session 6: cancer genetics & treatment Flashcards

Question 1

Q

what is AML?

Answer

A

clonal expansion of myeloid progenitors (blasts) in the peripheral blood (PB), bone marrow (BM) or other tissue.

Question 2

Q

how is AML diagnosed clinically?

Answer

A

at least 20% blasts are present in PB or BM UNLESS:
- molecular diagnosis
- myeloid sarcoma present - tumour mass consisting of blast cells
- erythrocyte leukaemia

Question 3

Q

what is breast cancer?

Answer

A

Heterogeneous group of neoplasms mostly arising from epithelial cells lining milk ducts. most common cancer in women (30% of new cancer cases). 1/8 risk. risk factors include, weight, age, genetics, alcohol and HRT but family history is strongest. 20% of cases are familial

Question 4

Q

Hereditary breast/ovarian cancer due to BRCA1 or BRCA2 pathogenic variants is suspected if?

Answer

A

early onset <50 years
2 or more breast primaries
both breast & ovarian cancer in single person
BrCa in close relatives from same side
at-risk populations eg. ashkenazi jewish
family member with confirmed mutation
male breast cancer
ovariant cancer at any age

Question 5

Q

what guidelines are there for BrCa diagnosis?

Answer

A

probability models eg. BOADICEA, Myriad II).
NICE 2013 lowered prior BC risk from 20 to 10% to access genetic testing meaning referrals of unaffecteds has increased
NICE 2018 recommends BRCA1 &2 testing for all <50 with TNC regardless of family history

Question 6

Q

what complications are there to clinical diagnosis of BrCa?

Answer

A

incomplete penetrance, sporadic cancer and phenocopies

Question 7

Q

what is HBOC syndrome ?

Answer

A

increased risk of early onset BR and Ov, pancreatic, prostate Ca and melanoma(BRCA2 only)
up to 7% of BrCa cases
incomplete penetrance - up to 87% risk of breast and 63 % risk of ovarian, 20% prostate, 7% pancreatic
BRCA1 accounts for 66% of cases and BRCA2 accounts for 34%

Question 8

Q

what is the role of BRCA1and 2 proteins?

Answer

A

DNA repair including homologous recombination repair of ds-breaks and nucleotide excision repair.
- BRCA1 forms complex with BARD1 and colocalises with BRCA2 and RAD51 at DNA damage site and BRCA2/RAD51 mediate homologous recombination
- BRCA1 also involved in cellular pathways controlling cell cycle progression and check-point control, gene transcription and regulation

Question 9

Q

describe the mutation spectrum of BRCA1 and 2 genes?

Answer

A

majority are LOF in coding regions of both genes
20% are VUS
10% are large rearrangements
founder mutations eg. 1.40 ashkenazi jewish have one of the three founder mutations

Question 10

Q

what is the testing strategy for BRCA1/2 testing?

Answer

A

NGS sequence anlaysis for all coding exons and intron boundaries
dosage analysis
targeted sanger for founder mutations
sanger for familial testing
NGS screen for unaffecteds where there is no DNA from affected family member
may test FFPE from affected if no other sample available but sensitivity may differ
VUS - segregation and tumour DNA from FFPE can be examined for LOH (WT allele deleted increases pathogenicity)

Question 11

Q

what ethical issues should be considered with BRCA testing?

Answer

A

counselling for diagnostic and predictive testing
PNT and minor testing not usually offered
age of onset and severity very variable - make sure patient understands
family member may not share result. also a family members result may be inferred through testing other family members

Question 12

Q

what treatments are available for BRCA?

Answer

A

surgery, chemo, radiotherapy, endocrine therapy, targeted drugs: Herceptin for HER2+ or tamoxifen for ER+
mastectomy reduces BC risk by 90%
oopherectomy reduces ovarian cancer risk by 53%
Tamoxifen used for ER+ BC. 25% of BRCA1 and 80% of BRCA2 breast cancers are oestrogen-receptor + . Drug reduces risk of BC by up to 50%
PARP inhibitors

Question 13

Q

how do PARP inhibitors work?

Answer

A

Parp is an enzyme that repairs ss DNA breaks by base excision repair. In BRCA null cells, double stranded breaks are not repaired and PARP inhibition leads to cell apoptosis

Question 14

Q

what surveillance strategies are there for BRCA families?

Answer

A

self-examination, clinical examination, mammography and MRI,

Question 15

Q

which cancer predisposition syndromes give an increased risk of breast cancer ?

Answer

A

Li-Fraumeni syndrome (LFS) -TP53 60% risk of BC by age 45
· Cowden Syndrome PTEN - increased risk of malignant tumours including BC
· Neurofibromatosis type I - NF1 - moderately increased risk

Question 16

Q

which genes can give rise to predisposition to familial breast cancer?

Answer

A

ATM - · Ataxia telangiectasia (AR) and heterozygous pathgenic variants cause 52% BC risk
CHEK2 - Li-Fraumeni type 2 but c.1100delC in particular has been associated with an 25-39% risk of breast cancer
PALB2 - 45% risk of BC, increased risk of prostate cancer. biallelic variants cause Fanconi anaemia
RAD50, BARD1
SNPs have been identified which each have small risk but PRS can be calculated for associated SNPs which could have significant impact on BC risk

Question 17

Q

what percentage of CRC are sporadic and what % are inherited?

Answer

A

85% are sporadic
15% inherited (3-5% lynch syndrome, >1% FAP and >10% other inherited cancer)

Question 18

Q

what causes lynch syndrome

Answer

A

AD mutation in a mismatch repair gene MLH1, MSH2, MSH6, PMS2 and EPCAM followed by secondary somatic loss of remaining copy of gene (LOH)
1/250 affected and gene dependent & age-related penetrance with variable expressivity.

mean age of onset is 45 years but higher in MSH6 and PMS2 cases and frequently non-penetrant (mutation frequencies for these likely to be higher as often missed)
cumulative Incidences of cancer (up to 70):
MLH1 (GI cancers) and MSH2 (greater variety of cancers) = 72%
MSH6= 54%
PMS2 = 18%

Question 19

Q

what % of germline mutations are found in MLH1 and MSH2, MSH6, PMS2 and 3’ deletions in EPCAM upsteam of MSH2? what other types of mutations account for lynch syndrome?

Answer

A

MLH1 and MSH2 = 90%
MSH6 = 10%
PMS2 (&PMS1) = <1%
EPCAM 3’ dels upstream of MSH2 = 3%

creating EPCAM-MSH2 fusion transcripts resulting in epigenetic hypermethylation of the MSH2 promoter and loss of MSH2 expression
- 10Mb inversion on 2p disrupts MSH2 and is cause of unexplained lynch
- LINE-1-mediated retrotranspositional insertion in PMS2 not identified by MLPA and sanger
- germline methylation of MLP1 promoter is heritable cause of lynch

Question 20

Q

what is the testing strategy for lynch syndrome?

Answer

A

Amsterdam & Bethesda criteria (more sensitive less specific)
FFPE testing for IHC and MSI prior to mutation screening
MLH1 pm
BRAF mutation (V600E)
Germline testing

Question 21

Q

how is IHC used for lynch testing?

Answer

A

antibodies test for presence/absense of MLH1 MSH2 MSH6 PMS2 proteins
95% senseitive for DNA MMR deficiency
concurrent loss of MLH1-PMS2 or MSH2 and MSH6 (heterodimers)
If IHC shows protein loss we do not test for MSI as we expect there to be MSI
loss of MLH1/PMS2 > MLH1pm studies on DNA extracted from tumour before mutation testing
V600E done in combination with MLH1pm as this combo is stronger indication that the tumour is sporadic
patients with normal IHC undergo MSI as may be missense mutation that results in a non-functional but present protein (drawback of IHC) (5% of cases)
• Loss of MSH2 function manifests as absent IHC expression of MSH2 and MSH6
• Loss of MLH1 function (deleterious mutation or promoter hypermethylation) is detectable as absent IHC expression of MLH1 and PMS2
• Isolated absence of MSH6 or PMS2 protein suggests mutation in the respective gene
• LS-MLH1 type is frequently caused by missense mutations – resulting in altered, non-functional protein, but the mutant protein may be expressed and retain its immunoreactivity. Therefore MSI-H with normal or weak expression of MLH1 in association with loss of PMS2 protein could be due to an inactivating mutation in PMS2, or a missense mutation in MLH1

Question 22

Q

how is MSI used for lynch testing?

Answer

A

DNA extracted from tumour tissue and tested for length alterations to microsatellites
tests 5 microsatellite markers
2 or more altered markers between tumour and germline are MSI-H - indicates MMR gene defect
MSS = not MMR defect
1/5 warrants further investigation
may have tissue mosaicism

Question 23

Q

how is MLH1 pm used for lynch testing?

Answer

A

• Hypermethylation of the MLH1 promoter has been shown in a high proportion of sporadic cancers (approx. 15%). Results in absence of MLH1 protein on IHC
- any IHC showing loss of MLH1 need MS-MLPA for MLH1pm. The kit tests for abnormal methylation at 5 sites in the MLH1 promoter, 4 sites in MSH2 promotor (indicative of 3’EPCAM deletions) and other MMR promoter regions
- samples with no abnormal methylation should be tested for germline MLH1 mutation
- hypermathylation is a somatic change in tumour but has rarely been seen in blood as a heritable mutation so blood can be tested at same time as tumour

Question 24

Q

how is BRAF (V600E) testing used in lynch syndrome?

Answer

A

associated with MLH1 methylation = sporadic cancer
V600E occurs in non MSI-high tumours and used as a screen to avoid unnecessary MMR gene screening
BUT also occurs in MMR germline mutation carriers at 1% frequency. SO it is done at same time as MLH1pm as a stronger indicator that tumour is sporadic
-• Constitutional MLH1 promoter hypermethylation is not associated with V600E

Question 25

Q

how is germline testing used in lynch syndrome?

Answer

A

NGS panel for MMR gene sequencing and dosage. PMS2 is challenging due to pseudogene. The 3’ end of the gene is non-amenable to NGS analysis and required long-range nested PCR to amplify PMS2 only
large rearrangements detected by NGS dosage, MLPA or array

Question 26

Q

how do EPCAM dels silence MSH2?

Answer

A

EPCAM dels account for 2% of lynch cases. dels of 3’ EPCAM are associated with methylation of MSH2. Dels may extend into MSH2 promotor or 5’ coding region and result in loss of the EPCAM termination codon and 3’ UTR causing transcriptional read-through from EPCAM into MSH2. smaller dels cause methylation of the MSH2 promoter and transcriptional silencing. MLPA kits available for EPCAM 3’dels and UTR/MSH2 promoter. Dels that do not extend to MSH2 coding sequence are assumed to be pathogenic by causing MSH2 pm > confirmed by MS-MLPA. 3’ EPCAM deletions are heritable and risk of CRC similar to that of MSH2 mutation carriers.

Question 27

Q

what is the testing pathway for lynch syndrome?

Answer

A

Evaluation of tumour tissue for MSI/ IHC of the four MMR proteins. The presence of MSI in the tumour alone is not sufficient to diagnosis Lynch syndrome because 10%-15% of sporadic colorectal cancers exhibit MSI. IHC testing helps identify the MMR gene that most likely harbours a germline mutation.
• If the MLH1 immunohistochemistry result is abnormal, testing of the tumour for methylation and/or somatic BRAF mutations to help identify those tumours more likely to be sporadic than hereditary. First do a BRAF V600E test, if neg do methyation. If neg do sequencing.
Molecular genetic testing of the MMR genes (depending which is lost on IHC) to identify a germline mutation when findings are consistent with Lynch syndrome.

NICE recommends Cascade testing of relatives should be employed where appropriate as it is cost-effective

Question 28

Q

what is the function of MMR genes?

Answer

A

recognises errors that escape DNA polymerase proofreading. without repair, random mutations occur.
MSH2-MSH6 repair single base mismatches = MutSa

In the absence of MSH6, MSH2 pairs with MSH3 = MutSb. MSH2-MSH3 recognises large loop out errors

MLH1-PMS2 = MutLa heterodimer

in the absence of PMS2, MLH1 pairs with PMS1 (MutLb).
MSH6 and PMS2 are unstable in the absence of their dominant partners.

Question 29

Q

what syndrome results from germline homozygous or compound het MMR gene mutations?

Answer

A

mismatch repair cancer syndrome (MMRCS) – a rare childhood cancer predisposition syndrome with 4 main tumour types; haematological malignancies, brain/central nervous system tumours, colorectal tumours and multiple intestinal polyps.

Question 30

Q

what treatment/prevention is there for CRC?

Answer

A

colectomy, routine colonoscopy to prevent and removal of precancerour polyps.
Chemoprevention = aspirin for people at high risk of CRC

Question 31

Q

what is familial adenomatous polyposis?

Answer

A

AD
thousands of colonic polyps - most common polyposis syndrome (1/8500 births). 95% of patients have polyps by 35 years
caused by APC mutations (tumour suppressor gene involved in WNT signalling)
normal APC regulates B-catenin degredation, mutant APC leads to accumulation of b-catenin which activates transcription factors resulting in expression of target genes such as proto-oncogenes
loss of APC protein causes adenoma (benign) > carcinoma (cancer originating in epithelial cells) sequence
10% of cases are de novo
symptoms include rectal bleeding, anaemia, abdominal pain, change in bowel habits and weight loss
100% risk of CRC if colectomy doesnt happen at early age
with screening, most patients are diagnosed before the development of CRC
annual colonoscopy screening begins from 10 years if APC mutation or at risk
certain mutations (such as 5’ or 3’) cause attenuated FAP (1-100 polyps)
MUTYH responsible for 18% of APC negative cases

Question 32

Q

what is the mutation spectrum of APC gene causing FAP?

Answer

A

80% are LOF SNVs with some frequent mutations
60% of mutations occur in a mutation cluster with highest number of polyps and younger onset
5’ or 3’ or final exon mutations may develop attenuated FAP with reduced adenomas (benign tumour that may grow) as protein doesn’t undergo NMD
missense mutations may have small increased risk of polyps
somatic mosaicism in 11% of de novo cases results in familial variability
promoter mutations cause silencing of the gene

Question 33

Q

how is FAP diagnosed?

Answer

A

family history and colorectal phenotype
genetic testing - CRC panel can detect 5% mosaicism(90% pick-up rate)
dosage/MLPA detects partial or whole gene deletion - 10% of APC cases
blood sequencing may fail to detect mosaic cases in singletons (de novo occurrence)
presymtomatic testing offered <16 years (rare) as early screening or reduces cost if no screening needed for negative cases

Question 34

Q

what treatments are available for FAP?

Answer

A

surgical resection when polyposis develops
chemoprevention: low toxicity, cheap and effective and can delay development of adenomas and colectomy. can prevent recurrence after surgery eg. NSAIDs or aspirin

Question 35

Q

what is attenuated FAP?

Answer

A

<100 polyps, older age of onset, milder
later diagnosis but increased risk for CRC (70% by 80 years)
5’, 3’ or last exon mutations where NMD doesn’t occur
underdiagnosed

Question 36

Q

what is MUTYH-associated polyposis?

Answer

A

AR caused by MUTYH (adenine base excision repair gene)
similar presentation to AFAP. increased risk of adenoma and cancer
average diagnosis at 48 years
40-100% lifetime risk of CRC
accounts for 1% of CRC

Question 37

Q

how is MUTYH-associated polyposis diagnosed?

Answer

A

genetic testing for those with 10-100 polyps and no APC mutation
testing offered >18 years as colonoscopy begins from then
FH consistent with recessive inheritance

Question 38

Q

what is the mutation spectrum for MUTYH-associated polyposis

Answer

A

99% are missense and 2 common mutations often tested prior to whole gene screen
full gene analysis offered if one mutation found
if only one mutation identified, there may be a rare deletion, mutation in another gene is responsible or polyposis may be due to non-hereditary factors. MLPA is available.
carrier testing can be offered to partners, may just be for two common mutations
many with polyposis tested for APC and MUTYH simultaneously on a panel

Question 39

Q

what causes CML?

Answer

A

t(9;22)(q34;q11), with the shortened chromosome 22,
designated as Philadelphia chromosome, 22q-
a juxtaposition of the ABL1 gene from chromosome 9
and the BCR gene from chromosome 22, resulting in a BCR–ABL1 fusion gene that codes for proteins with high tyrosine kinase activity and promotes cell proliferation

Question 40

Q

what are the progression stages of CML?

Answer

A

chronic phase (3-5 years)
accelerated phase (9 months) AND/OR Blast Crisis (6 months)

Without effective therapy, most cases of CML progress from CP to AP/BP within 3-5 years of diagnosis

Question 41

Q

what are the presenting features of CML?

Answer

A

Fatigue - anaemia and bleeding
Night sweats
weight loss
shortness of breath
Splenomegaly

Question 42

Q

what proportion of CML patients have the BCR::ABL1 fusion gene ?

Answer

A

100%

90-95% have the characteristic t(9;22)(q34.1;q11.2) reciprocal translocation, whereas the remaining patients have either variant translocations involving a third (or even fourth chromosome) in addition to the chromosome 9 and 22, or a cryptic translocation involving 9q34.1 and 22q22.2 that cannot be identified by G-banded karyotype analysis.

how fusion is produced has No impact on outcome

Question 43

Q

what is the treatment for CML and how does it work?

Answer

A

imatinib (TKI)
competes with ATP for BCR-ABL1 binding site inactivating phosphorylation

Question 44

Q

why might CML TKI drug resistance emerge? what can be given instead?

Answer

A

subclones that have BCR-ABL1 mutations
second and third generation TKIs can be given

Question 45

Q

what should be done for CML patients on treatment with undetectable BCR::ABL1 transcripts ?

Answer

A

stop treatment and should remain in remission for at least a year
MRD monitoring detects disease at low levels and allows patients to resume treatment before any clinical presentation reappears

Question 46

Q

how are CML patients tested in the lab?

Answer

A

-G-banding analysis on blood or marrow of 10 cells and BCR/ABL1 FISH test for cryptic or variant translocations
-It is useful to carry out molecular analysis to confirm the presence of BCR::ABL1 gene rearrangement and to determine the nature of the rearrangement for future Minimal Residual Disease (MRD) analysis.
-Patients with a positive result will benefit from treatment with Imatinib
- also scan for secondary abnormalities including +Ph, +8, +19, +21, -Y, i(17q).

Question 47

Q

how is the BCR::ABL1 fusion transcript identified mollecularly?

Answer

A

Reverse Transcription-qPCR
- uses cDNA to amplify BCR:ABL1 and ABL1
- uses probe located in ABL1 with 5’ reporter fluorophore and 3’ quencher-during extension, polymerase cleaves probe causing fluorescence which is directly proportional to the amount of target present
- known standards are used for comparison allowing exact amount of transcript to be determined

Question 48

Q

during treatment, how often is RT-qPCR undertaken for CML cases?

Answer

A

every 3 months until MMR is achieved

Question 49

Q

what is the prognosis for CML?

Answer

A

Most people with CML will have a very good prognosis – particularly those diagnosed in the chronic phase. Recent evidence suggests that if you respond well to treatment, you could have a similar life expectancy to someone who doesn’t have cancer.

Question 50

Q

what is the most common mechanism of resistance in CML?

Answer

A

point mutation in BCR-ABL kinase domain
Second and third generation TKIs e.g. Nilotinib, Dasatinib and Ponatinib have been developed to try to overcome this TKI resistance

T315I mutation was the most frequently detected - poor prognosis

Question 51

Q

what is CML?

Answer

A

a myeloproliferative neoplasm originating in stem cells in the bone marrow

Question 52

Q

what is ALL?

Answer

A

clonal expansion of lymphoid progenitor cells in the bone marrow (BM), lymph nodes, thymus, or spleen, leading to an accumulation of immature blast cells

Question 53

Q

what % of childhood leukaemia does ALL account for?

Answer

A

85% (85% B-cell and 15% T cell)

Question 54

Q

what are the symptoms of ALL?

Answer

A

fatigue
weight-loss
shortness of breath
bruising
infections
splenomegaly
anaemia
bone and joint pain

Question 55

Q

what is the priority for infant <1 year with ALL?

Answer

A

urgent priority (14 days) as survival considerably worse than for older children. 20% will relapse

Question 56

Q

what is the testing strategy for < 1 year old with ALL?

Answer

A

FISH analysis

MLL (poor prognosis)
ETV6/RUNX1 (good)
BCR-ABL1 (poor)

Also do G-banded analysis on bone marrow or blood and analyse 10 (abnormal) or 20 cells if normal

Question 57

Q

what is the most common (50%) ALL abnormality in infants <1 year?

Answer

A

MLL translocation t(4;11)(q21;q23)
MLL (chr11) AF4(chr4)
poor prognosis

Question 58

Q

what is the most common ALL abnormality in 1-25 years? what is the prognosis?
what would the dual fusion probe show (red and green)?
If the dual fusion showed loss of green ETV6 signal 2F1R what does this indicate loss of and how does this affect prognosis?
give 2 other chromosomal abnormalities in ALL that could be detected with the ETV6-RUNX1 probe?

Answer

A

t(12;21) ETV6-RUNX1 t(12;21)(p13;q22)

good prognosis (>90% cure rate)

results in fusion protein with dominant negative effect - interferes with function of RUNX1 transcription factor

2Fusion1Red1Green

loss of ETV6 gene = poor prognosis
iAMP21 - ≥5 copies of RUNX1 , corresponding to ≥3 extra copies of gene on 1 copy of chromosome 21. poor prognosis

-extra copies of ETV6 and RUNX1 - polysomy of chr 21 and 12

Question 59

Q

after MLL rearrangements and ETV6-RUNX1, what is the 3rd-line test for B-ALL in < 1 years?

what is the prognosis

Answer

A

1) t(9;22)(q34;q11) BCR-ABL1 rearrangement (4%)

poor prognosis

Question 60

Q

what is the most common rearrangement in T-ALL?

Answer

A

TCR (T-cell receptor) rearrangements - usually placed next to transcription factors
unknown prognosis

Question 61

Q

what is the most significant genetic prognostic factors for adult ALL?
what is the prognosis?
what is the treatment?

Answer

A

t(9;22)(q34;q11) BCR-ABL1 Philadelphia chromosome - 30% of adult ALL cases

poor prognosis

TKI therapy enhances long-term outcome

Question 62

Q

what is the second-line test in adult B-ALL? what is the prognosis?

Answer

A

t(4;11)(q21;q23)
MLL (chr11) AF4(chr4)
poor prognosis

Question 63

Q

other than (4;11)(q21;q23) MLL ; AF4 what other common rearrangements involving MLL are found in ALL?

Answer

A

o t(9;11)
o t(11;19)

Question 64

Q

other than BCR:ABL1, MLL rearrangements and ETV6-RUNX1 what other common rearrangements are found in ALL?

Answer

A

high hyperdiploidy (51-56 chromosomes) - good prognosis
hypodiploidy <44 chromosomes
TCF3 rearrangements
IGH rearrangements
Dic (9;12)
Dic(9;20)
Abn/del(9p)

Answer 65

A

normal marrow may outgrow leukaemic clone
high failure rate
poor quality
crytic and subtle rearrangements

minimum 2 cultures should be set up

Answer 66

A

interphase FISH for high hyper diploidy (good prognosis)

Answer 67

A

identifies exact breakpoint so molecular monitoring can be undertaken during treatment

can be used to identify gene fusions

Answer 68

A

identify karyotype evolution or secondary malignancy

Answer 69

A

fatigue
shortness of breath
anaemia
joint pain
bruising
bleeding
increased infections
splenomegaly
rapid and fatal within weeks or months

Answer 70

A

new clinical, prognostic, morphologic, immunophenotypic, and genetic data that have emerged since the last edition

morphology = blood and marrow smears morphologically examined using Giemsa stain. blast count >20%

immunophenotyping = examine expression of cell-surface markers using flow-cytometry

cytogenetics - chromosomal abnormalities in 55% of adult AML cases

molecular genetics- screening for mutations in
a) NPM1, CEBPA, and RUNX1 genes
b) FLT3 - ITD, WT/M ratio and D835 and I836 tyrosine kinase domain mutations
3)TP53 and ASXL1 - poor prognosis

RT-PCR for recurrent gene-fusions. initiated when cytogenetics fails. following identification of a translocation by karyotype and for cryptic gene fusions where cytogenetic abnormality not present. also used to monitor MRD.

Answer 71

A

t(8;21)(q22;q22) - RUNX1T1-RUNX1
inv(16)(p13q22)
t(15;17)(q24.1;q21.2) - PML- RARA

Answer 72

A

t(9;22)(q34;q11)
5q abnormalities

Answer 73

A

ALL: t(12;21)(p13;q22) ETV6-RUNX1 good prognosis
ALL: iAMP(21) - poor prognosis
MDS/AML t(3;21)(q26. 2;q22) - Therapy-Related Disease Associated With Poor Outcome

Answer 74

A

examine 10 metaphases with full analysis
if no abnormality is detected, FISH/rt-PCR if there is a significant chance of replapse
there is a possibility of second malignancy in late relapse cases

Answer 75

A

abnormality linked to the WHO classification with ISCN
• the number of cells, within the current ISCN
• relationship of any abnormalities found to the referral reason, or other possible diagnosis
• association with prognosis if a robust association from multiple publications/international trials/trial protocols exists
presence of significant abnormalities detected only by FISH
test used and sensitivities
inclusion in clinical trial
additional abnormalities
literature references
relapse risk

Answer 76

A

activate signal transduction pathways and confer proliferative advantage

eg. FLT3 tyrosine kinase receptor. most mutations are internal tandem duplications that activate FLT3. poor prognosis. Also codons Asp835 and Ile836 TKD activates FLT3 - unclear prognosis. FLT3 mutations may be acquired or lost at relapse. ongoing trials to determine if FLT3 inhibitor therapy with chemo can reduce risk of relapse and improve survival

eg. PTPN11 - encodes cytoplasmic tyrosine phosphatase GOF. uncertain prognosis

Answer 77

A

affect transcription factors and block stem cell differentiation

eg. RUNX1 - uncertain prognosis - lesser overall survival in younger and older patients

MLL - causes DNA hypermethylation and epigenetic silencing of TSGs. poor prognosis

PML/RARA - acute promyelocytic leukaemia (APL)

Answer 78

A

NPM1 DNA repair gene- 50% of AML cases with normal karyotype have NPM1 frameshift mutations. good prognosis. commonly co-exists with FLT3 ITD

Answer 79

A

acquired UPD due to mitotic event

Answer 80

A

size - gene fusions are too large to detect with PCR due to introns
structure - multiple gene fusions due to alternate splicing
sensitivity - detects fusions that are transcribed, not just their presence
BUT more difficult to handle due to RNAses and mRNA transcripts are unstable with a short half life

Answer 81

A

allogeneic transplant, a person’s stem cells are replaced with new, healthy stem cells

Answer 82

A

molecular markers such as gene fusions or mutations are monitored
blood or BM are received at set intervals following diagnosis and the mutation compared to housekeeping gene ABL is determined by RQ-PCR
knowledge of the breakpoints or mutation is needed beforehand
relapse depends on mutation
flow cytometry can also be used

those with high transcript level at diagnosis have worse survival. significantly improved survival if reduction of transcript level after therapy. if transcript level increases following remission, this may indicate relapse. low -level transcript may be detected in patients even in long term remission as RQ-PCR is very sensitive.

Answer 83

A

ATRA ( a ligand for RARA) - t(15;17)(q24.1;q21.2); PML-RARA. good prognosis. binds to and degrades fusion protein.

Answer 84

A

discovery of novel leukaemia subgroups and of prognostic signature

Answer 85

A

expression patterns analysed using microarrays demonstrated that the expression of certain miRNAs correlates with the outcome of AML patients without cytogenetic aberrations. This may allow prognostic stratification based on miRNA expression patterns in the future.

Answer 86

A

activation of oncogene - typically encode cell proliferation and apoptosis controlling proteins
inactivation of TSG (both copies) - TSG’s prevent abnormal cell growth and stimulate cell death - once these are inhibited a malignancy arises. more important than oncogene activation

Answer 87

A

recessive at cell level (2nd hit is somatic) but show dominant inheritance in familial cancer syndromes. inactivation of 2nd copy may be by mutation, methylation or LOH.

Answer 88

A

gatekeeper - encodes proteins to control cell cycle and regulate cell proliferation eg. TP53, APC
caretaker - maintain and protect integrity of the genome. involved in DNA repair and preventing mutations eg. MLH1, MSH2

recent ‘landscaper’ group to create environments that control cell growth eg. PTEN

Answer 89

A

restrain cell growth eg. TP53 controls progression through G1 and TGF-b hormone receptor inhibits proliferation
genome integrity - MLH1 MMR
stimulate cell death - TP53

Answer 90

A

transcription factor that is part of signalling pathways that are essential for cell growth, regulation and apoptosis
in normal cells TP53 level regulated by binding of proteins eg. MDM2 that causes TP53 migration to cytoplasm and degradation. MDM2 upregulated by TP53 in a regulation loop that keeps levels low
In stressed cells, TP53 is inactivated meaning MDM2 is not upregulated and TP53 levels rise (transcription factor). Increased transcription of genes relating to apoptosis, inhibition of metastasis and Angiogenesis add repair arrest
TP53 stops cell cycle at G1 checkpoint - triggers CDK inhibitor which block activity and allows time for DNA repair. it also activates DNA repair enzymes or apoptosis if non-reparable

Answer 91

A

mutations in genes upstream of Tp53 such as those involved in radiation induced DNA damage repair pathway that prevent activation eg. ATM in Ataxia-telangiectasia
TP53 mutations - somatic mutations account for 50% of all tumours. point mutations via dominant negative manner and GOF eg. 25% breast cancers, 50% ovarian, Li-Fraumeni syndrome caused by TP53 germline mutation - multiple primary tumours.
downstream mediator mutations - PTEN is a TSG and is regulated by TP53. germline mutations cause cowden syndrome

Answer 92

A

can function as TSGs
Can use as markers for diagnosis, prognosis and therapeutic targets

Answer 93

A

APC Familial adenomatous polyposis - CRC, >100 colonic polyps, involved in cell division, DNA damage, cell death. negative regulator of b-catenin
MLH1, MSH2 & MSH6 MMR and cell cycle regulation. MLH1 heterodimerises with PMS2 to form MutL alpha- a component of the post-replicative DNA MMR system and MSH2 heterodimerises with MSH6 to form MutS alpha. Binding to dsDNA mismatch assembles the MutL-MutS heteroduplex complex which activates endonuclease activity of PMS2. introduces ssDNA breaks near mismatches followed by degredation. MSI seen in 15% of CRC but most are sporadic and caused by MLP1pm.
BRCA1/BRCA2 - involved in DNA repair and recombination.
NF1/NF2 - nerve tumours. RAS-mediated signal transduction, cell differentiation, cell division, developmental processes
TP53 - Li-Fraumeni syndrome, breast cancer, somatic pancreatic cancer, CRC

Answer 94

A

controls cell proliferation
GOF mutations can transform normal cells into tumour cells by uncontrolled growth or inhibiting apoptosis

Answer 95

A

point mutations eg. BRAF v600e found in malignant melanomas and can also be seen in metastatic colorectal cancer
amplification - over-production of coding protein such as HER2 found on normal breast cell. cancer cells have multiple copies and overproduction of the protein.
translocation to create novel chimeric gene - eg. Philadelphia chromosome (Ph) t(9;22)(q34;q11) seen in 90% of CML patients
Translocation into transcriptionally active region - oncogene moved next to high transcription regulatory elements leading to upregulated expression of oncogene eg. Burkitt lymphoma, t(8;14)(q24;q32); MYC oncogene is brought under the control of an immunoglobulin (IG) locus
DNA rearrangements such as inversions and deletions creating fusion genes
viral oncogenes insertion near cellular genes eg. hepatitis B in hepatocellular carcinoma

Answer 96

A

secreted growth factors: activation of growth factor gene induces cell proliferation
growth factor receptors eg. EGFR in NSCLC: many are altered and stimulate transducer signals for cell growth and proliferation. some mutations allow receptors to escape down-regulation providing a growth advantage
signal transducers eg. PIK3CA - mutations can lead to upregulation of oncogenic processes
apoptosis inhibitors
transcription factors - translocations leading to fusion protein and producing transcription factors which alter DNA repair genes

Answer 97

A

G0 = resting phase where cell has left cell cycle
G1 = growth phase where cellular contents except chromosomes are duplicated. proteins and RNA are synthesised. At G1 check-point (restriction point) the cell moves to S phase
S phase - synthesis. DNA is replicated and each chromosome consists of two sister chromatids.
G2 = cell continues growing and cell double-checks the duplicated chromosomes for errors. G2 checkpoint ensures enough cytoplasmic materials for mitosis.
M= nuclear division (mitosis) and cell division (cytokinesis). metaphase checkpoint in middle ensures cell is ready to complete division.

Answer 98

A

cyclin = form the regulatory subunit and have no catalytic activity
cyclin-dependent kinases = proteins which are inactive in the absence of a partner cyclin. They are the catalytic subunit of an activated heterodimer which phosphorylates target proteins to orchestrate coordinated entry into the next phase of the cell cycle.

overexpression of cyclins is detected in cancers
cyclin kinase inhibitor genes inactive CDKs. mutations in these genes result in cell proliferation.

Answer 99

A

blocking MDM2 expression with gene silencing - prevents TP53 degradation
inhibit MDM2-TP53 binding -
stop ligase activity of MDM2 to prevent negative regulation of TP53

Answer 100

A

greater efficacy
fewer side effects
greater therapy tolerance
reduced dependence
better compliance
improved quality of life
fewer hospital admissions and mortalities
reduced cost

Answer 101

A

DPYD metabolizes 5FU (chemo drug - prevent cell proliferation) used in treatment of CRC, breast, pancreatic solid tumours
-DPYD variants may cause poor metabolism and so dose-management is critical to prevent toxicity. -heterozygotes usually asymptomatic but homozygotes may have epilepsy and motor dysfunction.
4 known variants affect protein function and lead to increased toxicity
the 4 variants are given different activity values
depending on whether hom, het or compound het the DPD activity is classified into normal, intermediate or poor metabolisers
intermediate metabolisers should reduce dose by 50%. dose is then adjusted depending on toxicity
NHS england provide this service to all patients prior to 5FU chemo

Answer 102

A

warfarin prevents clotting in thromboembolism
metabolised by CYP2C9
polymorphisms in CYP2C9 contribute to variability in dose requirement (15% variation in warfarin response)
overdosing leads to excessive bleeding
limited genotyping in the UK

Answer 103

A

non genetic factors influence response eg. age, height, body mass, other therapies, comorbidities
complex metabolic pathways limit efficacy of single gene testing
GWAS is better than genotype-phenotype association data
population databases lack ethnic diversity
drug-drug interactions need to be studied

Answer 104

A

heterogeneous hematologic malignancies characterized by clonal expansion of bone marrow myeloid cells with impaired differentiation, increased risk of transformation to AML and development of peripheral cytopenias (low levels of RBC, WBC or platelets) due to ineffective haemopoiesis and dysplasia (presence of abnormal cells) = hallmark of MDS

blasts <20%, when >20% becomes AML
- considered a pre-malignant condition
- median age of diagnosis is 70 years
- may be de novo or due to prior cytotoxic chemotherapy

Answer 105

A

chemo, therapy, transfusions, bone marrow stimulation and cytotoxic chemotherapy

bone marrow transplant is the only curative treatment

FDA has approved 3 therapeutic agents.

If low risk MDS - growth factors and lenalidomide
If high risk - hypomethylating agents and bone marrow transplant

Answer 106

A

-5q deletion (15% of cases) - good prognosis if isolated finding. low risk of transforming to AML. TP53 mutation testing recommended in WHO as presence of mutation and del(5q) predicts poor response to lenolidomide
-monosomy 7 - poor prognosis (more common in therapy-related MDS)
- trisomy 8- intermediate
- complex (3 or more abnormalities often TP53 locus) 90% of therapy related-MDS - poor prognosis

Answer 107

A

Revised International Prognostic Scoring System (IPSS-R, 2012) is the most recent one, however can only be used at the time of MDS diagnosis
The World Health Organization (WHO) Prognostic Scoring System (WPSS) is dynamic and can be used at any time during the course of the disease.
both of these exclude patients who received prior therapy and so the global MDACC was developed - evaluate patients at any time during their disease without a needed WHO classificaition
karyotype of BM cells is one of the most important prognostic markers in IPSS and WPSS
70% of patients have recurring chromosome abnormalities

a major terminology change in WHO 2017 removed the term “refractory” as in “refractory anaemia/cytopenia” as it relies more on degree of dysplasia and blast percentages

Answer 108

A

90% of MDS patients have a DNA mutation
genes frequently mutated include spliceosomal genes, epigenetic modifiers, cohesins, transcription factors (eg. RUNX1, ETV6 - poor prognosis) and signalling molecules (Jak2, FLT3, NF1, NRAS, PTPN11)

Answer 109

A

-1/3
- due to acquisition of two driver mutations resulting in proliferation of subclonal populations

Answer 110

A

karyotyping is gold standard
chr 5 and 7 abnormalities must be demonstrated by karyotype and not FISH or NGS (WHO guidelines)
array improves yield of chr abnormalities
NGS aids diagnosis -
SNParray - detects UPD (20% of MDS cases)

Answer 111

A

Measurement of the total number of non-synonymous mutations in the tumour exome per megabase of DNA analysed

MMR tumours have higher TMB

No consistent testing approach as yet. Platforms and thresholds differ massively between testing centres and for various cancer types.

WGS, WES and targeted panels can be used

Answer 112

A

higher tumour mutation burden means high chance of expressing new antigens - recognised by immune system and T cells kill cancer cells which lowers mutation burden
Immune checkpoint inhibitors boost the immune response
improve response and survival in multiple cancers - higher TMB correlates with increased response to immune checkpoint inhibitors

Answer 113

A

deamination causes C:G>T:A changes (uracil if unmethylated) which can result in an over-estimation of TMB.

deaminated cytosines (uracil) can be removed enzymatically prior to testing

Answer 114

A

neoplasia of melanocytes
skin cancer, may also be eyes, ears, GI tract, pancreatic, lymph nodes
highly lethal if not detected and treated - responsible for most skin cancer deaths
10% of melanoma cases are hereditary
45% of familial (inherited) melanoma associated with germline CDKN2A or CDK4 (2%)
other genes involved in melanoma are BRCA1, TERT (telomerase maintenance gene)
CDKN2A is a TSG involved in cell cycle regulation
CDKN2A variants also predispose to breast, lung cancers
CDKN2A has varied estimates of penetrance
if pathogenic variant detected in CDKN2A> skin surveillance and pancreatic screening

Answer 115

A

18bp in-frame deletion in exon 19 (c.2240_2257del) and a common point mutation in exon 21 (p.L858R) (90%). GOF activating mutations.

p.T790M mutation is the most common resistant mutation and is found in ~50% of patients that develop acquired resistance to tyrosine kinase inhibitors

Answer 116

A

V600E - 80% of BRAF mutations
V600K - common in melanoma, thyroid cancer, CRC and NSCLC

Answer 117

A

MEK inhibitor (trametinib)
RAF inhibitors (vemurafenib and dabrafenib)
Vemurafenib specifically targets the V600E mutant BRAF and triggers apoptosis

Answer 118

A

Cetuximab (monoclonal Ab) – targets EGFR

Answer 119

A

Gefitinib/Erlotinib/Osimertinib

. Osimertinib targets the T790M mutant EGFR

Answer 120

A

checkpoint failures - checkpoint activation controlled by kinases ATM and ATR
genotoxic agents (both endogenous and exogenous),
intrinsic biochemical instability of the DNA
error rate of DNA polymerase

Answer 121

A

mismatch repair (MMR)
base excision repair(BER)
nucleotide excision repair (NER)
direct repair

Double strand breaks are repaired by homologous recombination (HR) and non-homologous end joining (NHEJ)

Answer 122

A

Recognises erroneous insertions, deletions and mis-incorporation of bases that arise during DNA replication and recombination.

MutS (MSH2 and MSH6) and MutL,(MLH1) mutations found in lynch syndrome and sporadic MLH1-deficient colon cancers. Mutations affect genomic stability which can result in MSI

MLH1 heterodimerises with PMS2 to form MutL alpha and MSH2 heterodimerises with MSH6 to form MutS alpha. Binding to dsDNA mismatch assembles the MutL-MutS heteroduplex complex which activates endonuclease activity, introduces ssDNA breaks near mismatches followed by excision and repair.

Answer 123

A

repairs damaged base and ss breaks
DNA glycosylases recognise and remove damaged or wrong base
endonuclease breaks strand
DNA polymerase inserts new base
DNA ligase repairs strand

rare homozygous defects in MUTYH repair enzyme are associated with increased risk to colon cancer

Answer 124

A

removes thymine dimers (covalently bonded complex of two adjacent thymines on a single strand of DNA)
There are two classes

1.Global excision repair (GER) which repairs all DNA
2. Transcription-coupled repair (TCR) where DNA undergoing transcription is repaired.

unwinding of the DNA helix, excision of the damaged area and then repairs using low fidelity polymerases

defective in Xeroderma pigmentosum (XP) and Cockayne syndrom

Answer 125

A

-double strand breaks are directly repaired without the need for a homologous template
- utilizes short homologous DNA sequences called microhomologies to guide repair
- microhomologies are often present in single-stranded overhangs on the ends of double-strand breaks
- When the NHEJ pathway is inactivated, double-strand breaks can be repaired by a more error-prone pathway called microhomology-mediated end joining (MMEJ).

Answer 126

A

requires a homologous sequence to guide repair extensive sequence identity (>300 bp)
BRCA1 and BRCA2 are involved in homologous recombination
After DNA damage, BRCA1 is phosphorylated and re-localises along with RAD51 to the damaged regions activating homologous repair
Bloom syndrome BLM
Nijmegen breakage syndrome

Answer 127

A

BRCA1/2 - breast, colon, ovarian etc defect in double strand break repair
MUTYH-associated polyposis colon cancer - base excision repair
Lynch - MLH1 MSH2 MSH6 PMS2 mismatch repair colon cancer
Li - Fraumeni TP53 cell cycle defect + apoptosis - many different tumours
Ataxia Talangiectasia - AR ATM gene DNA strand breakage and cell checkpoin defects - acute leukaemias and lymphomas
Fanconi’s anemia cross-link repair. AML. FANCA, FANCC

Answer 128

A

targeting of treatments according to the characteristics shared by a group of patients
uses biological markers to separate patients into specific groups and treating a specified group by the inactivation of a well-defined target or biological pathway
step towards personalised medicine

Answer 129

A

safer, more effective
reduced side-effects and greater drug compliance
cost-effective
improved response rates
avoids treatments for those who don’t need it
improved diagnosis and prognosis
NGS allows prediction of resistance and identifying patients who could be considered for trials

Answer 130

A

Herceptin and HER2+ Breast cancer
BRCA1 and BRCA2 mutations ovarian and breast cancer detected via germline sequencing treated with PARP inhibitors - Olaparib and Talazoparib

Answer 131

A

HER2 is a receptor tyrosine kinase and a member of the EGFR family
testing on all new primary or metastatic breast cancer
amplification found in 30% of early stage breast cancer
HER2+ more aggressive, higher recurrence risk (up to 80%) and worse outcome
Herceptin is a monoclonal antibody given to patients with overexpression of HER2
testing performed with IHC and dual probe FISH (ratio of HER2 to CEP17 control gene) or multigene assay. IHC score 3+ = positive and 2+ = borderline in combination with FISH confirms result
FISH result ratio HER2:CEP17 >2 = amplified and the average HER2 gene copy number is ≥4.0 per tumor cell , <2 and <4 copies per tumour cell= negative
Herceptin has cardiac side effects
resistance is a major issue as tumours accumulate molecular changes during evolution eg. PIK3CA activating mutations in helical and kinase domains, PTEN loss

Answer 132

A

-PARP1 - repairs DNA breaks. recognises ssDNA breaks and is involved in base excision repair. (BER)
- PARP inhibition is lethal to mutated BRCA1/2 somatic cells (synthetic lethality - cell death)
- PARP inhibition occurs via siRNA or small molecules
- failure of PARP1 activity causes replication forks to stall during replication and dsDNA breaks to accumulate
- when BRCA1 or 2 is mutated, errors in DNA repair occur as error-prone NHEJ occurs rather than homologous recombination that eventually causes breast cancer
- with PARP inhibition, cells cannot be repaired and die. normal cells have efficient HR as still have WT copy of BRCA which allows them to survive PARP inhibition
- UK NICE recommends Olaparib for BRCA-positive cancer that has responded to first-line platinum chemo

Answer 133

A

up to 30% of NSCLC patients have EGFR mutation. 90% of these mutations are in-frame deletions in exon 19 or missense (Leu858Arg) in exon 21
NSCLC with EGFR mutations have better prognosis compared to those with wildtype
Gefitinib is a tyrosine kinase inhibitor, directed against EGFR, and functions by blocking the ATP binding site
EGFR mutation status is a likely indicator of drug response
exon 20 mutations trigger EGFR activation, and typically confer resistance to gefitinib
Gefitinib given to EGFR tyrosine kinase domain exons 18-21 mutations
DNA extracted from FFPE tissue, pyrosequencing or RT-PCR used to detect mutations in exons 18-21
resistance mutation = EGFR mutation T790M in exon 20 associated with relapse
MET amplification also observed in 15-20% of patients who develop EGFR-TKI resistance
Osimertinib 2nd line treatment can be given to EGFR+ and T790M EGFR inhibitor resistance lung cancers

Answer 134

A

KRAS activating mutations occur in 20% of NSCLC
poor prognosis
KRAS and EGFR are mutually exclusive so if a KRAS mutation is identified, molecular testing stops.
reduced responsiveness to EGFR TKI therapy
no targeted therapy for KRAS-mutant NSCLCs

Answer 135

A

EML4-ALK fusion occurs in 6% of lung adenocarcinomas
- EML4 is most common partner and due to different breakpoints, several variants have been described
- majority due to inv(2)(p21p23); del(2)(p21p23)
responsive to ALK TKI eg. alectinib as first-line therapy has improved efficacy over crizotinib
- usually independent of KRAS and WGFR mutations
- resistance to therapy =ALK kinase domain mutations
- novel 2nd generation ALK inhibitors are in development
- FFPE tumour - tested by FISH using ALK break-apart probe and and dual colour fusion specific for the inversion
- RT-PCR can detect EML4-ALK transcripts if all primer sets are included (unable to detect novel partners)
- ALK overexpression can be detected by IHC as screening strategy
-

Answer 136

A

MET amplification (crizotinib), MET exon 14 skipping
ROS1 rearrangements (ROS1 TKIs)
-RET rearrangements
BRAF mutations (V600E) - oral BRAF and MEK inhibitors dabrafenib plus trametinib
PD-L1 (Programmed Death Ligand 1)
HER2 mutations - herceptin
tumour mutation burden

Answer 137

A

1.chromosome instability - KRAS, NRAS, BRAF + PIK3CA
2. sessile serrated pathway - BRAF
3. MSI
4. hereditary LYNCH

all patients with metastatic CRC should have KRAS, NRAS and BRAF sequenced

Answer 138

A

up to 70% of CRC has KRAS activating mutations
90% of mutations occur in codons 12 and 13
patients with KRAS mutation should not be treated with EGFR TKI Cetuximab

cetuximab given to EGFR expressing, RAS wild-type metastatic colorectal cancer

Answer 139

A

account for 10% CRC
patients with BRAF V600E mutation unlikely to respond to cetuximab (EGFR) unless given BRAF inhibitor
BRAF mutations with MSS = poor prognosis
NRAS mutations occur in exons 12 and 13 and also should not be treated with EGFR TKI cetuximab

cetuximab given to EGFR expressing, RAS wild-type metastatic colorectal cancer

Answer 140

A

Activating mutations in PIK3CA occur in 10-30% of all CRC, and are most likely to occur in exons 9 and 20
- may also have BRAF and KRAS mutations

Answer 141

A

BRAF occurs in 50% of melanomas also causes CRC and lung tumours
BRAF is a member of the Raf kinase family of growth signal transduction protein kinases - it is a serine threonine kinase and plays a role in regulating the MAP/ERK signalling pathway kinase - cell division, differentiation and secretion. mutations lead to unrestrained cell growth and proliferation
v600E most common and leads to constitutive activation of Raf. V600K next common.
v600E mutations makes CRCs resistant to EGFR therapies such as cetuximab
MEK inhibitors and BRAF inhibitors together are best treatment
IHC screen for mutated v600E protein and then RT-PCR, pyrosequencing or panel testing
• Vemurafenib and Dabrafenib are BRAF gene mutation inhibitors

Answer 142

A

-neurotrophic tyrosine kinase (NTRK) gene family - responsible for normal development and function of CNS and PNS
- NTRK1 NTRK2 and NTRK3 code for tropomyosin receptor kinases
- NTRK fusions associated with tumour growth
- >80 different fusion partners
- NTRK gene fusions occur in 1% of solid tumours
- NTRK fusions are detected in ALL and AML <5% frequency
- treated with TRK-inhibitors (TKI inhibitors) such as larotrectinib and entrectinib
- test by FISH or FFPE RNA sequencing
second generation TKIs in development

Answer 143

A

partnership between NHS, government, CRUK and pharma companies to streamline and standardise genetic testing of tumour samples across UK
phase 1 = how NHS can test solid tumours routinely

phase 2 = creating a national genetic pre-screening programme and advancing treatment for people with late stage non-small cell lung cancer (NSCLC).

developed National Lung Matrix Clinical Trial - testing most likely beneficial drugs on patients after diagnosed by NHS.

Answer 144

A

released into the bloodstream from the primary tumour and may result in metastasis into distant organs
may be present at low concentrations, need enrichment to test for
Analysis of ctDNA can provide information on the genetic alterations present in a patient’s tumour, without needing a solid biopsy sample.
ctDNA could theoretically be used to screen for multiple cancers

Answer 145

A

IMMUNOLOGICAL: expression of protein markers - eg. anti-epithelial antibody for EPCAM marker (positively enriched) or CD45 antibodies to deplete leukocytes (negative enrichment)

physical properties - size & density (CTCs bigger than leukocytes) - eg. membrane filtration, centrifugation, electrophoresis

MOLECULAR: reverse-transcription PCR + liquid bead array. multiplex RT-PCR approaches have been often established, giving therefore the chance to screen at the same time for more than one single marker
Functional assays - mice models

Answer 146

A

NSCLC EGFR mutations - unable to provide solid biopsy but analysing liquid biopsy means patients can still access therapies that target the EGFR mutations
- as cancer progresses can take further ctc samples to monitor recurrence, look at evolution and resistance - no need to undergo repeat tumour biopsies
- NSCLC makes up approximately 87% of lung cancers, only around 10-15% of NSCLC patients have cancer with EGFR mutations

Answer 147

A

 Most cancers are sporadic and originate from clonal expansion of a transformed cell through the accumulation of serial somatic mutations

mutations include single nucleotide changes, small duplications, insertion or deletions, exon or whole gene copy number change and chromosomal aberrations such as translocations and aneuploidy

Answer 148

A

IHC, FISH, Q-PCR & digital (specific point mutations, RT-PCR to amplify mRNA of fusion gene)), pyrosequencing, sanger (rarely used as doesnt detect detect low allele frequencies and is sensitive to contamination and poor DNA quality), expression arrays, NGS + exome (multiple genes and hot spots)

Answer 149

A

be very heterogeneous - different mutations cause same tumour in different patients and different mutation status in same patient
percentage tumour present is variable - background of normal tissue. may need enrichment eg. macrodissection
Tumour tissue is not always available - may be insufficient material after cytology for molecular
FFPE - quality and quantity is variable. formalin degrades DNA and RNA

Answer 150

A

variable quality depending on size of resection, amount of time in formalin or whether recalcified (bone samples - unsuitable for testing)
extracted DNA is fragmented
more PCR artefacts due to deamination of cytosine>uracil - results in C>T/G>A transition sequencing artefacts
low tumour %
need specific technologies to detect low-level mutation
Tumour heterogeneity - mutation may only be present in part of tumour
test failure due to poor quality/quantity
is labour intensive - hard to have fast TATs (eg. EGFR testing in Lung cancer up to 2 weeks)

implementing fresh frozen tissue is better - done for 100k cancer

Answer 151

A

analysis of specific cell types, or separate clones as well as its cost, speed and the relatively small amount of material required

Answer 152

A

detection of both frequent and infrequent changes using short reads from random positions gives confident sequence reads
targeted NGS panels, to whole exome (WES) or whole genome (WGS) testing.
targeted panel testing for clinically actionable mutations is being used but WGS approaches are in the process of being implemented (WGS and WES on FFPE is still under development _
germline testing also required - Comparison of tumour and paired germline samples is necessary to identify a somatic event
Coverage and allele frequency is problematic in cases with low tumour content
high cost and TAT
high coverage providing enough sensitivity in situations of low tumour content, tumour heterogeneity and low-level mutation detection
may need to enrich DNA samples - Long Range PCR and multiplex amplicon sequencing eg. two primer target PCR

Answer 153

A

developing based on 100K Genomes Project - tumour and germline tissue testing and results returned to GLHs
fresh tumour and EDTA
leukaemia = PB or bone marrow + normal skin biopsy or saliva. important to make sure not germline and not use any germline carrier family members for transplant

Gel gave mutation signatures= (patterns of mutations) in a given tumour samples

tumour mutation burden= the total number of somatic non-synonymous small variants per megabase of DNA. . compared to tumours of same type in the project. predicts response to to immune checkpoint inhibitors

Answer 154

A

used for CNVs at high resolution in regions of interest
-failure to detect balanced rearrangements, different clones can not be distinguished, technical challenges and data interpretation difficulties.

Answer 155

A

detects CNVs and UPD/LOH
Acquired somatic UPD may lead to duplication of an activating somatic mutation or homozygosity for a disease prone minor allele present in the germ line.
UPD can result in increased/decreased gene expression due to the duplication of a particular methylation pattern.
variable coverage - uneven distribution of SNPs

Answer 156

A

quantify transcripts from mRNA and cDNA = gene signature
allow numerous regions of a single tumour to be investigated at the same time
gives info on Alternative splicing & The non-coding transcriptome eg. miRNA (used for prognosis)
high cost, not optimised
Tumour samples have to be analysed against other samples or standards whose classification is known,

Answer 157

A

Non-invasive blood tests to detect circulating tumour cells (CTCs) and fragments of tumour DNA that are shed into the bloodstream from the primary tumour and from metastatic sites
-circulating tumour DNA (ctDNA) is easier to assess and study, offering more potential for high throughput strategies of assessment
- could be used for prognosis, treatment, allows genomic and transcriptional analysis
- can be used for detection, monitoring and treatment.
- digital PCR, droplet testing, mass-spec, NGS (in development) can be used
- EGFR and BRAF mutations used clinically, EML4-ALK rearrangements in lung cancer in development
- Serial sampling gives a real time view of the tumour therefore can detect genetic changes within the tumour allowing monitoring of tumour burden, therapeutic responses and the emergence of treatment resistance

-Analysis is challenging as the fraction is small and exists on a background of normal cell free DNA

Answer 158

A

FFPE pros: diagnostic (presence of tumour known) easy storage
FFPR cons: poor quality, fragmented DNA, low quantity, need to re-biopsy if run out, invasive, one fixed time-point
ctc pros: non-invasive, can repeat, shows heterogeneity, detects low level
ctc cons: false negatives, limited conditions detected, short half life, low conentration

Answer 159

A

used for known mutations - SNVs, indels, and rearrangements
can be used for ct-DNA
extracted DNA is mixed with PCR reaction components, primers for genes of interest, uses probes specific for mutation or wild-type sequences
mix is emulsified into tiny droplets such that, on average, there is less than one haploid genome equivalent per droplet and is used for emulsion PCR
During ePCR, the fluorescent probes hybridize to amplified mutant or wild-type sequences present in the droplet and are cleaved during amplification to release the fluorophores
fluorescence detection for each individual droplet
use: NSCLC patients for EGFR testing, both where no tissue sample is available for testing (a limited mutation screen), and when patients have progressed on their EGFR TKI - deletions within exon 19, c.2573T>G (L858R), and the c.2369C>T (T790M) - resistance to first-line TKIs
For progression testing it is important to know the primary sensitising mutation as this can be tested in parallel which helps with interpreting results: is the T790M test negative because the mutation is not present or because there is no ctDNA in the sample
A RAS assay for colorectal cancer patients is also available that tests KRAS codons 12, 13 and 61 and NRAS codons 12 and 13.

Answer 160

A

low level cancerous cells that remain following treatment, and are only detectable by highly sensitive techniques.

• 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 (used to kill any cancer cells that may be left in the body)
• To determine prognosis after completion of standard treatment enabling early recognition of impending relapse
• Monitor disease burden in the setting of stem cell transplantation
• To spare toxicity and cost of SCT in those with low risk of relapse
• Assignment to maintenance therapy after completion of standard treatment

Answer 161

A

FISH - cytogenetically defined rearrangements, low sensitivity
RT-qPCR (uses RNA) - measures RNA expression of translocation products by quantitation of PCR products during the exponential phase eg. t(9;22) for BCR-ABL1, t(15;17) for PML-RARA. molecular breakpoints should be assessed at diagnosis to allow for MRD testing. • ABL is used as the most reliable control gene
Quantitative-PCR (uses DNA) - Less popular than RT-PCR as varying translocation breakpoints make assay design difficult
Tandem duplication PCR uses a pair of primers for amplification but is oriented in the opposite direction from standard PCR, therefore only allowing amplification when a duplication is present. Specifically used for FLT3-ITD detection
Immunological - fluorescently-labelled antibodies or flow-cytometry to identify cell-surface proteins
QF-PCR – of highly polymorphic markers is used to detect chimerism in which no other markers are available.
NGS in development - independent of the specific leukemic clone present and can also cope with disease progression and transformation
ddPCR - need to develop for specific mutations

Answer 162

A

mostly flow cytometry
•Gene fusions – certain gene fusions such as BCR-ABL1, MLL-AF4 t(4;11)(q21;q23) , and ETV6-RUNX1

Answer 163

A

reallocation of patients to favourable or unfavourable categories based on MRD status instead of molecular and cytogenetic findings at diagnosis.

1) fusion-gene monitoring - t(8;21) RUNX1-RUNX1T1, inv(16) CBFB-MYH11, t(15;17) PML-RARA and mutated-NPM1 ALL GOOD PROGNOSIS

2) Tandem duplication PCR - FLT3-ITD status POOR PROGNOSIS

3) WT1 monitoring - overexpressed at the mRNA level in 80–90% of AML cases at diagnosis in both peripheral blood (PB) and bone marrow (also causes DSD)

4) flow cytometry

Answer 164

A

karyotype Philadelphia chr -At least 20 metaphases needed 5% sensitivity
RT-QPCR - BCR-ABL1 fusion transcript level. high sensitivity, PB or bone marrow
Interphase FISH - BCR-ABL1 fusion can detect cryptic translocations. quick but targeted

Answer 165

A

post BMT is used to determine the success of the engraftment
1) • Sex mismatched BMT: XY FISH
2) • Sex matched BMT: PCR using short tandem repeat microsatellite markers.

Answer 166

A

divided into 4 tiers where tier 1 and 2 are clinically actionable, tier 3 is VUS and tie 4 is benign. evidence includes:
disease guidelines, tumour databases eg. cosmic, in-house databases, in-silico tools, pop frequency, clinical trial data, functional studies and literature.

tier 1 and 2 likely to be Frameshift, splice site or nonsense in LOF gene, previously reported as pathogenic, low freq, in-silico predicts pathogenic, functional studies + mutation hotspot.

a pathogenic variant does not mean that the variant is implicated in tumour development. The variant may be involved in drug resistance for example

Answer 167

A

Confer growth advantage on the cells carrying them and have been positively selected during the evolution of the cancer. Non-recurrent variants are unlikely to be drivers otherwise they would more than likely been seen previously.

Answer 168

A

AF - germline is 0.5 or 1 , somatic is variable
heterogeneity - high in tumours, homogenous in germline
Low AF - can discount in germline but may be real or sequence artefact in cancer
linkage - can do family studies in germline, cant use linkage in somatic
interpretation - in germline just need to decide if pathogenic, in tumour need to know if a driver, prognosis, treatable etc

Answer 169

A

• A number of genes on somatic cancer panels are also associated with familial cancer syndromes
(e.g. BRCA, TP53, RUNX1, CEBPA)
- implications to family members
- • Identifying, confirming and reporting potential germline variants will be recommended in the
forthcoming UK somatic variant interpretation guidelines.
• Confirmation of germline status needs to be performed on appropriate tissue. Referral to Clinical
Genetics for appropriate counselling and to facilitate screening of the family is highly
recommended.

Answer 170

A

fresh tumour tissue - Invasive malignant nuclei ≥30%

Brainscape's Knowledge GenomeTM

session 6: cancer genetics & treatment Flashcards

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