiency is the most common mechanism of pathogenicity for heterozygous deletions where loss of one allele resulting in a single normal allele at a particular locus is inadequate for normal function resulting in a phenotype. Regions of copy number loss containing established HI genes are more likely to be pathogenic. TS is where an additional copy of a gene/genomic region results in a phenotype. Fewer genes appear to exhibit TS than HI and therefore copy number gains are more likely to result in a milder phenotype or to be benign than the reciprocal deletion.

Answer 71

Inherited breast and ovarian cancer: intragenic CNVs BRCA1; BRCA2; PALB2; ATM; CHEK2; RAD51C; RAD51D. Lynch syndrome: Intragenic CNVs in MLH1, MSH2, MSH6, PMS2 Usually tested for by NGS or MLPA

Answer 72

Myelodysplastic syndrome (MDS): genome wide screen of copy number changes including -7/del7q, -5/del5q, i(17q)/t(17p), -13/del13q, +8; +19. Usually tested for by G banding or SNP arrays Non small cell lung cancer (NSCLC): MET amplification. Usually tested for by FISH or NGS

Answer 73

Used historically as diagnostic pre-screening tool when DNA sequencing was labour intensive. This is now largely superseded by NGS and arrays and the scanning techniques below are no longer used routinely in diagnostic genetics laboratories.

Answer 74

Pyrosequencing is a sequencing method where pyrophosphate release during nucleotide incorporation generates a light signal that indicates the sequence.

Answer 75

DNA is labelled with biotin and amplified During sequencing, each addition of a dNTP releases a pyrophosphate which is coverted to ATP Luciferase uses ATP to produce light This is measured and produces a trace where the peaks are proportional to the number of nucleotides added

Answer 76

Pros include fewer steps and better detection limits (~5%). Cons include shorter read lengths (100-400 bp) and issues with homopolymers, potential for mutation masking due to suboptimal dispensation

Answer 77

Methylation analysis with bisulphite conversion (can be used for MLH1 promoter hypermethylation) Detecting KRAS mutations and quantifying mutant alleles, which helps in selecting patients for TKI therapy in NSCLC.

Answer 78

Electrophoresis is a technique used to separate DNA fragments according to size using an electric field running through a matrix. DNA is negatively charged, so it migrates through agarose gel to the positively charged anode.

Answer 79

PAGE is more sensitive than agarose gels and can resolve fragments less than 50 bp in length.

Answer 80

Capillary electrophoresis uses fluorescent tags to size DNA fragments within 1 bp of each other.

Answer 81

Capillary electropheresis It performs size fractionation and quantification of DNA, RNA, or protein samples using nanofluidics, electrophoresis, and flow cytometry

Answer 82

Pyrosequencing Combined Bisulphite Restriction Analysis (COBRA) Methylation-sensitive melt curve analysis MS-MLPA NGS Arrays

Answer 83

An epigenetic event that alters gene expression by the addition of a methyl group to the 5-carbon of cytosine in a CpG dinucleotide, catalyzed by DNA methyltransferase (DNMT).

Answer 84

DNA polymerase cannot distinguish between methylated and unmethylated cytosines during PCR, so DNA must be modified to preserve methylation information.

Answer 85

To convert unmethylated cytosines to uracil, allowing differentiation between methylated and unmethylated cytosines. Sodium bisulphite deaminates unmethylated cytosine to uracil The methyl group on 5-methylcytosine protects the amino group from the deamination.

Answer 86

Uses bisulphite coversion first Unmethylated regions will show C to T changes and methylated regions will retain C

Answer 87

The differences in sequence between methylated and unmethylated DNA after bisulphite modification can lead to the creation of new methylation-dependent restriction sites (e.g. conversion of CmCGA to the Taq I recognition site TCGA) or the maintenance of restriction sites in a methylation-dependent manner. The COBRA assay, which relies on the separation of digested PCR products followed by hybridisation of fluorescently labelled probes. Bisulfite conversion of non-methylated cytosines to uracils, Locus-specific PCR amplification of converted DNA, Restriction digestion, Analysis of restriction patterns on the gel, and the quantification of these restriction patterns using ImageJ or a similar program

Answer 88

not all sequence changes resulting from bisulphite modification result in the formation/abolition of a commercially available restriction enzyme site. Technique with restriction digestion post-PCR is prone to error due to the formation of heteroduplexes between the converted methylated and unmethylated DNA. This heteroduplex will not be cleaved by restriction enzyme.

Answer 89

RQ-PCR method for Methylation ommercial TaqMan assays using MethyLight technology are available for CDKN2A, ERBB2 and others Uses methylation insensitive TaqMan probes which target regions in between methylation-specific primer sites (2 different primer pairs are used in separate reactions, one specific for methylated and converted DNA, the other specific for unmethylated and converted DNA).

Answer 90

Bisulphite modification of DNA Whole-genome multiple displacement amplification via random hexamer priming and Φ29 DNA polymerase, which has a proofreading activity resulting in error rates 100 times lower than the Taq polymerase. Enzyme shearing. Hybridisation and single step extension Fluorescence staining and chip scanning Repeated round of staining of hapten-labelled dNTPS Scanning and data analysis

Answer 91

Illumina currently market The HumanMethylation27 BeadChip which allows researchers to interrogate 27,578 CpG loci, covering more than 14,000 genes. The HumanMethylation450 BeadChip offers coverage of >450,000 methylation sites, and is marketed as being high throughput and low price, therefore suitable for screening genome-wide association study (GWAS) populations, or tumour samples for tumour-wide methylation status.

Answer 92

Use of a methylation-sensitive restriction enzyme HhaI Two reactions: undigested for CNV and digested for methylation Hybrids of probes and unmethylated sample DNA are digested by the HhaI enzyme. Digested probes cannot be amplified during PCR and hence will not produce a signal during capillary electrophoresis. In contrast, if the sample DNA is methylated, the DNA-probe hybrids are protected against HhaI digestion and the ligated probes will generate a peak.

Answer 93

Long read sequencing: nanopore

Answer 94

Hypermethylation of the hMLH1 promoter, which results in transcriptional silencing of hMLH1 and is commonly seen in sporadic colon and endometrial cancers with microsatellite instability (MSI) is now routinely offered in diagnostic labs. routinely offered in diagnostic labs. Analysis of tumour DNA can aid discrimination between those individuals likely to have sporadic forms of colon and endometrial cancer and those likely to benefit from mutation screening of Lynch Syndrome genes.

Answer 95

Hypermethylation of the MGMT promoter is associated with chemosensitivity and is seen in a wide spectrum of tumours, including gliomas. Therefore, use of this kit can enable identification of those tumours likely to respond to treatment with alkylating agents

Answer 96

WHO 2022 includes methylation status in classification of CNS tumours DNA methylation has been shown to stratify meningiomas into methylation classes that more accurately than histopathology identify patients at high risk of recurrence. Molecular classification of meningiomas based on copy number variation, point mutations, methylation, and transcriptomic and proteomic data stands out as a future diagnostic work-up of meningiomas.

Answer 97

Important in lynch (reduces germline screening) and CNS and classification of many tumours Techniques can involve lab standard protocols such as PCR and may not require additional equipment Techniques can be flexible and designed for specific targets Some techniques such as RT-PCR can be used for a wide range of samples e.g. fresh, frozen, or formalin-fixed paraffin-embedded tissues and remote samples, such as serum, plasma, and urine

Answer 98

Techniques can be time-consuming, prone to contamination, and difficult to interpret in non-binary samples.

Answer 99

Real time quantitive PCR Patient RNA converted to cDNA with reverse transcriptase A fluorescently labelled probe for the region of interest has a quencher molecular and binds to the region on cDNA cDNA is amplified with taq polymerase, this digests the probe releasing the quencher This increases the fluorescence with each round of amplification This is measured in real time and the ct value is taken (the number of cycles taken to pass background fluorescence). This is directly proportional to the amount of template present ct value is then compared to standard curve of known concentrations for absolute quantification Normalised to housekeeping gene (ABL1)

Answer 100

Pair of fluorescent probes labelled at 5' and 3' When bound these probes are end-to-end allowing a transfer of energy from one fluorophore to the other PCR extension cleaves the probe and it is unable to transfer energy This allows detection during annealing or extension phase Perform melt curve analysis

Answer 101

qPCR (commonly Taqman) is used for monitoring MRD CML – qPCR of BCR-ABL1 transcript levels is useful for the early detection of relapse of CML. During treatment, European LeukaemiaNet (ELN) currently suggests BCR-ABL1 RQ-PCR every 3 months until a major molecular response is confirmed, then every 3-6 months (PB is sufficient). ALL – to monitor gene fusions (e.g. BCR-ABL1, MLL-AF4, TCF3(E2A)-PBX1, and ETV6-RUNX1), or if absent, a rearrangement of immunoglobulin or T-cell receptor genes resulting in unique molecular signature. AML – to monitor gene fusions (e.g. PML-RARA, RUNX1-RUNXT1 and CBFB-MYH11), mutations (e.g. NPM1) and transcripts that are commonly unregulated in AML, particularly WT1. The MRC AML15 trial showed sequential monitoring by RQ-PCR for the PML-RARA transcript is the strongest predictor of relapse in APML.

Answer 102

Sample divided to 20,000 tiny droplets, allows thousands of independent amplification events in a single sample. Each droplet contains either a positive (fluorescent) or negative (non-fluorescent) template. This digital counting directly translates to the absolute number of target molecules A Taqman mastermix is prepared with fluorescent probes, primers, and the template nucleic acid. A control signal (ROX) indicates the presence of DNA in the droplet, while target and reference signals are typically FAM and VIC. The quantity of the target can be calculated either absolutely or relative to a reference.

Answer 103

The exact number of fragments per partition is not known – there could be none or more than one fragment present in each partition. The Poisson distribution is the statistical probability of each partition containing 0, 1, 2, 3, 4+ fragments of DNA – this is used to correct and account for fragment number variability. The equation used for this correction is A=– loge(1–P) where A is average number of fragments per partition, and P is the proportion of positive partitions.

Answer 104

The number of positive partitions is compared to the number of reference partitions e.g. variant allele v wild type allele. The quantity of the reference is normally known before e.g. a diploid cell is known to have 2 copies. The Poisson correction equation is applied first, then the quantity is normalised to the reference quantity to give relative abundance.

Answer 105

High sensitivity- the presence of a single target molecule within a droplet is sufficient for detection Highly accurate Reduced Bias: minimizes bias inherent in traditional PCR methods, such as competition between target and reference sequences and challenges associated with determining Ct values. Low input volume required 

Answer 106

Cost: ddPCR instruments and consumables tend to be more expensive compared to conventional PCR setups. Throughput: Due to individual droplet analysis, ddPCR has a lower throughput Technical Expertise: ddPCR workflows can be more complex Ultra-sensitivity means contamination is easier. Uniformity of partition size and correct starting dilution of DNA are essential for accuracy. Can only detect a single variant in each assay.

Answer 107

JAK2 V617F in MPN EGFR Thr790Met (T790M) testing of ctDNA in relapsing lung cancer patients HER2 copy number in Breast Cancer Patients KIT Asp816Val (D816V) for suspected systemic mastocytosis BCR::ABL1 monitoring in CML - mainly RQ-PCR used as specified by guidelines

Answer 108

A solution containing the primary antibody is added to the tissue section which binds to target protein Unbound and surplus antibodies are washed away and the secondary antibody is added. The secondary antibody, which carries a linker molecule with horseradish peroxidase (HRP) enzymes binds to the primary antibody, followed by another washing step. After this, 3,3' Diaminobenzidine (DAB) is added. The HRP enzyme transforms the DAB substrate into a brownish precipitate that is deposited in the tissue at the site of the reaction, thus producing a visual representation of where the primary antibody first bound its target.

Answer 109

Tissues are typically fixed with formalin and embedded in paraffin wax (FFPE) to preserve the epitopes and morphology. Frozen tissue sections can also be used

Answer 110

Formalin can mask protein targets. This step helps "unmask" them for antibody binding. The most common method for antigen retrieval in FFPE samples is to pressure-boil the tissue slides in an acidic citrate buffer for around 15-20 minutes

Answer 111

Polyclonal: Diverse antibodies targeting various regions of the protein. sensitive but potentially less specific Monoclonal: Highly specific antibodies targeting a single protein region. can still generate results that are hard to interpret if the target epitope is present in low abundance.

Answer 112

Enables assessment of protein expression and localization. Well-established and widely used in pathology labs. Provides spatial information within the tissue context. Rapid and cheap

Answer 113

Limited to known protein markers. Does not directly detect genetic mutations.

Answer 114

Sanger sequencing gives high-quality sequence for long stretches of DNA (900 bp). Gold standard- often used to confirm NGS

Answer 115

Template DNA denatured and primers annealed This chain-termination method makes use of chemical analogues of the dNTPs called dideoxy nucleotides (ddNTPs). The ddNTPs lack a hydroxyl group (OH) on the 3’ carbon of the sugar ring needed to form the phosphodiester bond between one nucleotide and the next during DNA strand elongation. This inhibits further strand extension. The chain ends with the dideoxynucleotide, which is marked with a particular colour of dye/radioactive label depending on the base (A, T, C or G) that it carries. Exonuclease I (Exo) digests residual single-stranded DNA primers and any extraneous single-stranded DNA fragments produced during the PCR process. Shrimp Alkaline Phosphatase (SAP) dephosphorylates remaining dNTPs (free nucleotides) from the PCR product so they do not interfere with the sequencing reaction As a result, different DNA fragments of varying length are produced. These differently sized fragments can be size-fractionated on a polyacrylamide gel or by capillary electrophoresis.

Answer 116

A commonly used program is Mutation Surveyor. The software translates the raw data into the corresponding nucleotide bases, and also assigns a quality score to each base, which predicts the probability of a base call error (i.e. how reliable the call is for any given base). Forward sequence shows clear and distinct peaks.

Answer 117

Cost-efficient sequencing of single samples In some cases, analysis of longer fragments (~1,000 bp in length) Less error prone so can confirm NGS or fill in the gaps needed for NGS Less reliant on computational tools Requires more DNA and higher quality

Answer 118

SNPs under probes can prevent amplification Poor sequencing close primer Sensitivity limited to 15-20% Requires high DNA input Low throughput Only analyse 1 target

Answer 119

High throughput massive parallel sequencing

Answer 120

DNA/RNA extraction Library preparation and enrichment Sequencing by synthesis

Answer 121

Fragmentation: Break the DNA/RNA into smaller fragments 300-400bp. Methods: Mechanical shearing (e.g., sonication), enzymatic digestion, or tagmentation (transposon-mediated fragmentation and tagging). End Repair and A-tailing: Prepare DNA ends for adapter ligation. Enzymatic treatments to produce blunt ends, followed by the addition of an adenine (A) overhang. Adapter Ligation: Attach platform-specific adapters to the DNA fragments and indexes. Ligase enzyme to attach adapters, which contain sequences necessary for binding to the sequencing platform. PCR Amplification: Amplify the library to obtain sufficient quantities for sequencing

Answer 122

Hybrid Capture - Enrich specific regions of interest from the genome by uses biotin labelled probes specific to target regions and using magnetic beads to pull down the probes along with the sequence. This is then eluted Amplicon based - Amplify specific regions of interest using PCR and primers for specific regions

Answer 123

Hybrid - Highly specific and uniform as no amplification - Scalable for large panels Amplicon - highly specific - quick and cost effective - but potential amplification bias

Answer 124

Flow cell contains probes complimentary to the library adaptors which binds the library fragments. This is extended and bends over and binds to another probe- bridge amplification. This results in clusters all across the flow cell. Each dNTPs are fluorescently labelled and as it is added it is excited and scanned. The consensus fluorescence at each position in each cluster is used to determine the nucleotide

Answer 125

Uses amplicon based enrichment Flow cell contains millions of wells with semi conductors. Sequencing by synthesis- as each base dNTP is added to a H+ ion is released changing the pH. The semi conductors can determine the base at each position based on the change in pH- massive parallel sequencing

Answer 126

Targeted panels Whole Exome Sequencing Whole genome sequencing Whole transcriptome sequencing Epigenomics

Answer 127

Cost-effectiveness, unprecedented sequencing speed, high resolution (detect SNVs, CNVs, indels) Highly accurate (although sometimes confirmed with sanger) Analyse large numbers of genes at once High throughput

Answer 128

Massive volume of data generation - creates problems with data storage - creates large amount of data which requires interpretation Short reads so is unable to call large structural variants Accuracy reduces towards end of reads- phasing pseudogenes and repetitive sequences are difficult to sequence

Answer 129

The average number of times a base pair is sequenced

Answer 130

By providing confidence scores for each base call, allowing for quality filtering during data mining

Answer 131

High GC content reads can be favoured in sequence leading to non-uniform coverage Nonuniform coverage can leave portions of the genome undersequenced, making it difficult to identify SNPs, point mutations, or structural variants. Deeper sequencing can help to compensate for this limitation

Answer 132

To customize health management based on an individual's genetic profile, improving disease prevention, early detection, and treatment. NGS has helped improve this

Answer 133

An NHS initiative to decode the human genome in people with rare diseases and cancer to predict disease development, diagnose conditions, and identify treatments.

Answer 134

Identifies the most appropriate tests for each clinical indication and the testing methodology by which it should be delivered. Through expert panels including clinicians, scientists, health economists, policy experts, public representatives, and patient organizations.

Answer 135

The term generally given to technologies capable of sequencing single DNA molecules without amplification.  Also known as long-read sequencing (LRS). Removes the need for PCR-based amplification or the requirement to halt between read steps

Answer 136

Small amount of starting material Higher throughput –hundreds to millions of sequencing reactions can be carried out asynchronously. Lower projected cost per base Longer read lengths (10,000-100,000bp)- allowing enhanced de novo assembly; mapping certainty; haplotype detection; chromosome phasing; CNV detection; recognition of insertions, deletions, and translocations Sequencing of repetitive elements Provide discrimination between genes and pseudogenes. Produces more uniform coverage of the genome- less sensitive to GC content than SGS (dependent on tech). Potential to detect epigenetic modifications such as methylation. Redundant sequencing will result in higher consensus accuracy to enable rare variant detection and simplified data analysis. No amplification so less bias

Answer 137

Higher error rate per read; fewer reads per run; more expensive per base (more relevant to PacBio than ONT) limited standardisation raises challenges for use in clinical diagnostics.

Answer 138

PacBio Single molecule real time (SMRT) sequencing Oxford Nanopore FRET sequencing (Life Technology)

Answer 139

This method is based on monitoring polymerase activity while incorporating differently labelled nucleotides into the DNA strand. Each nucleotide carries a base-specific fluorescent label on its phosphate group, which is released when being incorporated by the polymerase. dNTP incorporation on each single molecule template per well is continuously visualised, with a laser and camera that records the colour and duration of emitted light DNA sequencing is performed on SMRT cells, which contain tens of thousands of ZMWs. SMRT technology utilises zero-mode waveguide (ZMW) – a hole measuring 30-70 nm in diameter, fabricated in 100nm thick metal film deposited on a silicon dioxide substrate. It can detect the activity of a single molecule amongst a background of thousands of labelled nucleotides

Answer 140

Fast – DNA polymerase continues to incorporate bases at a speed of tens per second. Circular consensus sequence- reduce error rates to from 1-5% to 0.1-0.5% (>Q30). Discriminate between methylated and unmethylated bases- The polymerase pauses for longer at modified vs. unmodified bases, which can be detected by measuring a metric called the interpulse duration.

Answer 141

Limited throughput: Limited number of ZMWs that can be read per SMRT cell. Also, longer molecules take longer to pass through the polymerase in the ZMW. Expensive ~$1000/Gb.

Answer 142

3rd gen sequencing ssDNA molecule is electrophoretically driven through a nanoscale pore (a very small hole through a thin membrane submerged in salt buffer solution). When an electrical potential is applied to solutions on either side of the pore, it drives dissolved salt ions through the pore, establishing electrical current. Molecules passing through the pore can measurably change the ionic current, and the amplitude and duration is determined by the molecule Due to the length of the pore the shift in voltage due to the presence of a DNA molecule is caused by the string of bases (called a k-mer) within the pore rather than by an individual nucleotide- this detects the composition of the DNA molecule

Answer 143

Can be biological or synthetic (synthetic are more stable but only biological have been used) Biological - α-hemolysin- This is an exotoxin secreted from Staphylococcus aureus. - MspA- mycobacterium smegmatis porin A channel is 1-1.2nm at its narrowest point. - Bacteriophage phi29.

Answer 144

There are more than 1000 possible signals instead of just 4– one for each k-mer (which is increased if modified bases are taken into account). This results in a large error rate of up to 30%, particularly with indels, and homopolymers longer than the k-mer length can be difficult to identify. Can also miss SNPs due to the speed that DNA passes through the pores

Answer 145

MinION - Portable - Max time 72 hours GridION - 5 flow cells PromethION - 48 flow cells

Answer 146

FRET fluorescence resonance energy transfer. DNA polymerase is tagged with a fluorophore that when brought into close proximity to a nucleotide, tagged with an acceptor fluorophore, would emit a fluorescence resonance energy transfer (FRET) signal. After incorporation, the fluorophore label on the nucleotide can be released gDNA is chemically attached to the coverslip of a standard TIRF-based (Total Internal Reflection) microscope. Universal primers are annealed to the gDNA. Nanoparticle-labelled DNA polymerase is added to the slide. Dye attachment to the nucleotide is via the terminal phosphate, released into solution after base-incorporation.

Answer 147

Mapping technologies determine the large-scale sequence structure of DNA without sequencing every base Irys system from BioNano Genomics Dovetail Genomics cHiCago protocol

Answer 148

Primary analysis – involves the alignment of the short reads of the sequencing data (FASTQ) to a reference sequence, with the resulting alignments typically stored in binary alignment/map (BAM) file format. Secondary analysis – is the calling of the somatic variants in the BAM files which creates an output as a variant calling file (VCF) Tertiary or downstream analysis – involves the interpretation and annotation of the somatic variants called in the VCF as well as identifying mutational signatures, tumour mutation burden (TMB), HRD, MSI

Answer 149

Tumour sample Germline sample- to determine which variants are somatic and which are germline Record of discussion (consent form) Referral form (TOF)

Answer 150

samples (tumour and normal) are not contaminated by DNA from another patient. sequencing data are of sufficient quality with uniform coverage, the DNA fragment has a length of ~400 bp, there is a high mapping rate of sequenced reads to the reference genome and there is a low number of chimeric fragments. tumour content (purity or cellularity) is above the limit of detection for somatic variants (>30%) tumour in normal contamination (TINC) level for haematological samples is low. High TINC can result in inappropriate subtraction of true somatic variants resulting in false negatives.

Answer 151

Interpretation of somatic mutations can be particularly challenging as some tumour types can carry tens or even hundreds of thousands of somatic mutations, yet only a small number justify clinical review. Therefore, variant prioritisation is essential, but requires careful design and implementation to ensure that clinically relevant variants are not missed. The virtual panel used is dependent on the sample type.

Answer 152

Domain 1: Variants in a virtual panel of potentially actionable genes related to tumour type are reported in domain 1 Domain 2: Variants in a virtual panel of cancer-related genes (536 genes, listed in the Cancer census genes) Domain 3: Small variants in genes not included in domains 1 & 2 are reported in domain 3.

Answer 153

Circus plot key: Track 1 (innermost track): chromosomes. Tracks 2 and 3: number of somatic single nucleotide variants (in red) and indels (in green) Track 4: ratio of normalised depth of coverage for tumour versus normal Copy number losses are indicated in red, CNVs indicated Track 5: absolute depth of coverage in tumour sample. Structural variants are indicated by arcs inside the plot

Answer 154

Tier 1: pathogenic or likely pathogenic variants conferring susceptibility to the relevant clinical indication using a tumour-type specific panel. For genes with a biallelic mode of inheritance, only homozygous or potential compound heterozygous variants are reported. Tier 3: Variants are prioritised to tier 3 using a broad gene panels spanning cancer susceptibility genes in addition to the tumour-type specific panel.

Answer 155

Every single variant in the genome is identified by one test – may reduce need for multiple tests of same sample in the future. High resolution of WGS- potential to replace SNP arrays and allows identification of the precise location of fusion breakpoints (in frame) Distinguish between somatic or germline variants. Identifies biomarkers with emerging clinical actionability (e.g. TMB, mutational signatures for HRD and MSI etc) Reanalysis for any targets

Answer 156

Lower read depth coverage compared with targeted NGS panels means reduced sensitivity so low-level variants could be missed. Tumour in normal contamination (TINC) - a risk of an increased number of false negative somatic variants as true somatic variants may be inappropriately subtracted in the analysis (most commonly observed in haematological cancers) Long TaT FFPE is not suitable for WGS due to poor quality, uses fresh frozen Germline sample difficult to ensure cancer free in HO conditions- needs to be post treatment blood or skin/saliva (dependant upon condition) Complex consent process

Answer 157

Does not replace standard of care all paediatric cancers, neurological and sarcomas and acute leukaemias presenting at any age,

Answer 158

cfDNA are fragmented DNA that have been released from cells and are circulating in the blood stream This is found in both healthy and cancer populations Cancer populations have a higher level of cfDNA in the blood due to the presence of circulating tumour DNA (ctDNA) which has been released from tumour cells Different tumours shed different amounts of ctDNA

Answer 159

It is not possible to be sure that a sample has ctDNA in it- limitation of the test Presence of cancer specific abnormalities may help determine It is thought that ctDNA tends to be shorter fragments than cfDNA which can be used to help determine ratio e.g. with tapestation

Answer 160

Tumour cells dying though necrosis and apoptosis release ctDNA from tumour cells DNA released from apoptosis is normally around 160-200bp and necrosis is larger Found to be poorer integrity than cfDNA

Answer 161

Normally extracted as cfDNA, not specifically ctDNA Extracted from plasma portion of EDTA whole blood sample which has been separated by centrifugation Number of extraction kits available e.g. QIAamp, cobas, genexus purification system Bead based methodologies such as MagMax are thought to favour short fragments and help select for ctDNA

Answer 162

Blood storage - If using EDTA tubes, ctDNA needs to be processed within 6 hours due to high clearance and degradation - Better to use cell stabilization tubes such as streck tubes- can stabilize for up to 7 days - No refrigeration- results in more cell lysis which will release gDNA and contaminate the ctDNA Patient history - different tumours shed different amounts - Smoking, diabetes, exercise can impact shedding

Answer 163

Requires highly sensitive test due to very low level variants Targeted PCR (small number of variants) - Roche cobas EGFR- 42 EGFR variants using RQ-PCR - Qiagen EGFR NGS (many potential targets) - Thermofisher OPA - Agilent - TSO500

Answer 164

Improved sensitivity/depth of coverage by he introduction of unique molecular identifiers (UMIs) 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

Answer 165

Not used widely and unlikely to completely replace tumour testing TD - NSCLC: EGFR (T790M) and ALK resistance variants - companion diagnostic for osimertinib if T790M detected - Future- ESR1 in breast for resistance hormone therapy FDA - colorectal for KRAS, BRAF and NRAS

Answer 166

Minimally invasive - for patients were tumour is inaccessible - allow repeat biopsy for monitoring - MRD and resistance variants Relatively cheap and quick - doesn't require tissue processing or going to pathology Accounts for tumour heterogeneity Could in theory allow earlier diagnosis of tumours in the future

Answer 167

Requires highly sensitive tests (0.5% VAF due to dlituion in cfDNA) - need high coverage test False negatives - Difficult to confirm ctDNA actually present and at what burden- may be below LOD False positives - 10% of actionable variants are thought to actually be CHIP - May also detect incidental germline findings Not gold standard - tissue is gold standard which has standardised processing - No standardised method for ctDNA extraction or processing - doesn't go to pathology so doesn't have additional tests e.g. morphology Requires storage in specific ctDNA tubes to prevent degradation- e.g. streck tube Can't confirm result is from that tumour - concurrent primaries or unknown HM

Answer 168

Circulating tumour cells (CTC’s) can be released into the bloodstream from the primary tumour and may result in metastasis into distant organs. Occur at very low concetrations and require enrichment to detect

Answer 169

Liquid Biopsy: CTCs can be detected in a blood sample, offering a non-invasive method for early cancer detection, potentially before tumors are visible through imaging. Prognostic value: The number of CTCs can correlate with prognosis. Higher CTC counts often indicate more aggressive disease Tracking Changes: Regular monitoring can provide real-time information on disease progression or response to treatment Personalized Therapy: Analyzing the genetic and molecular characteristics

Answer 170

Size-Based Filtration: Utilizes filters to separate CTCs from blood cells based on their larger size. Density Gradient Centrifugation: Isolates CTCs based on their different densities compared to other blood cells. Immunomagnetic Separation: Uses magnetic beads coated with antibodies specific to surface markers on CTCs (e.g., EpCAM) to capture and isolate them or beads specific to the depletion of other cells e.g. CD45+

Answer 171

Immunocytochemistry (ICC): Stains CTCs with fluorescent antibodies against specific markers (e.g., cytokeratins) and uses microscopy to identify them. Flow Cytometry: Labels CTCs with fluorescent antibodies and uses a flow cytometer to count and sort the cells. RT-PCR: Amplifies and detects specific RNA markers that are characteristic of CTCs. Next-Generation Sequencing (NGS): Analyzes the genetic material of CTCs for mutations and alterations, providing detailed molecular information. Microfluidic Devices: Use specialized chips with microchannels and antibodies to capture and isolate CTCs efficiently from blood samples.

Answer 172

The need to perform cellular lysis, which prevents cell counting The possibility of false positive results due to illegitimate gene transcription in non-tumour cells The amplification of cell free nucleic acids potentially present in blood The possibility of false positive results derived from the use of unspecific markers The presence in the blood of non-malignant epithelial cells, released for example after an invasive procedure The possibility of false negative results due to low PCR sensitivity connected to limited expression of the tumour marker or to the presence of PCR inhibitors SNPs affecting PCR primer binding

Answer 173

CellSearch System: The only FDA-approved method for CTC detection, combining immunomagnetic separation and immunofluorescent staining to identify and count CTCs. CTC-chip: an array of 78.000 microposts coated with anti-EpCAM antibodies. Whole blood is pumped through the chip and EpCAM positive cells are captured and detected by cameras recognising their morphology, their viability and the expression of tumour markers

Answer 174

Low Abundance: CTCs are rare in the bloodstream, making detection technically challenging. Heterogeneity: CTCs can be highly heterogeneous, complicating the detection and analysis. Standardization: Need for standardized methods and protocols for consistent and reliable results

Answer 175

Fixation with 10% neutral buffered formalin to preserve tissues Tissue processing Consists of three steps; 1) Dehydration with increasing conc of alcohol, 2) Clearing with xylene, 3) Infiltration (lasting between 8-12 hours) Tissue embedding into paraffin Sectioning with a microtome Deparrafinisation and rehyrdration - Xylene and ethanol to disolve parafine - Heat to melt parafin DNA/RNA extraction

Answer 176

Preserves tissues irreversibly in a stable and reproducible state, through chemical interactions between fixative and cellular components This prevents autolysis (digestion of cellular components by digestive enzymes) and putrefaction (digestion by bacterial enzymes) This retains the living state as much as possible and allow visualization through staining The most widely practiced methods for clinical sample preservation and archiving. Allows storage of samples for years

Answer 177

Fixation Extraction method Fragmentation Deamination artefacts Tumour heterogeneity Presence of normal tissue

Answer 178

Detrimental to testing Fragmentation Deamination artefacts Formaldehyde-induced crosslinks

Answer 179

- lead to low amounts of amplifiable template for PCR amplification and increases sequence artefacts - Associated with poor coverage - low pH formalin over time increases the rate of apurinic/apyrimidinic site formation and eventually decomposition and fragmentation

Answer 180

- Hydrolytic deamination of cytosine to form uracil (or thymine if the cytosine is methylated). This results in non-reproducible C>T/G>A transition sequencing artefacts - Occur to some degree in every sample - Can result in false positives

Answer 181

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).

Answer 182

Formaldehyde is readily oxidized to formic acid in the reaction with atmospheric oxygen. The formation of formic acid reduces the pH of formalin The N-glycosidic bonds of the purine bases to the sugar backbone are susceptible to hydrolysis at low pH, generating abasic sites in the DNA. Thus, fixation of tissues in unbuffered formalin will significantly lower the amount of amplifiable DNA templates. DNA polymerases have generally low bypass efficiencies and can lead to sequencing artefacts (SNVs and deletions)

Answer 183

Presence of normal tissue will dilute the tumour DNA down and mean that variants are present at a lower frequency. This may push them below the limit of detection for an assay Tumour content requirements vary between assays- often 20-30% for detection of variants at 5% VAF

Answer 184

Fresh frozen tissue (requires institutional change) Cell free tumour DNA (liquid biopsy).

Answer 185

No- often overestimates as lots of the DNA will be fragmented and will not be amplified

Answer 186

Base Excision Repair removes damaged bases that do not significantly alter the overall structure of DNA, Nucleotide Excision Repair: can recognise and repair the bulky, structurally altering ssDNA lesions Mis-Match Repair: functions as a method of proofreading after DNA replication to catch any mismatched base pairs Non-Homologous End Joining: a form of repair template-independent repair and thus is an error-prone mechanism Homologous Recombination Repair: is considered to be an error-free method of DSB repair .

Answer 187

Present in ovarian 50%, breast 15%, pancreatic 8%, and prostate cancer 5%. Predictive biomarker for repsonse to platinum based chemotherapy Predictive biomarker for PARP inhibitors: exploiting and inducing cancer-specific synthetic lethality.

Answer 188

BRCA1/2 - Germline or somatic BRCA-like tumours - ARID1A, ATM, PALB2, RAD51, RAD54, ATR - Mutations in other HR genes

Answer 189

PARP inhibitors block PARP enzyme which is essential for base excision repair, leadinf tot he accumulation of DNA breaks Synthetic Lethality: In HRD-positive cells, the inability to repair single-strand breaks (due to PARP inhibition) leads to the formation of double-strand breaks, which these cells cannot efficiently repair due to their deficient HR pathway. This results in cell death, selectively targeting HRD-positive cancer cells.

Answer 190

Restoring BRCA 1/2 activity. Recently BRCA1/2, RAD51C/D and PALB2 reversions or secondary mutations have been described to re-store homologous recombination.

Answer 191

BRCA, HRD variants Genomic scarring (panels, WGS, myriad) - Looks for the consequence rather than the cause - The level of chromsomal abnormlaities correlates with HRD status - Loss of heterozygosity (LOH)- regions of intermediate size >15MB and

Answer 192

Mutational signatures are patterns of somatic passenger mutations that arise during tumorigenesis and can provide insights into the underlying causes of individual cancers. HRD predominantly manifests as small indels and genome rearrangements due to abnormal double strand break repair but also in the form of this base substitution signature. Single-base substitution Signature 3 (SBS3): SBS3 is strongly associated with germline and somatic BRCA1 and BRCA2 mutations and BRCA1 promoter methylation- can predict HRD

Answer 193

Resistance pathways would not remove the existing “scar” of prior HR. FFPE may overestimate HRD Most HRD assays have not been validated in a clinical trial of PARP inhibition now myriad not widely used Lack of standaridation of assays and HRD thresholds/ guidelines A negative HRD score does not exclude HRD Tumour heterogeneity

Answer 194

‘driver’ mutations -confer growth and survival advantage to the cell, promote oncogenesis. Most are ‘passenger’ mutations- do not confer growth advantage but accumulate as a consequence of inherent genomic instability of cancer cells.

Answer 195

External environmental exposures e.g. smokers with lung cancer Internal biological processes e.g. defective DNA repair

Answer 196

1. Single Base Substitution (SBS) Signatures- SNVs 2. Doublet Base Substitution (DBS) Signatures- concurrent modification of two consecutive nucleotides 3. ID Signatures-Small insertions and deletions (ID) Signatures- are defined as the incorporation or loss of small fragments of DNA (usually between 1 and 50 base pairs) in a specific genomic location. 4. Copy Number Variations (CN) Signatures 5. Structural Variations (SV) 6. RNA Single Base Substitution (RNA-SBS)

Answer 197

Sequencing - NGS or WGS Classification of signature with BI - Signature Extraction Algorithms: Tools such as MutationalPatterns, SigProfiler, and DeconstructSigs analyze sequencing data to extract and identify mutational signatures. - COSMIC provides a reference set of mutational signatures identified in different cancer types.

Answer 198

Tissue availability/low tumour purity: impacts the ability to detect somatic sequence alterations near the limit of detection Need germline sample to filter our variants Artifacts: from tissue preservation, sequencing, oxidative damage and formalin artifacts Poor validation of mutational signatures and clinical based correlations Lack of standardised reporting. Some mutational signatures exhibit overlapping features e.g colorectal cancers arising from MUTYH-associated polyposis exhibit an enrichment of C>A transversions, and the resulting mutational signature shares features with the mutational signature associated with tobacco smoke.

Answer 199

At present, largely used in research setting, only available through WGS . Diagnosis: in CUP e.g. in melanoma Assessment of HRD – PARPi indications if HRD identified in ovarian Assessment of MMR deficiency - Immunotherapy: use in guiding treatment options, since MMR-deficient tumours demonstrate increased sensitivity to immune checkpoint inhibitors. - Screening tool for Lynch syndrome Public Health: Identifying mutational signatures from prior exposures from environmental toxins (such as smoking and UV light) can help quantifying the relative cancer risk of potential genotoxins.

Answer 200

Minimal residual disease (MRD) - low level cancerous cells that remain following treatment, and are only detectable by highly sensitive techniques.

Answer 201

High-resolution determination of efficacy of therapy To 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 SCT in those with low risk of relapse Assignment to maintenance therapy after completion of standard treatment

Answer 202

FISH - Low sensitivity RQ-PCR - Real time measuring of fusion transcripts Fragment analysis for tandem duplications - Used for FLT3-ITD, can use allelic ratio Flow cytometry - monitoring of leukaemic associated immunophenotype (LAIP) QF-PCR - highly polymorphic markers is used to detect chimeris

Answer 203

Flow - LAIP - Can relaspe with different immunophenotype RQ-PCR - IG/TCR clonal rearrangments - gene fusions such as BCR-ABL1

Answer 204

RQ-PCR - Fusion genes and NPM1 Fragment analysis - FLT3- ITD status Flow - LAIP WT1 - WT1 is overexpressed at the mRNA level in 80–90% of AML cases - ELN found that reduction is an independant prognostic factor

Answer 205

RQ-PCR - BCR-ABL1 FISH

Answer 206

RC-PCR - Ig/TCR clonal rearrangmenets - Gene fusions e.g. IGH-BCL2

Answer 207

Sex mismatched BMT: XY FISH, Amelogenin-XY marker on PCR. Sex matched BMT: PCR using short tandem repeat microsatellite markers.