extra CSM

Question 1

copy number variation

Answer 1

definition
- gain or loss of part of chromosome
- include loss of heterozygosity (LOH)
existential differences
- copy number polymorphism (CNP) - recurrent in population
- copy number variation (CNV) - specific in sample

Question 2

large deletions or insertions detected by

Answer 2

SKY chromosome painting e.g. breast cancer
normal SKY chromosomes are not mutlicoloured
chromosomes in breast cancer appear multicoloured due to an exchange in genetic material

Question 3

structural variants with NGS

Answer 3

adv: more data and context than SNP array
limitations
- mapping systematically biased/lower coverage/high variability on coverage/exome data provides incomplete reference/cost
tools e.g. CNVHitSeq/OncoSNP-SEQ

Question 4

CTCs/ctDNA as a cancer detection method

Answer 4

tumours shed both cells and DNA into the circulation
amounts are proportional to size/stage of tumour
can be detected as part of a liquid biopsy
CTCs harvested by physical/immunochemical capture

Question 5

immunotherapy

Answer 5

chimeric antigen receptor T cells (CAR T cells)
1. T cell collection
2. T cell transfection
3. T cell adoptive transfer
4. patient monitoring

Question 6

CTCs uses

Answer 6

CTCs could be analysed for protein and RNA biomarkers plus the full-range of genomic changes and epigenetic changes
- inform of tumour biology and treatment
- select therapies based of the sensitivity of CTCs to drugs in vitro

Question 7

ctDNA detection e.g. point mutation by Taqman PCR

Answer 7

point mutation detection by Taqman PCR
- 1 pair of primers
- 1 probe complimentary to wt sequence
- 1 probe complimentary to mutant sequence

Question 8

ctDNA detection e.g. Next Generation Sequencing

Answer 8

Next Generation Sequencing (NGS)
- millions of short sequences determined in parallel
- individual ctDNA molecules with mutations should be detectable in a vast excess of wt DNA

Question 9

cfDNA

Answer 9

as DNA is fragmented simply ligate sequencing adapters and amplify and introduce barcodes with a few cycles of PCR (8-15 cycles)
whole genome sequencing = expensive
shallow WGS = inexpensive used to detect copy number changes

Question 10

unique molecular identifiers (UMIs)

Answer 10

UMIs allow NGS to detect extremely low level of ctDNA and an excess of normal cfDNA
short sequences introduced prior to main PCR
families of reads can be traced back to individual cfDNA molecules
more sequencing (cost) and can be difficult to implement

Question 11

CA-125 as a screening tool in ovarian cancer

Answer 11

CA-125 = cancer antigen 125
actually is MUC16 a 22kDa protein that has elevated levels in epithelial ovarian cancer
problems are:
- ovarian cancer is generally asymptomatic in women until it reaches an advanced stage
- CA-125 doesn’t have 100% sensitivity for ovarian cancer
- levels can be elevated in other cancers
- levels can be elevated in benign disease

Question 12

assessing treatment efficacy: survival time

Answer 12

overall survival time - time from start of treatment to date of death
disease-free survival time - time prior to tumour relapse after radical treatment
progression-free survival time - survival time prior to tumour progression

Question 13

challenges of cancer recognition by the immune system

Answer 13

cancer cells look extremely similar to normal cells
T cells that recognise self cells are destroyed during development - immune tolerance

Question 14

cancer’s ways to evade immune recognition

Answer 14

1. immunosuppressive cytokines
   e.g. TGF-beta/IL-10
   the tumour micro environment is complex and often very immunosuppressive
2. down regulation of MHC
3. down regulation of components of antigen processing pathways
4. exploiting T cell checkpoints
   prevent T cell activation by interfering with T cell check points

Question 15

cancer vaccine types

Answer 15

HPV vaccine - good to prevent getting cancer
cancer immunotherapy to help those with cancer
- checkpoint blockade therapy - reawakening naturally occurring anti-tumour T cells
- CAR T cells - giving patients T cells that have been genetically engineered to recognise cancer

Question 16

chemotherapy: clinical uses

Answer 16

1. for advanced disease
   - where no treatment exists
2. adjuvant chemotherapy
   - systemic treatment following local radiotherapy or surgery
   - to control microscopic metastases
3. primary or neo-adjuvant chemotherapy
   - chemotherapy as initial therapy for locally advanced cancer: renders cancer more amenable to subsequent surgery/improve cosmesis/function/control micrometastases

Question 17

chemotherapy drugs type for the cell cycle phases

Answer 17

G1/G0: micro-tubule inhibitors/topoisomerase inhibitors/alkylating agents
S: anti-metabolites/topoisomerase inhibitors
G2: platinum analogues
M: micro-tubule inhibitors

Question 18

what are the benefits of delivering chemotherapy drugs at an increased intensity

Answer 18

delivering treatments at a greater rate (dose density) could optimise chemotherapy efficacy
- minimising the regrowth of cancer btw doses of therapy
- inc. the cumulative cell kill
- achieving greater clinical benefit

Question 19

chemotherapy and toxicity

Answer 19

low therapeutic index
toxicity to normal cells is major limiting factor
careful dose calculation
- body surface area/derived from renal function
- individual dose adjustment based on prior dose toxicity

Question 20

myelosuppression

Answer 20

kill normal cells and cancer cells in the bone marrow. This lowers the number of normal red blood cells, white blood cells, and platelets in the blood and bone marrow.

Question 21

mucositis

Answer 21

when your mouth or gut is sore and inflamed

Question 22

combination chemotherapy uses drugs with

Answer 22

• with different mechanisms of action
• with different dose-limiting toxicities to minimise damage to and one organ system
• in optimum dose and schedule
• with minimum interval between cycles
• monitoring response, performance status and toxicity

Question 23

new signal transduction inhibitors

Answer 23

growth factor antibodies - bevacizumab anti VEGF
growth factor receptor antibodies - trastuzumab anti HER2/cetuximab anti HER1
receptor anatgonists - imatinib/gefitinib/erlotinib

Question 24

drug discovery: target selection occurs by

Answer 24

understanding the mechanism of disease
genome based approaches

Question 25

drug discovery: lead identification

Answer 25

lead compounds, chemical entities active against the target that could potentially be turned into a new drug hits generated by screening programme are analysed in more detail - hit to lead retest hits good hits = have selectivity for the target, good solubility and other favourable properties

Question 26

drug discovery: lead optimisation

Answer 26

hits become lead compounds may lack sufficient activity - need to be refined pharmacokinetics are also important : Absorbtion Distribution Metabolism Excretion (ADME) desired properties vary depending on intended usage - BBB - drugs for the CNS require a lower molecular weight/lower total polar surface area/lower efflux transporters e.g. P-glycoprotein

Question 27

drug discovery: preclinical development

Answer 27

safety adssessment is preformed 4 main types of adverse reactions 1. exaggerated effects of drug 2. side effects unrelated to the drug pharmacology 3. toxic side effects unrelated to the drug pharmacology 4. idiosyncratic reactions not uncommon for drugs to be withdrawn for safety reasons after licensure

Question 28

drug discovery: clinical development

Answer 28

pharmacokinetics - short term toxicity and initial dose finding toxicology analysis pharmacokinetics - absorption and elimination of the drug and the typical dose achieved in the body pharmacodynamics may be explored pre-formulation studies are also preformed

Question 29

luminal A: SERMs

Answer 29

selective oestrogen receptor modulators e.g. Tamoxifen not steroids but have a similar tertiary structure which allows them to bind to oestrogen receptor main endocrine therapy for breast cancer reduces recurrence by 42% with 5 years treatment improves survival in early breast cancer reduces risk of developing contralateral primary tumour

Question 30

the tumour immune microenvironment 3 groups: infiltrated-inflamed

Answer 30

infiltrated-inflamed - adaptive anti-immune mechanisms may be overcome by e.g. anti-PD1 checkpoint blockade

Question 31

the tumour immune microenvironment 3 groups: infiltrate excluded

Answer 31

infiltrate excluded - immune cells trapped at the tumour:stroma interface - respond poorly to immunotherapies - effector cells can't enter the tumour

Question 32

the tumour immune microenvironment 3 groups: infiltrated-tertiary lymphoid structures

Answer 32

infiltrated-tertiary lymphoid structures - TLS present at invasive margin of tumour may suggest ongoing immune priming - associated with better prognosis

Question 33

why the body provides cancer with it's essential stroma

Answer 33

tumours are wounds that never heal wound healing - hemostasis - humoural inflammation - cellular inflammation - angiogenesis - generation of mature connective stroma wound healing is self limiting - tumours continuously secrete factors that cause persistent angiogenesis and stroma formation

Question 34

extracellular matrix

Answer 34

network of proteins and proteoglycans = ECM basis for cell anchorage collagen = most abundant structural protein in the body to allow for tumour growth the ECM must be remodelled, this also allows for tumour dissemination proteases e.g. MMPs breakdown the ECM allowing for remodelling in cancer high levels of MMPs correlates with a more invasive phenotype and worse outcome components of the ECM also signal to cells

Question 35

oxygen levels and anatomical location

Answer 35

O2 levels vary with anatomical location as distance from the vessel increases there is - increased acidity - increased lactic acid - decreased ATP - decreased O2 hypoxia is the key driver of angiogenesis

Question 36

stromal cells contribute to tumour angiogenesis

Answer 36

VEGF is released from ECM and inflammatory cells FGF2 is produced by cancer associated fibroblasts macrophage recruitment is associated with increased vessel density

Question 37

inhibition of angiogenesis

Answer 37

- inhibit the production of angiogenic factors - interferon alpha/trastzumab/herceptin - neutralising antibody - bind to the angiogenic factor and stop it from binding to its receptor - soluble receptor - antibody binds to the soluble receptor instead of the one on the target cell - blocking antibody - blocks the receptor and stops the antibody from binding small molecule inhibitor/RTK inhibitor - targets the intracellular RTK domain and stops signalling

Question 38

VEGF signalling inhibition

Answer 38

monoclonal antibodies - Bevacizumab/Avastin soluble receptor - VEGF trap/aflibercept small molecule inhibitors of VEGFR - Sunitinib/sutent, sorafenib/nexavar antibody blocking binding of VEGF to receptor - ICM 1C11

Question 39

VEGF in renal cell carcinoma

Answer 39

tumour suppressor VHL frequently lost in RCC increased expression of VEGF promotes tumour growth sorafenib, sunitinib and pazopanib are used at 1st line therapies used as monotherapies sorafenib = 1st line treatment - targets both tumour cells and tumour vessel formation - targets both VEGFR/PDGFR and Raf

Question 40

potential mechanisms of resistance to VEGF mediated therapy

Answer 40

production of angiogenic factors, hypoxia tolerance or inc. invasiveness of more malignant and hypoxic tumour cells - FGFs/chemokines/PIGF vessel lining by tumour cells/ECs derived from putative CSCs, vessel co-option or intussusception - vessels lined by tumour cells pericyte-covered tumour vessels BMDC recruitment or CAF activation - macrophages/fibroblasts-PDFG/CD11b+GR1+ myeloid cells-BV8

Question 41

nanoparticle accumulation in abnormal tumour vasculature

Answer 41

accumulation of nanoparticles via enhanced permeation and retention poor lymphatic drainage promotes retention of nanoparticles leaky vessels and poor lymphatic drainage allow accumulation of nanoparticles in the tumour

Question 42

luminal A: genomic action of oestrogen E2

Answer 42

ER located in the nucleus associated with HSP90 E2 = steroid passes through membrane causes dimerisation and phosphorylation increases binding of co-activators and releases co-repressors transcriptional activator factor (TAF) 1 and 2 regions within receptor activated increases transcription of genes (e.g. PR/IGF/TGFalpha) non-oestrogen bound state - bound by a co-repressor to keep the receptor in an non-active state inside the nucleus oestrogen reaches the cell and passes through the outer and nuclear membranes causes the receptors to dimerise release of the co-repressors recruitment of the co-activators allows for interaction with the DNA and transcription

Question 43

luminal A: tamoxifen uses

Answer 43

20mg per day - no benefit of an inc dose 1st test by IHC for presence of ER used for - neo-adjuvant treatment of locally advanced disease adjuvant systemic therapy - reduces risk of metastatic spread - metastatic disease - 40% response rate often used in combination with chemotherapy - better than chemo alone

Question 44

luminal A: tamoxifen mechanism of action

Answer 44

selective modulation - alters the conformation (blocking TAF-2 activity) - alters co-regulator binding competitive binding: TAF-2 activity is blocked, TAF-1 is still active - no gene transcription blocks G1 (cytostatic) tamoxifen blocks AF-2 activation partially inactivated transcription (reduce rate of tumour cell division)

Question 45

luminal A: aromatase inhibitors (AIs)

Answer 45

prevents peripheral conversion of androgens to oestrogen in the tissues by aromatase enzymes particularly important in post-menopausal women e.g. letrozole/anastrazole/exemestane

Question 46

HER2 over-expressing breast cancer

Answer 46

ER and PR absent responsive to herceptin

Question 47

basal like breast cancer

Answer 47

generally triple negative (ER/PR/HER2 -ve) high metastasis rate young associated with BRCA1 germline mutations more sensitive to chemotherapy PARP inhibitors

Question 48

the warburg effect: cachexia

Answer 48

large tumour burdens use so much nutrients that the plasma levels are depleted the body goes into a starvation like mode in which it catabolises the amino acids that from the muscle mass

Question 49

current chemotherapies that target cancer metabolism

Answer 49

5-fluorouracil (5-FU) disrupts nucleotide synthesis by blocking thymidylate synthase (TS) TS is part of the metabolic pathway to synthesise dTTP elicit a DNA damage response by being incorporated into the DNA and RNA side effects affect fats dividing ells e.g. hair loss/GI disturbances/dry skin/etcv

Question 50

novel approaches e.g tumour acidification, glycolysis, PK etc

Answer 50

targeting tumour acidification - target carbonic anhydrase - may cause issues in the kidneys/other tissues target glycolysis - result in death as no 2-deoxyglucose pyruvate kinase - energy generating step of glycolysis - isoforms M1/2 - M1 expressed in differentiated/non-proliferating cells, M2 expressed in proliferating/embryonic cells - target M2 LDH - required for NAD+ regeneration for continued glycolytic activity - ubiquitous LDHA/LDHB expression - side effects: muscle activity/brain function/kidney function

Question 51

metastasis is an inefficient process because

Answer 51

at every stage at which the cells enter a new environment the cells must learn to survive lots of tumour cells die whilst attempting to metastasise

Question 52

selectins

Answer 52

carbohydrate binding cell adhesion proteins (L/P/E-selectin) carbohydrate ligands for selectins are covalently linked to mucins + proteoglycans on the surface of tumour cells selectins trigger biochemical signals that stop rolling of tumour cells of the endothelium - activation of integrins - inc chemokine production - inc production of MMPs

Question 53

pRB regulation by p16^ink4a Cdk inhibitor

Answer 53

in response to cellular stress p16^ink4a binds to Cdk4/6 and Cyc D to inhibit the phosphorylation of pRB causing growth arrest and cellular senescence p16^ink4a is inhibited in cancer via mutation/epigenetic silencing - cause pRB to be phosphorylated allowing for passage through the restriction point and G1-S progression

Question 54

TRFs-Shelterin complex function

Answer 54

telomere repeat binding factors (TRFs)-Shelterin bind telomeric duplex DNA TANK, TRF1, TRF2, POT1 and TIN2 are altered in some tumours

Question 55

TRF2 and POT1 function

Answer 55

protect telomeres from DNA damage surveillance TRF2 inhibits ATM which causes a DNA damage signal and causes NHEJ repair POT1 inhibits ATR which causes a DNA damage signal and causes NHEJ repair

Question 56

regulation of telomeric chromatin by pRB and SUV39H

Answer 56

regulation of telomeric chromatin by pRB and SUV39H

Question 57

bioinformatics

Answer 57

large data sets allow for the identification of driver/passenger mutations across several tumour types e.g. 100000 genomes project/cancer genome atlas high read exon depth or whole genome sequencing of tumour/normal pairs to identify all the drivers in a cancer - need to look at the mutations at each point in the cancer's darwinian evolution software used: MutSigCV/Genome MUSIC/Intogen - use multiple packages at once - may lose sensitivity

Question 58

bioinformatics - issues

Answer 58

cancer background mutation rate - difficult to differentiate noise from signals - some tumours are more hyper mutated than others over come by - identifying genes that are more mutated than the background mutation rate - identifying genes that have a functional impact - identifying mutations that cluster together in regions of high impact

Question 59

causes of apoptosis

Answer 59

DNA damage signalling causes activation of the transcription factor p53 p53 promotes apoptosis if DNA damage is not repaired p53 activates pro-apoptotic genes: PUMA/BAX (intrinsic pathway) and FAS (extrinsic pathway) many chemotherapy agents aim to cause DNA damage by inducing apoptosis by p53 activity active p53 promotes apoptosis by repressing Bcl-2 expression MDM2 prevents apoptosis by exporting p53 form the nucleus and thus blocking its activity

Question 60

apoptosis: cancer

Answer 60

faulty apoptosis can lead to accumulation of cancer-causing mutations in DNA faulty apoptosis: - over activation of anti-apoptotic genes e.g. Bcl2 - over activation of pro-survival pathway genes e.g. PI3 kinase - down-regulation of death receptors - mutations in p53 (>50% all human cancers) many chemotherapy agents fail due to the absence of functioning p53 alternative methods - stimulate death receptors - down-regulate Bcl2 - introduce wild-type p53 into cells

Question 61

autophagy: functions in health and disease

Answer 61

maintain healthy cells in stress/starvation conditions inhibition causes neurodegenerative disease/liver disease/cancer hyper activity causes neurodegenerative disease/skeletal and cardiac muscle degeneration/cancer and inc. resistance to chemotherapy

Question 62

autophagy: cancer

Answer 62

active autophagy has pro-tumour effects - tumour cells can cope with stress and resistant to treatment active autophagy has anti-tumour effects - prevents chromosomal instability early cancer stages = anti-tumour effects/late stages = pro-tumour effects success of cancer treatment = dependent on autophagy

Question 63

TSGs and genome integrity

Answer 63

bi-allelic loss of function - no immediate impact long term increase in accumulation of mutations acquisition of mutations in TSGs/oncogenes leading which dysregulates cell growth and accelerates cancer progression eg. p53/ATM/BRCA1/BRCA2

Question 64

oncogene activation: point mutations e.g.

Answer 64

receptor tyrosine kinase activating mutations result in increased kinase activity/ligand independent activity EGFR - in frame deletion aa747-752 -> changes to protein conformation and prolongs active dimer configuration/missense mutation L858R (leu>arg) -> inc. kinase activity 50-fold EGFR/RAS freq mutated in cancers RAS mutations at G12/G13/Q61 - cause a decrease in GTP hydrolysis and lock RAS in the active GTP-bound state mutations in hotspots that encode for important functional domains mostly missense mutations resulting in GOF

Question 65

oncogene activation: amplification of genomic DNA e.g.

Answer 65

MYCN in neuroblastoma amplification can be used as a prognostic factor MYCN associated with aggressive disease, metastatic potential, therapeutic resistance and poor patient outcome EGFR amplification in glioblastomas

Question 66

oncogene activation: chromosome translocation to a different promoter e.g.

Answer 66

Burkitt lymphoma (BL) translocations involving c-Myc from chromosome 8 to 1 of 3 immunoglobulin gene loci t(8;14)(q24;q32) t(8:14) translocation places IgH enhancer adjacent to c-Myc which drives c-Myc protein overexpression c-Myc causes cell growth/proliferation/ribosomal + protein synthesis/metabolism/energy generation BCL2-IGH translocations in follicular lymphoma - leads to overexpression of BCL2, a pro-survival gene that counteracts apoptosis

Question 67

oncogene activation: chromosome translocation joining 2 genes e.g.

Answer 67

chronic myeloid leukaemia (CML) t(9:22) translocation = a hallmark of CML creates the Philadelphia chromosome creates a hybrid BCR-ABL fusion protein, which has novel TK activities constitutively activates the ABL TK activity leads to increased proliferation, survival and therefore overproduction of granulocytes leading to CML

Question 68

cancer causing viruses

Answer 68

EBV and KSHV - big viruses HBV and HCV - small viruses HTLV1, HPV16/18/24, MPV associated but not causing = HIV

Question 69

Epstein Barr Virus and cancer

Answer 69

asymptomatic infection during childhood long latency period 95% of adults infected causes endemic Burkitt lymphoma, age 4-7, 100% EBV sporadic Burkitt lymphoma, age 11-30, % EBV AIDS associated burkitt lymphoma Hodgkin lymphoma, ages 20-24 or 70-80 % EBV

Question 70

control of EGFR signalling

Answer 70

switched off by removal of extracellular signal switch off activated receptor TKs by protein tyrosine phosphatases (PTPs) dephosphorylation of target proteins by serine/threonine phosphatases PTEN removal of PIP3 signal if not tightly controlled - leads to cancer predisposition

Question 71

developing antibody-drug conjugates

Answer 71

Trastuzumab-(DXd/DM1) humanised ab - specific to minimise off target effects, ab-antigen conjugates leads to receptor internalisation linker - conjugates to ab payload - cytotoxic compound many mechanisms of resistance e.g. loss of PTEN/amplification of AKT/activating muations in PI3/ increased expression - EGFRorErb3R/decreased HER2 expression

Question 72

the receiver - determining a threshold for a positive test in SM

Answer 72

the receiver - operator curve that models lots of scenarios, enables us to pick an optimum score for a +ve test

Question 73

e.g.s of stratifies

Answer 73

prognostic - CMS in colorectal cancer predictive - DDRD stratification assay in breast cancer

Question 74

breast cancer progression

Answer 74

epithelial proliferation -> lobular/ductal -> high/low grade

Question 75

define tumour heterogeneity

Answer 75

once established cells within a tumour will continue to divide and may acquire mutations/epigenetic changes that result in the formation of subclones - important in tumour progression