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Question 1

What genes have been linked with hereditary breast and ovarian cancer?

Answer 1

There are two primary genes linked with the majority of HBOC families; BRCA1 and BRCA2. There are also other genes which have been linked to an increased risk of developing breast and other cancers including TP53, PTEN, CDH1, ATM, CHEK2 or PALB2. The genes currently tested as part of the national test directory are BRCA1, BRCA2, PALB2, ATM and CHEK2

Question 2

What is the inheritance pattern of BRCA1 and BRCA2 germline mutations and associated familial risks?

Answer 2

HBOC follows an autosomal dominant pattern of inheritance. Therefore a mutation need only occur in one copy of the gene to have an increased risk of cancer. Parents may pass along a copy of the gene with a mutation with the child having a 50% chance of inheriting the change. A sibling or parent will also have 50% chance of inheriting the mutation, however if the parents test negative for the mutation, the siblings risk will significantly decrease but still may be higher than an average risk.

Question 3

What is the incidence of hereditary breast and ovarian cancer?

Answer 3

Most breast and ovarian cancers are sporadic. Currently it is estimated that <1% of the general population have a mutation in BRCA1 or BRCA2 and up to 10% of women and 20% of men who develop breast or ovarian cancer will have a mutation in one of these genes. 10-30% of women younger than 60 diagnosed with triple negative breast cancer, cancer which are negative for HER2, progesterone and oestrogen, have a BRCA1 or BRCA2 mutation. Others may have mutations in other associated genes.

Question 4

Which sub-population has an increased risk of hereditary breast and ovarian cancer and why?

Answer 4

Families with Ashkenazi Jewish ancestry have an increase chance through the occurance of three ‘founder mutations’; 185delAG in BRCA1, 5382insC in BRCA1 and 6174delT in BRCA2. It is estimated that 1 in 40 people of Ashkenazi Jewish ancestry will have one of these three mutations.

Question 5

What is the estimated risk of breast and ovarian cancer for women with a BRCA1 or BRCA2 germline mutation?

Answer 5

Cancer risks for women with HBOC:
• Lifetime risk of breast cancer – 45-75%
• Lifetime risk of ovarian cancer – BRCA1 25-40%, BRCA2 10-20%
• Developing a second breast cancer – 20-40%

Question 6

What is the estimated risk of breast and ovarian cancer for men with a BRCA1 or BRCA2 germline mutation?

Answer 6

• Lifetime risk of breast cancer – BRCA1 1-2%, BRCA2 6%
• Risk of prostate cancer – BRCA1 some increased risk, BRCA2 – 20%
• Men with a BRCA2 mutation have significantly increase risk of developing more aggressive prostate cancer before the age of 65 and therefore screening should begin at age 40

Question 7

What are the clinical features of neurofibromatosis type 1?

Answer 7

Multiple light brown patches of skin pigments (café au lait spots), skinfold freckling, visible neurofibromas under the skin and small modules in the iris

Question 8

What type of mutations in NF1 cause neurofibromatosis type 1?

Answer 8

Point mutations are responsible for 90% of NF1 patients with a single exon or whole NF1 gene deletion associated with the remaining 5-7%.

Question 9

What type of mutations in NF1 cause neurofibromatosis type 1?

Answer 9

Point mutations are responsible for 90% of NF1 patients with a single exon or whole NF1 gene deletion associated with the remaining 5-7%.

Question 10

What are the clinical features of neurofibromatosis type 2?

Answer 10

NF2 patients typically develop schwannomas on the bilateral vestibular portion of the eight cranial nerve and on other cranial nerves, spinal roots or peripheral nerves. In addition NF2 patients often develop multiple meningiomas and ependymomas at an early age. NF2 patients often experience hearing loss, balance problems, flesh coloured skin flap and muscle wasting. Visual impairment is likely due to cataracts, optic nerve meningiomas and retinal hamartomas.

Question 11

What is the most frequent type of mutation in NF2 which causes neurofibromatosis type 2?

Answer 11

Truncating mutations are the most frequent germline event and cause the most severe disease. The presence of truncated protein is also associated with a younger age at diagnosis and a higher prevalence of meningiomas, spinal tumours and cranial nerve tumours.

Question 12

Selumetinib is what type of inhibitor and what is it used to treat?

Answer 12

Selumetinib is a MEK inhibitor which is approved by NICE for NF1 for treating symptomatic and inoperable plexiform neurofibromas (PN) associated with type 1 neurofibromatosis (NF1) in children aged 3 and over.

Question 13

What drug can cause toxicity in patients with TPMT mutations?

Answer 13

Mercaptopurine, Azathioprine and Thioguanine.
mercaptopurine and azathioprine are generally used for non-malignant immunogenic disorders and thioguanine for myeloid leukaemias.

Question 14

What is the frequency of TPMT mutations causing TMPT deficiency?

Answer 14

• 1%

Question 15

What are the four TPMT deficiency alleles which account for 95% of TMPT deficiency cases?

Answer 15

o TPMT 2* (238G>C) – 100x decrease in activity (very low)
o TPMT *3A (contains two SNPs, *3B and *3C) – No detectable enzyme activity
o TPMT *3B (460G>A) – 4x decrease in activity (very low)
o TPMT *3C (719A>G)) – 1.4x decrease in activity

Question 16

What starting does alterations for azathioprine are carried out according to TPMT testing?

Answer 16

o Homozygous wild-type – Normal dose
o Heterozygous – one functional and one non-functional – reduce dose by 30-70%
o Homozygous / Compound het – Two non functional alleles – Consider alternative treatments for non-malignancy. For malignancy start at a drastically reduced dose.

Question 17

What type of treatment does DPD efficiency cause toxicity to?

Answer 17

Treatment with fluoropyrimidines - 5-fluorouracil (5FU) and capecitabine

Question 18

What does the dihydropyrimadine dehydrogenase (DPD) enzyme do and what happens when DPD is deficient?

Answer 18

DPD is the first step and rate limiting enzyme for the catabolism of fluoropyrimidines. Reduced DPD activity results in reduced clearance and increased half life of fluoropyrimidines (5FU and capecitabine) and can cause profound dose related toxicities.

Question 19

What are the four clinically relevant DPYD variants and their associated impact on DPD function?

Answer 19

o c.1905+1G>A, activity 0
o c.1679T>G, activity 0
o c.2846A>T, activity 0.5
o c.1129-5923C>G (c.1236G>A), activity 0.5

Question 20

What variant is in perfect linkage disequilibrium with the DPYD c.1129-5923C>G variant?

Answer 20

The synonymous variant c.1236G>A
This can be used as a proxy when testing

Question 21

What percentage of the European population is estimated to have a clinically relevance DPYD variant?

Answer 21

7%

Question 22

What are the proven biomarkers for response to immunotherapy?

Answer 22

High Tumour Mutational Burden
MSI High / dMMR
POLE or POLD1 mutation
PD-L1 expression on IHC

Question 23

Why is PD-L1 expression a predictor of immunotherapy response?

Answer 23

PD-L1 is a protein which helps to keep immune cells from attacking non harmful cells in the body. Some cancers have high amounts of PD-L1 which allows the cancer to ‘trick’ the immune system and avoid being attacked as a foreign, harmful substance.

Question 24

What is tumour mutational burden (TMB)?

Answer 24

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 25

What tumour features can impact TMB calculations?

Answer 25

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

Question 26

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

Answer 26

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 27

How do immune checkpoint inhibitors work?

Answer 27

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 28

Why is TMB a marker for ICI response?

Answer 28

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

Question 29

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

Answer 29

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

Question 30

What methods can be used to detect TMB?

Answer 30

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 31

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

Answer 31

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 32

What are the two indicators of a homologous recombination deficient tumour?

Answer 32

1) Mutation in a HRD gene (e.g. BRCA1/2, RAD51C)
2) Identification of an HRD signature (e.g. HRD Scar, Genomic Instability Score, COSMIC Sig 3)

Question 33

What types of variants are the feature of an HRD scar?

Answer 33

Loss of heterozygosity (LOH)
Telomere allelic imbalance (TAI)
Large Scale State Transitions (LST)

Question 34

What is telomeric allelic imbalance?

Answer 34

Telomeric allelic imbalance, is defined as the number of regions with allelic imbalance extending to the sub telomere but not crossing the centromere

Question 35

What is a large scale state transition?

Answer 35

Chromosomal breakage that generates 10 Mb or larger fragments. The quantification of these breaks can be used as a surrogate measure for genomic instability

Question 36

What is the main limitation of genomic scar assays?

Answer 36

A major limitation of the genomic scar assays is the potential lack of representing of the current HRD status when analysing archival tissue. Tumour cells previously shown to be HR deficient may have restored their proficiency by resistance mechanisms such as reverse mutations in HRD genes.

Question 37

What techniques can be used to detect HRD?

Answer 37

• Single nucleotide polymorphism (SNP) arrays
• Whole Genome Sequencing
• Targeted Panel Sequencing

Question 38

What are the names of some bioinformatics tools which can be used to generate an HRD score form WGS?

Answer 38

HRDetect and scarHRD

Question 39

What test method is approved in the UK for detecting HRD and what does it test?

Answer 39

Myriad myChoice assay
- Tests BRCA1/2 for likely pathogenic/pathogenic variants
- Generated a genomic instability (GIS) score from loss of heterozygosity, telomere allelic imbalance and large scale state transitions. a GIS >= 42 or BRCA1/2 +ve is considered positive for HRD

Question 40

What is the main difficulty in implementing HRD scoring in clinical practice?

Answer 40

Scores which are used between different bioinformatics tools or assays are not comparable which makes validation and threshold determination difficult.

Question 41

How is deparaffinisation of FFPE samples carried out?

Answer 41

The solvent xylene is typically used to remove all paraffin from the tissue sections attached to the slides/curls. Prior to deparaffinisation, slides are heated to melt the wax, they are then washed multiple times with xylene to solubilise and remove the paraffin. Next xylene is removed by washed with xylene and ethanol. Finally the sample is rehydrated with graded concentrations of ethanol in water ending in a final rise of pure water. Xylene is hazardous and non-hazardous alternatives are available to de-wax the sample.

Question 42

What are the four types of damage caused by formalin fixation?

Answer 42

Formaldehyde induced crosslinks
DNA fragmentation
Abasic Sites
Deamination of cytosine bases

Question 43

What is the basic procedure for DNA extraction and the enzymes involved?

Answer 43

Basic Procedure:
1) Cell lysis to break the cell membranes and expose DNA along with the cytoplasm
2) Lipids from cell membranes broken down with detergents and surfactants
3) Proteins broken down with protease (optional) using proteinase K
4) RNA broken down with RNAse (optional)
5) Solution treated with a concentrated salt solution (saline) to make debris such a broken proteins, lipids and RNA clump together.
6) Centrifugation of the solution to separate clumped debris from the DNA
7) DNA purification from detergents and debris. Most commonly used is
a. Ethanol precipitation usually by ice cold ethanol or isopropanol. Since DNA is insoluble in these alcohols It will aggregate together and form a pellet on centrifugation.
b. Phenol-choloform extraction in which phenol denatures proteins in the same.
8) DNA is precipitated in a slightly alkaline buffer (usually a TE buffer)

Question 44

What are the two methods for DNA quantification and name associated commercial example?

Answer 44

Spectrophotometry - NanoDrop Spectrophotometer (Thermo Fisher)
Fluorometry - Quantus (Progema)

Question 45

How is spectrometry used to evaluate DNA concentration and quality?

Answer 45

DNA concentration can be determined by measuring the absorbance at 260nm(A260) in a spectrophotometer using a quartz cuvette. The ratio of readings at 260nm and 280nm provides an estimate of the DNA purity with respect to contaminants that absorb light.

Question 46

How is fluorometry used to evaluate DNA concentration?

Answer 46

Fluorometry allows specific and sensitive measurement (more than spectrophotometry) of DNA concentration by use of a florescent dye, a common dye is Pico Green. The fluorescently labelled DNA can then be run on an agarose gel alongside a DNA ‘ladder’ consisting of known DNA quantities (e.g. Agarose gel electrophoresis or capillary gel electrophoresis). The amount of DNA can then be estimated by comparison of the band intensity with the standards either visually or using a scanner or imaging system.

Question 47

Pathogenic mutations in which genes are causative of lynch syndrome and what is the function of this group of genes?

Answer 47

MLH1, MSH2, MSH6 and PMS2

DNA mismatch repair genes (MMR)

Question 48

What are the main barriers to introducing a pharmacogenomics service?

Answer 48

• Clinically urgent
• High volume of samples
• Clinical Relevance

Question 49

What is the name for the DPD enzyme and what is its function?

Answer 49

Dehydropyrimidine hydrogenase (DPD) is an enzyme which catabolises thymine and uracil. DPD catalyses the first step In the 5-FU degradation pathway, converting around 80% of 5-FU to its inactive metabolite.

Question 50

How does 5-FU (& capecitobine) work?

Answer 50

• 5-FU works as an antimetabolite, blocking the enzyme thymidylate synthase and therefore preventing the thymidine formation required for DNA synthesis and thereby preventing cell proliferation

Question 51

What are the clinical considerations for germline focussed tumour analysis?

Answer 51

On tumour / Off tumour
Actionability (penetrance/risk, severity, preventative measures, strength of evidence)
Consent & Patient Education - Explicit consent is not routine, requested at dif stages of the pathway, counselling & confirmation)

Question 52

What are the lab considerations for germline focussed tumour analysis?

Answer 52

Tumour heterogenity / contamination (VAFS)
Analysis (ACMG))