extra CSM Flashcards
copy number variation
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
large deletions or insertions detected by
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
structural variants with NGS
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
CTCs/ctDNA as a cancer detection method
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
immunotherapy
chimeric antigen receptor T cells (CAR T cells)
1. T cell collection
2. T cell transfection
3. T cell adoptive transfer
4. patient monitoring
CTCs uses
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
ctDNA detection e.g. point mutation by Taqman PCR
point mutation detection by Taqman PCR
- 1 pair of primers
- 1 probe complimentary to wt sequence
- 1 probe complimentary to mutant sequence
ctDNA detection e.g. Next Generation Sequencing
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
cfDNA
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
unique molecular identifiers (UMIs)
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
CA-125 as a screening tool in ovarian cancer
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
assessing treatment efficacy: survival time
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
challenges of cancer recognition by the immune system
cancer cells look extremely similar to normal cells
T cells that recognise self cells are destroyed during development - immune tolerance
cancer’s ways to evade immune recognition
- immunosuppressive cytokines
e.g. TGF-beta/IL-10
the tumour micro environment is complex and often very immunosuppressive - down regulation of MHC
- down regulation of components of antigen processing pathways
- exploiting T cell checkpoints
prevent T cell activation by interfering with T cell check points
cancer vaccine types
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
chemotherapy: clinical uses
- for advanced disease
- where no treatment exists - adjuvant chemotherapy
- systemic treatment following local radiotherapy or surgery
- to control microscopic metastases - 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
chemotherapy drugs type for the cell cycle phases
G1/G0: micro-tubule inhibitors/topoisomerase inhibitors/alkylating agents
S: anti-metabolites/topoisomerase inhibitors
G2: platinum analogues
M: micro-tubule inhibitors
what are the benefits of delivering chemotherapy drugs at an increased intensity
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
chemotherapy and toxicity
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
myelosuppression
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.
mucositis
when your mouth or gut is sore and inflamed
combination chemotherapy uses drugs with
- 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
new signal transduction inhibitors
growth factor antibodies - bevacizumab anti VEGF
growth factor receptor antibodies - trastuzumab anti HER2/cetuximab anti HER1
receptor anatgonists - imatinib/gefitinib/erlotinib
drug discovery: target selection occurs by
understanding the mechanism of disease
genome based approaches
drug discovery: lead identification
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
drug discovery: lead optimisation
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
drug discovery: preclinical development
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
drug discovery: clinical development
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
luminal A: SERMs
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
the tumour immune microenvironment 3 groups: infiltrated-inflamed
infiltrated-inflamed - adaptive anti-immune mechanisms may be overcome by e.g. anti-PD1 checkpoint blockade
the tumour immune microenvironment 3 groups: infiltrate excluded
infiltrate excluded - immune cells trapped at the tumour:stroma interface - respond poorly to immunotherapies - effector cells can’t enter the tumour
the tumour immune microenvironment 3 groups: infiltrated-tertiary lymphoid structures
infiltrated-tertiary lymphoid structures - TLS present at invasive margin of tumour may suggest ongoing immune priming - associated with better prognosis
why the body provides cancer with it’s essential stroma
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
extracellular matrix
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
oxygen levels and anatomical location
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
stromal cells contribute to tumour angiogenesis
VEGF is released from ECM and inflammatory cells
FGF2 is produced by cancer associated fibroblasts
macrophage recruitment is associated with increased vessel density
inhibition of angiogenesis
- 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
VEGF signalling inhibition
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
VEGF in renal cell carcinoma
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
potential mechanisms of resistance to VEGF mediated therapy
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
nanoparticle accumulation in abnormal tumour vasculature
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
luminal A: genomic action of oestrogen E2
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
luminal A: tamoxifen uses
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
luminal A: tamoxifen mechanism of action
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)
luminal A: aromatase inhibitors (AIs)
prevents peripheral conversion of androgens to oestrogen in the tissues by aromatase enzymes
particularly important in post-menopausal women
e.g. letrozole/anastrazole/exemestane
HER2 over-expressing breast cancer
ER and PR absent
responsive to herceptin
basal like breast cancer
generally triple negative (ER/PR/HER2 -ve)
high metastasis rate
young
associated with BRCA1 germline mutations
more sensitive to chemotherapy
PARP inhibitors
the warburg effect: cachexia
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
current chemotherapies that target cancer metabolism
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
novel approaches e.g tumour acidification, glycolysis, PK etc
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
metastasis is an inefficient process because
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
selectins
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
pRB regulation by p16^ink4a Cdk inhibitor
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
TRFs-Shelterin complex function
telomere repeat binding factors (TRFs)-Shelterin bind telomeric duplex DNA
TANK, TRF1, TRF2, POT1 and TIN2 are altered in some tumours
TRF2 and POT1 function
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
regulation of telomeric chromatin by pRB and SUV39H
regulation of telomeric chromatin by pRB and SUV39H
bioinformatics
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
bioinformatics - issues
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
causes of apoptosis
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
apoptosis: cancer
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
autophagy: functions in health and disease
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
autophagy: cancer
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
TSGs and genome integrity
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
oncogene activation: point mutations e.g.
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
oncogene activation: amplification of genomic DNA e.g.
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
oncogene activation: chromosome translocation to a different promoter e.g.
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
oncogene activation: chromosome translocation joining 2 genes e.g.
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
cancer causing viruses
EBV and KSHV - big viruses
HBV and HCV - small viruses
HTLV1, HPV16/18/24, MPV
associated but not causing = HIV
Epstein Barr Virus and cancer
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
control of EGFR signalling
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
developing antibody-drug conjugates
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
the receiver - determining a threshold for a positive test in SM
the receiver - operator curve that models lots of scenarios, enables us to pick an optimum score for a +ve test
e.g.s of stratifies
prognostic - CMS in colorectal cancer
predictive - DDRD stratification assay in breast cancer
breast cancer progression
epithelial proliferation -> lobular/ductal -> high/low grade
define tumour heterogeneity
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
