CSM Flashcards
What is the evolutionary view of cancer?
cancer occurs when a somatic cell divides out of control and potentially destroys the germline. To prevent cancer, there are multiple systems for DNA repair and immune control of transformed cells.
What are some protective measures against cancer?
- selection of tumour suppressor genes
- DNA repair mechanisms
- immune control of transformed cells
the development of cancer
occurs when there is damage to the genetic material
cancer cells acquire characteristics - the hallmarks of cancer
a combination of genetic and environmental factors determine if an individual gets cancer
Germline - each individual carries a level of risk of developing cancer, genetic disorders can predispose to cancer e.g. A-T and FA
Environment - common epithelial tumours can be prevented by changes in lifestyle e.g. smoking, high BMI, infection
the development of cancer therapy
development of chemotherapy
alkylating agents identified through toxic effect of mustard gas
folate antagonists developed from anaemia treatment
folic acid as a treatment for anaemia, given to individuals with leukaemia cause an acceleration of symptoms
folic acid antagonists developed as a chemotherapy
What are some risk factors for breast cancer?
-lack of pregnancies, alcohol consumption and obesity
the treatment has improved
- surgery (less extensive)
- radiotherapy (addition of lumpectomy)
- adjuvant chemotherapy
- hormonal therapies (best prognostic factor = ER+ can use tamoxifen)
What is the Philadelphia chromosome?
The Philadelphia chromosome is a genetic abnormality that characterises chronic myeloid leukaemia .
What is imatinib used for?
Imatinib is used to block the action of the abnormal fused protein caused by the Philadelphia chromosome in chronic myeloid leukemia.
What are some future prospects for cancer management?
prevention - lifestyle/risk factors/diet/vaccination
detection - screening/determining genomic risk
treatment - personalised therapy (base on genome sequence)/targeted drug combinations/cost + side effects considered
What is a biomarker?
biomarker is a measurable indicator of a biological state, process, or condition that can be used to assess a patient’s health status or response to a treatment.
What are the features of a good biomarker?
A good biomarker should be sensitive, specific, reproducible, and cheap.
How do you measure a good biomarker?
measured using statistical determination of accuracy, sensitivity, specificity, positive predictive value, and negative predictive value.
How does the predictive value of a biomarker depend on the prevalence of the disease?
The predictive value of a biomarker depends on the prevalence of the disease, with more common diseases having a higher predictive value.
Receiver operator characteristic curve (ROC) shows how sensitivity and specificity vary with the threshold, how does it do this ?
The ROC curve shows the trade-off between sensitivity and specificity. It shows that the standard test is still the most accurate test (AUC=0.9125) in identifying men with prostate cancer, and biomarker B (AUC=0.5123) is the worst and shouldn’t be considered as a test for identifying men with prostate cancer.
What are the different types of biomarkers?
simple - single gene
complex - multi-gene/multiple types
predictive - of response to therapy
prognostic - predictive of survival/disease progression
What is the CMS classifier of colorectal cancer
The CMS classifier is a prognostic tool used in RNA microarray datasets to identify distinct clusters of patients with the same gene expression pattern in colorectal cancer
finding stratifiers
2 cohorts
1st - discovery - small sample, whole genome
2nd - validation - large sample, focussed
choose the cohort with the extremes of the disease e.g. no/response to radiotherapy
What is the DNA damage response and repair deficiency (DDRD) stratification assay in breast cancer?
The DDRD stratification assay is a predictive biomarker used in RNA microarray datasets to identify distinct clusters of patients with the same gene expression pattern, which can predict survival and response to chemotherapy
How can biomarkers be found?
Biomarkers can be found through two cohorts, discovery, and validation, by comparing cohorts with extremes of disease, i.e., response/no response.
What is the role of tumour immunity in colorectal cancer?
tumour-infiltrating lymphocyte density, is prognostic and predictive in colorectal cancer, and immune infiltration has been strongly associated with outcomes.
What are the different types of immunotherapies being investigated in CRC?
adoptive cell therapy, cancer vaccines, and checkpoint blockade.
What is FOCUS4?
FOCUS4 is a molecularly stratified clinical trial that aims to identify the most effective treatments for colorectal and oesophageal cancer patients by carrying out large-scale molecular characterisation using next-gen sequencing.
What are some developing biomarkers for the future?
Developing biomarkers for the future include stratification in colorectal cancer and oesophageal cancer clinical and molecular stratification studies designed to carry out large-scale molecular characterisation of cancers using next-gen sequencing.
What is neoplasia?
It is a new growth characterized by abnormal and continuous growth of cells no longer subject to the homeostatic controls that maintain the appropriate number of cells.
cellular basis of neoplasia
1 cell with a beneficial mutation/epigenetic change proliferates until it forms a group of identical cells/ a clone. Cells within the clone may go on to acquire advantageous mutations/epigenetic changes that further enhance the cancer growth and survival.
cell w/ mutation -> hyperplasia -> dysplasia -> in situ cancer -> invasive cancer
What is HPV?
human papilloma virus
99% of all cervical cancers
causes vulval and vaginal warts/pre-cancerous lesions
HPV vaccine introduced in 2006
HPV infection and the formation of squamous neoplasia
normal cells -> HPV infection -> low grade SIL -> de-regulation of E6/7 -> high grade SIL -> invasive cancer
at each stage advances in telomerase activation, inhibition of apoptosis, genetic changes and immune response occur
How does cancer arise?
A cell with a beneficial mutation or epigenetic change may continue to divide until a collection of identical cells or clone is formed.
Cells from this clone may acquire new genetic and epigenetic changes which further enhance their growth and survival.
Cell with mutation → hyperplasia → dysplasia → in situ cancer → invasive cancer.
What is in situ cancer and invasive cancer?
In situ cancer is not able to invade the basomembrane stroma muscle or blood vessels.
Invasive cancer has invaded the basomembrane.
What is tumour heterogeneity?
it is the presence of multiple subclones, each with differing properties
what are cahracteristics of tumour cells in vitro and in vivo and in general
In vitro: abnormal morphology/shape and can multiply wihtout attachement to a substrate
In vivo: transformed cells can form tumours in immune com, promised hosts
- genetic modification: abnormal chromosomes numbers and gene amplification
- dont require growth factors
- dont senescence
- invasive behaviour
What are the key morphological prognostic factors?
Tumour size
Nodal status
Tumour grade
What is the staging system for colon cancer?
Dukes A-D
A - 5 yr survival > 90%
B - 5 yr survival 55%-85%
C- 5 yr survival 20-55%
D - 5 yr survival < 5%
What is carcinogenesis?
It is the process where environmental agents that damage DNA such as chemicals, radiation, and viruses lead to DNA damage in normal cells.
This leads to mutation in somatic cells which can lead to activation of growth-promoting oncogenes, impaired apoptosis, and inactivation of tumour suppressor genes.
These all lead to altered gene products such as abnormal structural and regulatory proteins that lead to a malignant tumour.
What is cancer grading?
system of classifying cancer based on the degree of similarity to tissue of origin and other factors.
Grade 1 is well-differentiated, Grade 2 is moderately differentiated, Grade 3 is poorly differentiated, and Grade 4 is anaplastic.
What are the key prognostic factors?
Grade - how bad do the cells look? The degree of similarity to tissue of origin grade 1-3
Stage - how far has the cancer spread? TNM staging = Tumour, Nodes, Metastases
What is a predictive marker?
It is the measurement associated with response to a given therapy.
What is a prognostic factor?
A situation or condition, or a characteristic of a patient, that can be used to estimate the chance of recovery from a disease or the chance of the disease recurring (coming back).
What are the key histological classifications of cancer?
Cytology cells such as in a pap smear
Benign vs malignant histology (tissue)
Prediction versus prognosis
What percentage of cancers are caused by infectious agents, and what percentage of those are caused by viruses?
Infectious agents are associated with 16% of cancers, and 12% of those are caused by viruses, which is equivalent to 2,230,000 new cases per year.
What are some examples of cancer-causing viruses?
Epstein-Barr virus (EBV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Kaposi’s sarcoma herpes virus (KSHV), Human T-cell lymphotropic virus T1, and Merkel cell polyomavirus.
Is HIV a cancer-causing virus?
HIV is not a cancer-causing virus. It suppresses the immune system, but it does not directly lead to cancer.
What are some of the global differences in the distribution of cancers associated with infectious agents?
In developing nations, the cancers associated with infectious agents are breast, lung, bladder, non-Hodgkin’s lymphoma, cervix uteri, liver, prostate, colorectal, esophagus, stomach, and oral cancer.
In low-income countries, 26% of cancers are caused by viruses, while in high-income countries, only 8% are caused by viruses. This is due to differences in monitoring systems, access to vaccination, amount of HIV suppressing the immune system, exposure to pathogens, and access to treatment.
Why is it difficult to establish a link between a virus and cancer development?
long latency period between primary infection and tumor development, only a small percentage of virus-infected individuals develop the tumor, and there are no animal models of human virus-associated cancers.
What is the Epstein Barr virus and what cancers is it associated with?
The Epstein Barr virus (EBV) is a herpesvirus with enveloped dsDNA, 172 kb. It is associated with nasopharyngeal carcinoma, Burkitt’s lymphoma, and Hodgkin’s lymphoma, among others.
long latency period btw primary infection and tumour development
only a small % of virus infected individuals go on to develop cancer
NO animal models for virus associated cancer
How is the link between a virus and cancer proven?
virus infection always precedes cancer development, the virus is present in the malignant cells and not the surrounding non-malignant cells, and by using techniques like representational difference analysis to identify the virus.
What are some ways that viruses can indirectly cause cancer?
the virus must be present along with other factors e.g. immunosuppression/chronic inflammation/UV exposure/genetic changes/other infections
e.g. EBV + malaria + cMyc translocations - endemic Burkitt lymphoma
Kaposi sarcoma + HIV
HPV + UV + mutations
HBV + hepatocellular carcinoma
Can you provide some examples of viruses that can indirectly cause cancer?
EBV, malaria, and cMyc translocation leading to endemic Burkitt lymphoma; Kaposi’s sarcoma in immunosuppressed patients; skin cancer in epidermodysplasia verciformus patients with HPV-5/8 and UV light exposure; and HBV causing hepatocellular carcinoma.
How do direct carcinogens contribute to cancer cell transformation?
encode viral oncogenes that directly contribute to cancer cell transformation, inhibit a tumor suppressor gene, or directly activate a cellular oncogene.
Viruses as direct carcinogens?
introduce a viral oncogene into the host cell which directly contribute to cancer cell transformation e.g. HPV E6/7 or EBV LMP1 or MCP Large T
directly activates a cellular oncogene
directly inhibits a cellular TSG
viral genes that control cell proliferation/survival, immune regulations = important in cancer development
clonal integration of the viral DNA into the host
Large DNA viruses
- encode proteins necessary and sufficient to replicate themselves
- Do not need a host cell DNA replication machinery
- establish latency in slow-replicating or non replicating cells
Small DNA viruses
- Do not encode proteins to replication themselves
- require host cell DNA replicaition machinery to replicate DNA
- must deregulate the growth control of the infected cells
- consequences for the host can be serious
Human papillomavirus
genome divided into - early (non-structural)/late (structural)/non-coding regions
RNA splicing to generate multiple RNAa - dependent upon host cell for replication and transcription
How is the HPV lifecycle and cell cycle control
reliant on the induction of host cell replication factors and cell cycle regulators
Early proteins E6/7 stimulate growth
HPV encodes E7 which drives differentiating cells into S phase - E7 binds to RB - releases the E2F1 TF - causes the transcription of S phase genes inducing cyclin E and P53
P53 - removes virus infected cells via cell cycle arrest/apoptosis
HPV encodes E6 which recruits cellular E6AP (cellular ubiquitin ligase) to degrade P53 - causes an accumulation of chromosomal mutations - including oncogenic mutations
drives cervicale cancer
What is RB and how does it relate to cell cycle progression to S phase?
RB is a transcription factor that binds to E2F1 transcription factor for the S phase genes. Growth factor binds to the surface of the cell to cause activation of cyclin D which phosphorylates RB that releases E2F1 transcription factor to activate S phase genes.
What is Merkel cell polyomavirus (MCV) and what are some of its characteristics?
cause Merkel cell carcinoma - skin cancer of Merkel cells
higher incidence in immunosuppressed/elderly
tumours appear on head and neck
What is a Merkel cell and where is it found?
Sensory-mechanoreceptor located in the stratum basale. It is a light touch receptor found in the skin and is abundant in fingertips. It makes synaptic contacts with somatosensory afferent nerve fibers.
What is the role of large T antigen in Merkel cell polyomavirus replication?
Large T antigen plays a large role in Merkel cell polyomavirus replication by binding to the origin of replication of the viral DNA, acting as a helicase to unwind DNA, recruiting host DNA polymerase complex to this site, and initiating DNA replication.
To replicate DNA, they use host DNA synthesis machinery. These proteins are produced in the S phase of the cell cycle, and polyomavirus manipulates resting cells to enter the cell cycle.
What are some methods of controlling infection with oncogenic viruses?
vaccination
screening
What are some non-genetic factors that contribute to the development of cancer?
tobacco use, diet, obesity, alcohol consumption, occupation, radiation exposure (both UV and ionizing), and infections.
air pollution components
PM
sulphur dioxide
ozone
carbon monoxide
nitrogen dioxide
polycyclic aromatic hydrocarbons (PAHs)
What are some examples of exposure carcinogens?
chimney smoke, vinyl chloride, benzene, radium, arsenic, and other harmful chemicals.
What is the link between nicotine addiction and lung cancer through tobacco smoke cigarettes?
contains over 60 carcinogens that can cause various types of cancer, including lung, oral, nasal, and esophageal cancer. The carcinogenic process is central to the DNA adducts induced by tobacco products.
What is asbestos, and how does it cause cancer?
aturally occurring fibrous silicate material. Prolonged inhalation of asbestos fibers can lead to fatal illnesses, such as pleural mesothelioma, which is caused almost exclusively by exposure to asbestos.
What is asbestos, and how does it cause cancer?
aturally occurring fibrous silicate material. Prolonged inhalation of asbestos fibers can lead to fatal illnesses, such as pleural mesothelioma, which is caused almost exclusively by exposure to asbestos.
How does obesity contribute to the development of cancer?
altered adipokine productions
subclinical chronic inflammation
altered steroid metabolism
insulin resistance
altered metabolic microenvironment
altered microbiomeExcess weight can increase the risk of developing certain types of cancer, such as endometrial and renal cancer, due to factors such as insulin resistance, increased estrogen levels, pro-inflammatory cytokines, and changes in the microenvironment of the stomach.
What are some prevention strategies for reducing the risk of developing cancer?
education
vaccination
control of occupational hazards
reduction to sunlight exposure
how does alcohol cause cancer
chronic ethanol consumption leads to production of acetaldehyde and induction of oxidative stress and conversion of pro carcinogens to carcinogens
what are phytochemicals
Non-nutritive substances found in vegetables that appear to have a positive effect on health, possess anti-carcinogen and anti-mutagenic properties
What is intracellular signalling?
set of linked biochemical events that connect a specific biological stimulus with a specific cellular response.
What are the major growth factor signalling pathways?
cyclic AMP - PKA
Ca2+ - CaM-kinase/PKC
Ras MAP-kinase
PIP3 - PKB
What is the role of phosphorylation in cell signaling?
biochemical changes - changes in protein shape/charge/size
functional changes - localisation/enzyme activity/complex formation/stability
What are the major types of cell surface receptors?
tyrosine kinase receptors (TKR) and G-protein coupled receptors (GPCR).
G protein coupled receptors
7 transmembrane domain
activates downstream signalling through the activation of G proteins
energy source = GTP
e.g. beta-adrenergic Rs/prostaglandin E2 Rs/rhodopsin
What is the EGF receptor family?
EGFR1-4, which form homodimers and heterodimers with other family members. EGFR1 is activated by a number of ligands including EGF and TGFa, while HER2 (EGFR2) is an orphan receptor and is activated by heterodimerisation with other family members. Overexpression/amplification/mutation of EGFR family members are implicated in about 30% of cancers.
What is the role of phosphatases in cell signalling?
Phosphatases can remove phosphate groups from proteins, allowing for switchable mechanisms to take proteins from one functional state to another by regulating the activity of kinases and phosphatases in cells. This is a form of post-translational modification.
What is the importance of cell signalling pathways and networks?
Controlling cell growth and proliferation, and abnormalities in these pathways can lead to cancer predisposition. Understanding these pathways is important for developing therapy approaches and understanding therapy resistance mechanisms
How do cells communicate with their environment?
Through intracellular signalling pathways, which connect a specific biological stimulus with a specific cellular response. Signalling molecules bind to specific receptors on the cell surface or inside the cell to elicit a response, such as migration, gene expression, or metabolism.
What is RTK dimerization and activation?
binding of a signalling molecule initiates receptor dimerisation
kinase domains can now initiate autophosphorylation on multiple tyrosine residues
which creats high affinity binding sites for proteins e.g. Grb-2 (MAP kinase pathway) and PI 3 kinase
What is the Ras/MAP kinase pathway?
Signalling pathway that regulates cell growth and differentiation.
It involves the activation of RAS, which is an oncogene, leading to the activation of RAF, MEK, and ERK, which promotes cell cycle progression and gene transcription needed for cell survival and proliferation
Ras/MAP kinase pathway complex
cascade of phosphorylations leading to gene expression regulation:
RAS acts as a switch in the MAP kinase pathway
GEF e.g. SOS stimulates exchange of GDP for GTP
RAS is a GTPase - catalyses the hydrolysis of GTP to GDP
the intrinsic activity of RAS is poor need a GAP
e.g. RASA1-3, NF1 - stimulates the intrinsic activity and hydrolysis of GTP
RAS binds to RAF and promotes dimerisation and activation
RAF activation leads to rapid signalling through phosphorylating events
MEK
phosphorylation occurs on an activation segment/loop in the kinase domain
which leads to remodelling of the active site into an open conformation - for ATP and substrate binding
phospho-ERK translocates into the nucleus and activates TF e.g. c-FOS and c-JUN by phosphorylation
leading to changes in expression profiles
What is the PI3K signalling pathway?
Signalling pathway that regulates cell growth, survival, and metabolism.
Ras–>
PI 3-kinase is activated by binding to phosphorylated Try residues on RTKs
PI 3-kinase catalyses the phosphorylation of P1P2 to PIP3
PIP3 acts as a docking site for PDK1 and AKT
upon binding PDK1 phosphorylates AKT
active AKT is released into the cytosol for downstream signalling
How is signalling switched off?
removal of extracellular signals
the inactivation of activated receptor tyrosine kinases by protein tyrosine phosphatases,
the dephosphorylation of target proteins by serine/threonine phosphatases,
removal of the PI(3,4,5)P3 signal by PTEN.
What is PTEN?
PTEN is a tumour suppressor gene that encodes for an enzyme called inositol lipid phosphatase, which removes phosphate from PI(3,4,5)P3 so that it no longer acts as a docking site for PDK1 and AKT. It is commonly lost in cancer, and inactivating mutations in PTEN can promote uncontrolled cell growth and survival.
What is stratified or personalized medicine?
grouping patients according to genetic lesions within individuals and targeting those lesions or the signalling pathway to which they contribute. This approach helps to improve treatment efficacy and reduce unnecessary side effects.
What are humanised antibodies, and what are their advantages?
antibodies that have been modified to decrease their potential for immunogenicity by replacing parts of the antibody with human sequences.
The advantages of humanized antibodies include increased potential for recruiting immune effector mechanisms and decreased potential for immunogenicity.
What are the potential mechanisms of action of anti-EGFR antibodies?
blockade of ligand binding to receptor, inhibition of receptor dimerization, receptor internalisation/degradation, and antibody-dependent cellular cytotoxicity (ADCC).
What are the mechanisms of resistance to trastuzumab?
Loss or downregulation of HER2 receptors: Trastuzumab, a monoclonal antibody, primarily targets the HER2 receptor, which is overexpressed in certain cancers. Resistance can occur when cancer cells undergo a loss or downregulation of HER2 receptors, making them less susceptible to trastuzumab’s effects.
Cancer cells may activate alternative signaling pathways that bypass the HER2 receptor, allowing them to continue proliferating and surviving despite trastuzumab treatment. This can involve the activation of other receptor tyrosine kinases, such as EGFR or MET, which can drive tumor growth independently of HER2.
PIK3CA mutations, can lead to resistance to trastuzumab. These alterations can activate downstream signaling events, rendering the tumor cells insensitive to the inhibitory effects of trastuzumab.
PTEN loss or inactivation:
Loss or inactivation of PTEN can lead to constitutive activation of the PI3K/AKT pathway, promoting cell survival and resistance to trastuzumab.
What is a proto-oncogene?
A normal cellular gene that controls processes such as proliferation, survival, and invasion.
What is an oncogene?
A mutated form of a proto-oncogene that expresses gene products with aberrant activity or overexpresses normal gene products, contributing to the hallmarks of cancer.
What is the dominant effect of an altered copy of a proto-oncogene?
Even if a cell has a normal allele, the presence of a mutated allele leads to altered protein expression due to dominant expression of the mutant gene.
What are the hallmarks of cancer?
- Sustaining proliferative signaling
- Evading growth suppressors
- Activating invasion and metastasis
- Enabling replicative immortality
- Inducing angiogenesis
- Resisting cell death
HM : sustaining proliferative signalling
chronic proliferation
mitogenic signals
mehcanisms:
- autocrine singalling
- elevating number of receptors(hyperresponsive)
- constitutive activation of components of sig pathways
- defects on negative feedback mechanisms such as PTEN
HM: Evading growth supressors
loss of function mutations in TSGs
usually need to deactivate both copies e.g RB
HM: Enabling replicative immortality
cancer cells dont obey the hayflick limit as they express telomerase no dont senescence
HM: Inducing angiogenesis
some oncogenes (Myc, Ras) can upregulate expression of angiogenic factors
HM: activating invasion and metastasis
crosstalk between cancer cells and cell of the neoplastic stroma
- mesenchymal stem cells in tumour stroma secrete CCL5, stimulates invasion
- Macrophages: Metalloproteeinases, EGF
- inflammatory cells: extracellular matrix-degrating enzymes
what are the emerging hallmarks of cancer ?
1) reprogramming energy metabolism, cancder cells limit energy metabolism to glycolysis- aerobic. it allows diversion of glycolytic intermediates into biosynthesis pathways
2) evading immune system, cancer cells may paralse infiltrating CTLs and NK cells, such as TGF-b
What are the mechanisms of oncogene activation?
point mutation -> protein with altered characteristics e.g. EGFR/RAS
amplification of a genomic DNA region that induces the proto-oncogene -> overexpression of the gene and increased amounts of protein e.g. MYCN/EGFR
chromosome translocations that bring a proto-oncogene into close proximity to a different promoter -> inappropriate gene and protein expression e.g. c-Myc/BCR-2
chromosome translocations resulting in the fusion of 2 genes together -> creates a chimeric fusion protein/gene with novel characteristics e.g. PML/RARA
PCM- EGFR
1st generation TKIs - competitive ATP-mimics, reversible binding, freq. drug resistance
2nd generation TKIs - irreversible binding in the ATP pocket
3rd generation TKIs - bind more avidly to EGFR T790M mutants than wild-type EGFR
PCM: BCR-ABL
STI571/Gleevec/Imatinib = specific inhibitor of the BCR-ABL TK
competitive inhibition of ATP binding to active site, inhibiting TK activity
What is the EGFR signaling pathway?
EGF takes a signal to EGFR that passes it down to RAS, RAF, MEK, ERK, and transcription of genes.
What is a receptor tyrosine kinase?
Proteins such as EGFR that exist as inactive monomers in the membrane and, when they bind to EGF, dimerize and activate the receptor. The receptor then has activated phosphorylated tyrosine that binds to other cellular proteins and kickstarts other signaling pathways.
What are some common EGFR mutations?
In frame deletion aa 747-752, which changes protein conformation and prolongs active dimer configuration
missense mutation L858R (leu>arg), which increases kinase activity 50-fold of EGFR protein.
What is RAS?
molecular switch involved in signal transduction
What are some oncogenic RAS mutations?
variably involve missense mutations affecting one of three codons (G12/13 and Q61) that block the hydrolysis of GTP to GDP, permanently turning on the molecular switch.
What are some characteristics of mutations in RAS and EGFR?
clustered in specific regions of the gene that encode important functional domains in the protein. Most mutations are missense mutations resulting in altered protein activity, and receptor tyrosine kinases (RTKs) are frequently mutated in cancer.
What are some characteristics of mutations in RAS and EGFR?
clustered in specific regions of the gene that encode important functional domains in the protein. Most mutations are missense mutations resulting in altered protein activity, and receptor tyrosine kinases (RTKs) are frequently mutated in cancer.
What is the mechanism of oncogene activation by amplification of the genomic DNA region that includes the proto-oncogene?
Too much production of mRNA and protein. This protein has normal activity, but overexpression leads to too much activity.
MYC gene
TF
- oncogeneic activations are related to upregulation
- required for cell rpoliferationand normally transiently expressed at low levels in the cell cycle
- can activate or repress genes
to be active forms a dimer with max
What is MYCN amplification and why is it important in cancer?
MYCN amplification is a genetic alteration in which the MYCN oncogene is amplified or duplicated in the genome of cancer cells. MYCN is a member of the MYC family of oncogenes and plays a critical role in regulating cell growth and division during development. However, when MYCN is amplified in cancer cells, it can lead to uncontrolled cell growth and contribute to the development and progression of several types of cancer.
What is Burkitt lymphoma and what chromosomal translocation is associated with it?
Burkitt lymphoma is a highly aggressive B cell lymphoma derived from germinal centre B cells. All BL tumour cells carry a chromosomal translocation involving the c-MYC gene on chromosome 8 and one of the three immunoglobulin gene loci. The T(8:14) translocation places the IgH enhancer adjacent to c-MYC, leading to overexpression of c-MYC protein.
What is the BCR-ABL fusion protein, and why is it significant in chronic myeloid leukemia?
BCR-ABL fusion protein is a novel protein resulting from a translocation between chromosome 22 and 9, known as the Philadelphia chromosome. It is significant in chronic myeloid leukemia as it encodes a novel BCR-ABL fusion protein with tyrosine kinase activities, which leads to uncontrolled myeloblast growth and the accumulation of granulocytes in the blood.
Why is genomic analysis of cancer cells important?
It provides a better understanding of the function of aberrant oncogenes, detailed information about the mutational landscape of cancers, enables improved cancer diagnostic tests, and can be linked to prognostic information. It is also necessary for designing new targeted cancer treatments.
What is precision cancer medicine?
type of treatment that therapeutically targets key oncogenes in a patient’s cancer, such as BCR-ABL in chronic myeloid leukemia, EGFR in lung cancer, and ALK in lung cancer.
what is chronic myeloid leukaemia
uncontrolled clonal proliferation of myeloid cells, leading to accumulation of granulocytes in the blood
What are tumour suppressor genes (TSG) and what is their function?
e.g. P53
induce apoptosis or senescence
accelerate ageing
may be selected to prevent cancer before the reproductive age but then reduce affect there after
gene that protects against cancer development
inhibitory functions acting as a break on cell proliferation, repairing DNA or inducing apoptosis
How are TSGs characterised in terms of mutations?
characterised by loss of function mutations. Inactivation of TSG leads to tumour development by eliminating a negative regulatory protein. Mutations in TSGs are usually recessive so both alleles must be mutated to have an effect.
What is the prototype tumour suppressor gene?
retinoblastoma gene (RB).
What is retinoblastoma and what are its two forms?
Retinoblastoma is cancer of the eye. Most cases of retinoblastoma are sporadic and affect only one eye (unilateral). Familial occurrence is seen in rare cases. Inherited retinoblastoma often affects both eyes (bilateral).
What is Knudson’s 2-hit hypothesis and how does it explain the relationship between hereditary and non-hereditary forms of retinoblastoma?
Cancer is caused by two mutational events. Non-hereditary retinoblastoma requires two hits to develop, while hereditary retinoblastoma is much more likely to develop disease and also to occur in both eyes over time. The two hits for retinoblastoma are the first hit (nonsense, frameshift, splice site, missense mutation) and the second hit (loss of heterozygosity through nondisjunction or mitotic recombination).
What type of mutations typically inactivate RB?
RB LOF mutations (1st/2nd hit) - confined to hotspots
1st hit = nonsense/frameshift/missense mutations
loss of heterozygosity (LOH) or 2nd hit
LOH = chromosome loss/non-disjunction/deletion/recombination
LOH more likely then 2nd hit
How does the cell eliminate both copies of RB?
Cancer only develops when the cell becomes homozygous for the mutant RB. The first hit is typically a nonsense, frameshift, splice site, or missense mutation. The second hit is loss of heterozygosity (LOH) which is the loss of the second allele through nondisjunction or mitotic recombination. These mechanisms are more likely than point mutations of the second allele.
What is the consequence of RB mutation?
RB1 controls the G1-S checkpoint in the cell cycle. Loss of RB1 would mean that TF E2F would be uncontrolled and the expression of S phase genes would increase, resulting in uncontrolled proliferation.
What is the function of APC in the Wnt signalling pathway?
pathway controls developmental processes and tissue homeostasis
involves the binding Wnt ligands to Frizzled (FZD) Rs
leads to activation of beta-catenin (TF)
frequently mutated in cancer
negatively regulating canonical WNT signaling, APC counteracts proliferation, promotes differentiation, facilitates apoptosis and suppresses invasion and tumor progression. APC further antagonizes canonical WNT signaling by interacting with and counteracting β-catenin in the nucleus.
Wnt/beta-catenin regulation of the cell cycle
Wnt OFF - proteasome able to degrade beta-catenin and prevent cell cycle progression
Wnt ON - proteasome binds to FZD preventing beta-catenin degradation and allowing gene transcription
What is the consequence of APC mutation?
leads to loss of destruction complex, stabilisation of beta-catenin, and expression of Wnt target genes linked to cell proliferation. Loss of APC tumour suppressor gene is common in cancer, for example, 70% of colorectal cancer.
What is the second category of tumor suppressor genes and what is their impact on cancer development?
The second category of tumour suppressor genes functions to preserve the integrity of the genome. Bi-allelic loss of function has no immediate impact, but in the long term, it increases the genomic mutation rate, accelerates the accumulation of mutations in oncogenes and other TSGs, dysregulates cell growth, and accelerates cancer progression. Examples of genes in this category include p53, ATM, and BRCA1.
TP53 - the guardian of the genome
DNA damage/loss of genomic integrity -> cell cycle arrest + DNA repair or apoptosis - elimination of damaged DNA -> both maintain genomic integrity
frequently mutated in cancer, mostly missense mutations - associated with advanced disease stage, metastasis and poor survival - that affect the DNA binding function of p53
TF function
dominant negative effect of p53 mutations - only 1/4 in the tetramer must be affected to cause impaired DNA binding
What is the dominant negative effect of p53 mutations?
P53 mutations predominantly affect DNA binding function, occur at hotspots, and are mainly missense mutations. P53 tetramers with even just one defective subunit are likely to have impaired DNA binding activity, resulting in a dominant negative effect.
TP53 and Li Fraumeni syndrome?
autosomal dominant disease caused by an inherited mutation of p53
increased lifetime risk of cancer - early onset
susceptible to multiple tumours
What is cellular senescence?
Cellular senescence is the process in which primary cells have a finite replicative capacity, undergoing growth arrest after around 50 cell divisions.
replicative senescence- ageing
progressive loss of telomeres [erosion] induces a DNA damage response and this triggers senescence
potent tumour suppressor
STASIS
Stress or Abberant Signalling Induced Senescence
NOT dependent on telomere shortening
increased expression of Cdk inhibitors at senescence e.g. p16/p21 these block cyclin/Cdk activities and cell cycle progression
telomere shortening and oncogene activation induce cellular senescence
what is quiescence
- cells stopped in G1 but can be stimulated to restart division (reversible)
- it occurs halfway through G1 phase
- less durable form of cell cycle arrest
- stimulated by depreciation of extracellular growth factors, contact inhibition, anti-proliferative cytokines
what stimulates senescence
- telomere shortening
- oncogene- induced senescence : chronic activation of tumour supressors (RB,P53)
- ROS and other DNA damaging agents
the hayflick limit
cells have limited proliferative potential in culture: growth arrest after approx. 50 cell divisions
replicating and senescent fibroblasts
replicating- replicative
don’t respond to growth signals
normal fibroblast morphology
senescent- post-replicative
don’t respond to growth signals
flattened morphology
metabolically active
altered gene expression
change in tissue homeostasis
PRB TSG
inherited form - 1 germline mutation, 2nd somatic mutation
non-hereditary - both mutations are somatic
nomenclature = gene: RB1, protein (gene product): pRB
inactivated in many somatic human cancers
pRB and cell cycle control in G1
- transcriptional repression (G0, G1)
- phosphorylation of pRB (early G1) - by Cdk4/6 + Cyc D causes unlinking of HDAC
- phosphorylation of pRB (late G1) - by Cdk2 + Cyc E causes unlinking of E2F/DP allowing for transcription of S phase genes
pRB and restriction point entry into S-phase
pRB in normal cells - E2F/DP TFs regulated by pRB resulting in regulated S-phase entry
mutated pRB - E2F/DP TFs constitutively active, bypass the restriction point causing uncontrolled entry into S-phase
What are the roles of pRB and p53 in cellular senescence?
pRB and p53 are tumor suppressor gene products that play a critical role in cellular senescence by negatively regulating cell growth and maintaining cell cycle checkpoints to ensure genomic integrity.
E2F transcription factor
induces expression of genes related to DNA replication such as cyclin E and S phase Cdks.
constitutively bound to DNA
E2F bound to RB results in E2F inhibition. when Rb is phosphorylated it releases E2F.
What is the role of telomeres in cellular senescence?
nucleoproteins structures 10-15kb (humans) in size that protect the chromosome ends
[TTAGGG]n
what does telomerase do
maintains the telomeres
reverse transcriptase [hTERT] and RNA Template-TERC
telomerase activity in normal cells is absent or low, it is induced in most cancers (>90% dependent upon the tissue)
telomerase action in cancer
prevents the critical shortening of the telomeres
extended life-span
induce telomerase (hTERT)
structure of telomeric ends
D loop
T loop
strand invasion of the G-strand overhang
restricts the access of telomerase into the telomere
What is the role of Shelterin protein complexes in telomere regulation?
Shelterin protein complexes, such as TRF1/2, play a critical role in telomere regulation by binding to telomeric duplex DNA, protecting telomeres from DNA damage surveillance, and maintaining telomeric chromatin structure
What is the role of Telomerase and ALT in cellular immortalization?
loss of TSGs and maintenance of telomeres by telomerase or ALT leads to cellular immortalisation
Alternative lengthening of telomeres (ALT)
- strand invasion of the template molecule and the formation of the HR intermediate structure
- copying
- dissolution of the HR intermediate
- possible filling in of the complementary strand
How is Telomerase considered a cancer therapeutic target?
it is activated in most cancers, contributing to cellular immortalization. Inhibiting telomerase can prevent cancer cell growth and potentially induce cell death.
How is the function of pRB and p53 inactivated in human cancer?
pRB and p53 function can be inactivated through loss of tumor suppressor gene function, leading to loss of growth suppressive properties and/or loss of cell cycle checkpoint control, which results in enhanced proliferative potential.
The cell cycle is a cyclin cycle ..
cyclins are expressed during the cell cycle and they interact with Cdks. Cdks are kinases always expressed and induce mitosis. when they bind to cyclins there is a conformational change and they become active.
cyclins determine Cdks substrate specificity
What is the role of p53 in the G1-S checkpoint control?
p53 regulates the G1-S checkpoint control by inhibiting cyclin D-CDK4/6 and cyclin E-Cdk2, preventing the G1 to S-phase transition in response to cellular stress and DNA damage.
What are the characteristics of cells that bypass M1 senescence?
Cells that bypass M1 senescence continue to erode telomeres, leading to unprotected chromosomes, chromosome abnormalities, genetic instability, and accumulation of mutations. This occurs in cells with inactive p53 and RB, where there is no check on proliferation.
replicative M1 senescence is induced by
damage signalling pathways
1+ “too short” telomeres induces a DNA damage signalling pathway - put a permanent holt on cell cycle progression
p53 nd pRB in M1 senescence
p53 activates p21 which induces senescence
p16 inhibits the Cdk4/6, Cyc D complex inhibiting the phosphorylation of pRB therefore causing senescence
loss of p53 and pRB causes a bypass of cellular senescence -> telomere shortening/dysfunction -> genomic instability -> mutations in critical genes -> early tumourigenesis
Crisis- M2 replicative senescence
2nd proliferative barrier
telomeres continue to erode
the telomeres become too short and can’t protect the chromosomes which triggers apoptosis
failure to protect the chromosomes leads to chromosome abnormalities - chromosome end to end fusions or changes in copy number
in cells with inactive pRB and p53 there is no check on proliferation - genetic instability and accumulation of mutations
premature aging syndromes?
cause accelerated telomere loss
mutation/deletion of components of the TRF2 complex result in accelerated telomere loss
A-T/Werner syndrome/bloom syndrome
characterised by defects in DNA response/repair pathways
What are some strategies for targeting telomerase in human cancer?
- oligonucleotides complementary to the TERC template - Imetelstat
- telomerase targeted immunotherapy - hTERT vaccines
- targeting telomerase expressing cells with a suicide gene/oncolytic viruses
Role of P53 in G1-S checkpoint control
P53 responds to stress and recruits protein complexes
activates ATM kinase that activates p43
It can phosphorylate p53 directly or check 2 kinase which then phosphorylated p53, can also inhibit MDM2 which is a neg regulator of p53.
p53 is then a transcription factor that inhibits cyclin D-CDK4
Loss of p53 transcriptional activity
leads to uncontrolled entry into S-pased though cyclin D-CDK4/6 and cyclin E-Cdk2
What are driver mutations and passenger mutations in the context of cancer?
Driver mutations are those that initiate or progress cancer, while passenger mutations occur as cancer progresses but are not necessary for its progression.
What is an example of a therapeutic intervention that targets driver mutations?
The use of vemurafenib in malignant melanoma, which inhibits the bRAF kinase to switch off melanoma.
What is the adenoma-carcinoma sequence in bowel cancer described by Vogelstein?
The adenoma-carcinoma sequence is a series of mutations that initiate and progress bowel cancer. It postulates that colorectal cancer starts with a mutation in the APC gene, followed by other mutations that progress the cancer.
Why is mutational context important in understanding cancer?
Different cancers tend to have different driver mutations, depending on the initial tissue type and the context in which the cancer arises
What are some challenges in distinguishing driver mutations from passenger mutations?
mutational changes may have different effects in different tissues. Methods of distinguishing include molecular (cell biology) and bioinformatics approaches.
What are tissue organoids and why are they useful in studying cancer?
Tissue organoids are primary cells from normal tissue maintained in culture by manipulating signaling pathways. They are useful as in vitro model systems for studying the effects of mutations on the tissue in question.
What is the role of mouse models in understanding driver mutations in cancer?
Mouse models, such as the APC-/- min mouse in colorectal cancer, help confirm the role of driver mutations by demonstrating the effects of specific gene knockouts on cancer development.
How do large datasets, like the Cancer Genome Atlas and the 100,000 Genome project, help in identifying driver and passenger mutations?
datasets contain high read depth exome or whole genome sequencing of tumor-normal pairs across multiple tumor types, allowing mutational patterns to emerge that help distinguish driver and passenger mutations.
What are some bioinformatics methods used to identify driver mutations?
- identifying genes mutated more frequently than the background mutation rate,
- identifying genes with a functional impact, and identifying mutations that cluster together in regions of high impact.
cell death types
necrosis - spontaneous cell death
apoptosis - programmed cell death
autophagy - recycling of cellular components
What is programmed cell death (PCD)?
apoptosis, an active process that follows a specific pattern and is as important for life as mitosis.
What are the differences between apoptosis, necrosis, and autophagy?
Apoptosis is an active, programmed cell death process,
necrosis is “accidental” cell death, and autophagy is the recycling of cellular components.
What are the important genes in apoptosis in C. elegans?
Important apoptosis genes in C. elegans include ced-3 and ced-4, which are required for cell death, and ced-9, which antagonizes ced-3 and ced-4.
What are the stages of apoptosis?
1) DNA fragmentation, 2) cell shrinkage and chromatin condensation,
3) membrane blebbing and fragmentation,
4) formation of apoptotic bodies, and
5) phagocytosis.
What are caspases and their role in apoptosis?
Caspases are a family of cysteine proteases that are the executors of apoptosis. They are inactive in cells as proenzymes, and their activation involves cleavage of their N-terminal polypeptide sequence.
What are the three pathways of apoptosis?
The three pathways of apoptosis are the extrinsic pathway, the granzyme pathway, and the intrinsic pathway. All three converge at the activation of Caspase-3 and execution of apoptosis.
What is the extrinsic pathway of apoptosis?
The extrinsic pathway of apoptosis involves ligands binding to and activating their death receptors (FAS), activation of the adapter protein (e.g. FADD), and activation of executioner caspases and the proteolytic cascade.
apoptosis: stage 1 DNA fragmentation adn protein degredation
DNA fragmentation by endonucleases/DNAses
enzymes are able to access the sites between the nucleosomes
protein degradation (nuclear and cytoskeletal) by protease enzymes
apoptosis: stage 2 cell shrinkage and chromatin condensation
cytoplasm begins to shrink following the cleavage of lamins and actin filaments
organelles become more tightly packed
the chromatin condenses following the breakdown of chromatin and nuclear structural proteins
nuclei take a “horse-shoe” appearance
apoptosis: stage 3 membrane blebbing and fragmentation
apoptotic cells undergo plasma membrane changes e.g. the transfer of the phospholipid phosphatidylserine from the inner leaflet of the cell to the outer surface
promotes phagocytosis by macrophages
apoptosis: stage 4 apoptotic bodies
separation of fragments into apoptotic bodies
apoptotic bodies will contain organelles, cytoplasm and/or DNA fragments
enclosed by an intact plasma membrane
apoptosis: stage 5 phagocytosis
apoptotic bodies allow for easy clearance by macrophages
macrophages recognise the phosphatidylserine on the cell membrane of the apoptotic bodies
once engulfed the apoptotic bodies are degraded within phagolysosomes
there is no inflammatory response because the the apoptotic cell contents is not released into the tissue
cause 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
What is the intrinsic pathway of apoptosis and what are some of its key components?
The intrinsic pathway is activated by intracellular stress stimuli, such as the absence of growth factors or radiation.
- increase expression of BAX/PUMA (pro-apoptotic)
- lead to formation of pores in mitochondrial memebrane to increase permeability
- released of cytochrone C
- lead to formation of apoptosomes
- then activation of casp-9 and then activation of executioner caspases
Bcl-2 anti-apoptotic so increased expression can lead to over survival of cells that carry damage
How does the granzyme pathway activate apoptosis?
The granzyme pathway is activated by cytotoxic T cells, which release granules containing perforin and granzyme. Perforin creates pores in the target cell membrane, allowing granzyme (A or B) to enter the target cell and degrade proteins or activate caspases.
how do cancer cells evade apoptosis
- increase expression IAPS
- increase expression Bcl-2
- mutations on P53
What are the differences between apoptosis and necrosis?
Apoptosis is an energy-dependent process triggered by internal or external signals, affecting single cells and causing cell shrinkage without an inflammatory reaction.
Necrosis is not energy-dependent, caused by ischemia, toxins, or radiation, affecting groups of cells, leading to cell swelling, content release, and a marked inflammatory reaction.
What is autophagy and how does it relate to cell survival and cell death?
Autophagy is a process of recycling and degrading cellular components in times of stress or starvation, providing energy to the cell. It can be both pro-survival and pro-death, depending on the context, and is involved in survival during short-term starvation, clearance of aggregate-prone proteins, and programmed cell death.
How do faulty apoptosis and autophagy contribute to disease, including cancer?
Faulty apoptosis can lead to autoimmune disease, neurodegenerative disease, and cancer, with over-proliferation of cells or decreased removal of cells causing the accumulation of cancer-causing mutations in DNA. Faulty autophagy can contribute to neurodegenerative disease, liver disease, cancer, and skeletal and cardiac muscle degeneration, impacting cell survival, longevity, and resistance to chemotherapy.
mechanisms of autophagy
mTORC1 connects the environmental conditions with metabolic processes
- nutrient rich conditions mTORC1 phosphorylates ATG13 inhibiting autophagy and promotes cell growth through the synthesis of proteins/lipids/nucleotides
- starvation conditions mTORC1 is inhibited and Atg proteins are activated to initiate autophagy
Atg proteins work to assemble the phagophore
cellular components are trapped within the phagophore to create the autophagosome a double membrane bound vesicle
autophagosome fuses with a lysosome making the autolysosome
lysosomal hydrolase enzymes degrade trapped cellular components and the inner membrane
macromolecules are released
free nutrient levels reach a threshold which activates mTORC1 to switch off autophagy
How do many chemotherapy agents aim to induce apoptosis in cancer cells?
activating the transcription factor p53, which can promote apoptosis if DNA damage cannot be repaired. However, the absence of functioning p53 may result in failure or necrosis, leading to inflammation and unpleasant side effects.
cancer metabolism
the metabolism of cancer cells has to change due to the alterations in physiology compared to the tissue from which they derive
- increased cell proliferation
- mutations in signalling pathways
- detoxification of anti-cancer therapies
- survival in inhospitable environments
What is the Warburg effect?
refers to high lactate production in the presence of oxygen, also known as aerobic glycolysis, which is observed in cancer cells.
How is the Warburg effect used for diagnosis?
produces an acidic microenvironment
used for diagnosis FDG-PET imaging
- [18F] 2-fluoro-deoxy-D-glucose (FDG) is taken up by cells using glucose transporters, phosphorylated and then trapped in the cell
What are some hypotheses for why cells use high rates of glycolysis in normoxic conditions?
1) Mitochondrial energy generation is irreversibly damaged,
2) Changes in enzyme expression due to different signaling, and 3) Changes in the requirements for macromolecule synthesis.
the warburg effect: mitochondrial energy generation is irreversibly damages
no TCA cycle
mutations in TCA cycle enzymes - familial cancer syndromes
- pheochromocytoma and paraganglioma defects in SDH
- leiomyoma defects in FH
Which dysregulated signaling pathways are responsible for changes in metabolic enzyme expression in cancer?
c-Myc, p53, HIF1, and PTEN.
How do cancer cells meet their altered metabolic requirements?
use glycolysis as a source for many important molecules like nucleotides, lipids, and amino acids. In some cases, cancer cells cannot meet these needs entirely and send signals to the body to provide additional resources
What happens when large tumor burdens use many nutrients?
The plasma levels are depleted, leading to the body going into a starvation-like mode, in which it catabolizes the amino acids that form the muscle mass.
How does 5-fluorouracil (5-FU) work as a successful cancer therapy?
5-FU disrupts nucleotide synthesis by blocking thymidylate synthase, which is part of the metabolic pathway to synthesize dTTP. It may also elicit a DNA damage response by being incorporated into DNA and RNA.
Why is hypoxia prevalent in tumors?
Tumors are often poorly vascularized or have dysfunctional vasculature, leading to low oxygen levels in large areas of tumors.
hypoxic phenotype:
- slower proliferation
- inc. cell migration
- inc. in survival factors
- silencing areas of chromatin
- changes in cellular metabolism
- reduced differentiation profile
What are the pros and cons of anti-angiogenic therapies?
Pros: Starving the tumor of nutrients should help fight the cancer.
Easy access to endothelial cells
Cons:Chemotherapeutic drugs may not be able to reach the core of the tumor, which can stabilize the tumor and help its progression..
bleeding, clots
cant reduce tumour just slow growth
How does hypoxia negatively interfere with chemotherapeutic treatment?
Hypoxia slows down cell division, increases cell migration, increases survival factors, silences chromatin areas, induces changes in cellular metabolism, and reduces differentiation profile.
What are potential issues with targeting tumor acidification?
Targeting enzymes involved in tumor acidification, such as carbonic anhydrase, Cariporide, or monocarboxylate transporters, can have severe side effects and impact other tissues, like the kidneys.
Why is targeting glycolysis difficult?
The difference between killing the tumor and killing the person is difficult to achieve when targeting glycolysis, as it is a metabolic pathway used by every cell in the body.
What are some examples of amino acids as therapeutic targets?
Glutamine, asparagine, and arginine.
How does arginine depletion affect pancreatic cancer growth?
injection with arginine deiminase, which degrades plasma arginine, reduced tumor growth by 50% in mouse models.
What are the three isoforms of isocitrate dehydrogenase (IDH)?
IDH1 (located in the cytosol), IDH2 (located in the mitochondria, not involved in TCA cycle), and IDH3 (involved in TCA cycle, never mutated in cancer).
How do IDH1 mutations affect cancer cells?
IDH1 mutations lead to changes in epigenetic mechanisms, redox alterations, and activation of HIF1, which promotes cell survival.
In which cancers have IDH1 and IDH2 mutations been identified?
IDH1 mutations are present in 75% of secondary gliomas, 13% of acute myeloid leukemia (AML), and more than 50% of chondrosarcomas. IDH1 mutations are also sporadically found in other tumors such as melanoma and pancreatic adenocarcinoma.
Name some other DNA cross-linking agents.
Mitomycin C, Cisplatin, Psoralens.
What is Fanconi Anaemia (FA) and what are its clinical phenotypes?
FA is a rare genetic disorder characterized by microcephaly, mental retardation, developmental delay, aplastic anaemia, predisposition to tumor development, and bone marrow failure. It is also associated with cellular/chromosome hypersensitivity to DNA inter-strand cross-linking agents.
What is the genetic basis for FA?
FA is an autosomal recessive disease with genetically heterogeneous causes. 22 different genes (FANCA-FANCW) have been identified as the underlying cause of FA. 85% of cases involve mutations in FANCA, FANCC, or FANCG, while about 10% of cases involve mutations in FANCD1, FANCD2, FANCE, FANCF, and FANCL. The remaining 5% are caused by mutations in the other 8 genes.
FA phenotype
phenotype
- microcephaly
- mental retardation
- developmental delay
- asplastic anaemia
- predisposition to tumour development
- bone marrow failure
cellular/chromosomal hypersensitivity to DNA cross-linking agents = diagnostic test for FA
- formation of chromosomal gaps/breaks, tri-/quadra-radial chromosomes, complex chromosomal rearrangement
FA tumourigenesis
many FA patients develop Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukaemia (AML)
older FA patients are at higher risk of developing head/neck, gynaecological and or GI tumours
carriers of the FANCD1/FANCN/FANCS mutations have a higher predisposition to the development of breast/ovarian cancer
biallelic mutations in FNACD1 predispose for development of AML/T-ALL/Wilms’ tumour/medulloblastoma/GBM
What are the steps in the ICL repair and FA pathway?
1) Crosslink recognition during S phase, recruitment of FA core complex.
2) Replication fork convergence at ICL, activation of FANC1 and FANCD2 through ubiquitination.
3) Recruitment of other proteins like nucleases, deubiquitination of FANC1 and FANCD2.
4) Rotation of crosslink out of helix and gap synthesis.
5) Repair of the other strand through homologous recombination involving BRCA1 and BRCA2.
How does the loss of ICL repair contribute to genome instability?
Loss of ICL repair leads to under-replicated DNA, which separates during mitosis and forms micronuclei. This results in loss of genetic information and mitotic catastrophe.
How do ICLs in FA cells contribute to chromosomal breakage?
Under replicated DNA and anaphase bridge formation contribute to chromosomal breakage at common fragile sites (CFS). DNA breaks caused by ICLs in FA cells are mis-repaired by NHEJ, leading to mitotic catastrophe.
What is the endogenous lesion repaired by the FA pathway?
likely caused by naturally occurring acetaldehyde (alcohol metabolism) or formaldehyde (histone demethylation, amino acid synthesis, tobacco smoke), which can induce DNA interstrand and protein-DNA crosslinks.
What are some examples of genotoxic agents used in labs to induce ICLs?
Acetaldehyde, a by-product of alcohol metabolism and intermediate of carbohydrate metabolism, and formaldehyde, an intermediate from histone demethylation and dealkylation of methylated DNA.
What do the reactive intermediates of genotoxic agents do?
Reactive intermediates can induce DNA interstrand and protein-DNA crosslinks.
What is the role of endogenously-induced ICLs in FA?
Endogenously-induced ICLs play a critical role in the development of FA. Without functional FA proteins, endogenously-induced ICLs cannot be repaired efficiently, leading to accumulation of DNA damage and ultimately to cell death. This is particularly problematic in rapidly dividing cells, such as those in the bone marrow, where the failure to repair ICLs leads to bone marrow failure and anemia. Moreover, the accumulation of DNA damage also increases the risk of cancer development.
What happened when Fancd2+/- mice were crossed with Aldh2+/- mice?
The resulting mice had increased embryonic lethality, severe developmental defects, and severe cell attrition in the bone marrow and bone marrow failure when exposed to alcohol during pregnancy.
What is the function of the Fanconi Anaemia pathway?
The Fanconi Anaemia pathway functions to maintain genomic integrity by promoting the repair of DNA inter-strand cross-links or DNA-protein cross-links.
metastatic cascade
a highly regulated process
1. formation of the pre-metastatic niche by primary tumour cells
2. primary cells escape from the pirmary tumour due to alterations in cell-cell adhesion/ECM reoranistion
3. enter the vasculature through the process of internalisation
4. travel along a gradient of chemokines to the target organ
6. interaction with the endothelial cells at the target organ through selectins/integrins
6. leave the vasculature via activation of integrins and MMP production
7. enter the new tumour environment where they either stay dormant or start proliferating
What are the effects of smoking and drinking alcohol during pregnancy?
Smoking and drinking alcohol during pregnancy can cause developmental abnormalities in the fetus, most likely due to failed repair of ICLs and DPCs.
What is the premetastatic niche?
The premetastatic niche is formed when tumor cells at the primary tissue secrete soluble factors and extracellular components, modifying the target organs and changing the behaviors of cells. This recruits bone marrow-derived mesenchymal cells, which further modify the target organ by producing specific chemokines and attracting chemo cells.
What is the role of the tumor microenvironment in malignant progression?
facilitating active communication between tumor cells and surrounding cells, such as fibroblasts and adipocytes. This communication leads to the formation of cancer-associated cells.
How do macrophages affect tumor cells?
Macrophages are actively recruited to epithelial cancer sites due to the production of CSF. They modify the behavior of tumor cells by producing EGF, making tumor cells more proliferative and invasive, which facilitates the metastatic cascade.
What is the self-seeding model of metastasis?
tumor cells invade the vasculature and some return to the primary tumor, where they act as changed entities and modify primary tumor cells.
What are the altered mechanisms required for metastasis?
Metastasis requires altered mechanisms of cell adhesion, proteolysis, migration, lymph-/angiogenesis, and homing to target organs
What happens during the transition to the invasive phenotype?
The transition to the invasive phenotype requires modification of cell-cell adhesion, such as the loss of intracellular adhesion, re-organization of the extracellular matrix, and modulation of cell-ECM adhesion
What is the role of E-cadherin in metastatic breast cancers?
E-cadherin loss is seen in most invasive lobular carcinomas and around 50% of ductal carcinomas. Reduced E-cadherin expression is associated with shortened disease-free survival.
How does cellular adhesion change in malignant cells?
In malignant cells, cellular adhesion changes as cells lose cell-cell adhesion, leading to scattering and a more spindle-shaped morphology.
What is the extracellular matrix (ECM) composed of?
meshwork of macromolecules
2 main classes
1. polysaccaride chains: glycosoaminoglycans
2. fibrous proteins: collagens/laminis/fibronectin/elastin
adhesion plaques
focal adhesions - acitn
hemidesmosomes - keratin
adherens junctions
calcium dependent
- E-cadherin
- beta/alpha-catenin links to actin
calcium independent
- nectin
- afadin links to acitn
cell- cell adhesion plaques
cytoskeleton (actin/keratin filaments)
linker proteins (catenins/desmoplakin)
adhesion receptors
interactions between tumour cells and the ECM
intergrins
- 24 heterodimers 18alpha/8beta subunits
- inside-out/outside-in signalling
- binding to talin = essential for integrin activation
surface proteoglycans
DDR proteins
What proteins control the interactions of tumor cells with the extracellular matrix?
The interactions of tumor cells with the extracellular matrix are controlled by integrins, surface proteoglycans, and DDR proteins.
mechanical barriers that must be overcome by tumour cells
- basement membrane - a complex of interacting proteins separating epithelial and endothelial cells from connective tissues
- stromal environment (connective tissue matrix) - collagen fibrils and extracellular proteoglycans
tissue classification
parenchyma
- tissues that confer function
- can be either epithelial or connective tissue
stroma
- everything else
- no organ can function without the mechanical and nutrient support of the stroma
in tumours
- parenchyma = malignant cells
- stroma = non-malignant supporting cells, it is essential for tumour growth
What is the mechanism of homing in metastasis?
The mechanism of homing in metastasis involves selective growth, selective adhesion to sites on endothelial cells at the site of organ homing, and selective chemotaxis of circulating tumor cells to the organ producing soluble attraction factors.
What are parenchyma and stroma tissues in organs?
Parenchyma comprises the tissues that confer function, while stroma is everything else and consists of non-malignant supporting cells.
What are some examples of parenchyma in different organs?
Kidney: epithelial tissue; spleen: connective tissue; heart: muscle tissue (cardiac muscle cells); brain: nervous tissue (nerve cell, glia).
Why is stroma essential for tumor growth?
Stroma provides mechanical and nutrient support for parenchyma, which is composed of malignant cells in the context of tumors.
What is the seed and soil hypothesis?
Proposes that metastasis is the result of favorable interactions between tumor cells (‘seed’) and the organ microenvironment (‘soil’).
What are the stages of wound healing?
- Hemostasis
2.Humoral inflammation (vascular permeabilization)
3.Cellular inflammation (immune cell infiltration)
4.Angiogenesis (formation of new blood vessels)
5.Generation of mature connective stroma
What are the physicochemical properties of the tumor microenvironment?
Hypoxia (low O2), acidosis, high interstitial fluid pressure (IFP), and extracellular matrix (ECM) remodeling.
What are the physicochemical properties of the tumor microenvironment?
Hypoxia (low O2), acidosis, high interstitial fluid pressure (IFP), and extracellular matrix (ECM) remodeling.
Interstital fluid pressure in tumours
Increased
- expanding tumour mass
- abnormal ECM
- fibroblasts contracting in matric
- lack lymphatic vessels
- leaky blood vessels
- increase IFP poor prognosis
- prevents fluid transport and drug delivery
Tumour microenviroment cells
- cancer stem cells
- endothelial cells can have tumour associated vasculature and lymphatic vessels present on peripheries of tumours
- pericytes are specialised mesenchymal cell type, stability for vascular integrity and function
- immune inflammatory cells e.g macrophages , lymphocytes, neutrophils, releases signal moleucles and can be pro and anti tumour
- cancer associated fibroblasts such as myofibroblast usually present on site of chronic inflammtion and wound healing. in tumours: proliferation, angiogenesis and invasion
What are Cancer Associated Fibroblasts (CAFs)?
CAFs are an important constituent of the stroma that can directly and indirectly promote cancer progression and metastasis through angiogenesis and attracting pro-inflammatory cells.
recruited by PDGF
secrete growth factors (VEGF,TGF-b) exm protines and proteases
tumour associated macrophages
monocytes can differentiate into 2 types of macrophages depending on growth factor stimulation:
M1: classical activation phagocytotic cells
M2: alternative activation tissue remodelling during wound healing
tumouirs secrete Il-4 that activated macrophaes and lead to production of matrix degrading enzymes.
How does extracellular matrix (ECM) remodeling contribute to tumor growth and dissemination?
Proteases such as matrix metalloproteinases (MMPs) break down the ECM, allowing remodeling, which in turn enables tumor growth and cell dissemination.
cells within the tumour microenvironment
complex mix of cells that changes as the tumour develops invades and metastasises to new sites
cancer associated fibroblasts (CAFs) = important constituent of the stroma
- directly produce factors that promote angiogenesis
- indirectly attract pro-inflammatory cells such as macrophages which stimulate cancer invasion
targeting the tumour stroma for cancer therapy
- tumour vasculature
VEGF and VEGR antagonists such as bevacuzumab
RTK inhibitors such as brivinab
EGFR neutralising ab - cancer associaetd inflammation:
inhibiting TAM, target chemokines that lead to M2 recruitment
cells within the tumour microenvironment
complex mix of cells that changes as the tumour develops invades and metastasises to new sites
cancer associated fibroblasts (CAFs) = important constituent of the stroma
- directly produce factors that promote angiogenesis
- indirectly attract pro-inflammatory cells such as macrophages which stimulate cancer invasion
What are the three broad groups of tumor classification based on the immune microenvironment?
infiltrated-excluded, infiltrated-inflamed, and infiltrated-Tertiary Lymphoid Structures (TLS).
What are the two main categories of immune cells?
The two main categories of immune cells are innate immune cells and adaptive immune cells.
What is the role of macrophages in the tumor microenvironment?
derived from circulating monocyte cells that enter tissues and develop into macrophages
macrophages are versatile and plastic - they modify their phenotype to suit their surrounding environment
divided into 2 subgroups M1 and M2
tumour associated macrophages (TAMs) are M2 likeomote angiogenesis, suppress other immune cells, and release factors that promote metastasis.
types of macrophages
M1
induces by LPS and IFN gamma
- pro-inflammatory/bactericidal
- promote CD4 T cell differentiation into Th1/17 cells that support cellular immunity
M2
induced by IL4/10/13
- are immunosuppressive
- promote CD4 T cells to become Th2 and T-regs
- unhelpful
TAMs
- poor prognosis in most studies
- promote angiogenesis
- infiltrate at an early stage - trigger the angiogenic switch
- suppress other immune cell function
- release factors that promote metastasis
What are Myeloid-derived suppressor cells (MDSCs) induced by, and what do they do in the tumor microenvironment?
MDSCs are induced by inflammation (IL1beta, IL6, and PGE2). They interfere with innate and adaptive immunity by inhibiting NK and DC function, inducing T reg cells, producing arginase, and causing depletion of arginine in the tumor.
NK cells
tumour cells frequently downregulate MHC expression - should make them a target for NK cells
intratumoural NK cells are often anergic - unable to release cytokines/kill targets
suppression of NK function caused by TGF-beta released from tumour cells
What are the consequences of low arginine levels in the tumor microenvironment?
Low arginine levels lead to decreased T-cell receptor signaling in T-cells by decreasing CD3zeta chain expression.
CD8+ T cells
cytotoxic effector cells
cytotoxic granules containing perforin and granzymes that trigger apoptosis in target cells
produce cytokines INF-gamma and TNF-alpha
What are the subtypes of CD4 T cells?
Th1, Th2, Th17, T-reg, T-fh, and Th9.
What are the main functions of CD4 Th1 cells?
CD4 Th1 cells support cytotoxic
CD8 T cells, activate macrophages, and can kill tumor cells in some cases.
What is the general effect of increased numbers of T-reg cells on cancer prognosis?
increased numbers of T-reg cells correlate with poorer prognosis, although some studies in other cancers suggest increased numbers are beneficial.
How do T-reg cells suppress other immune cells?
T-reg cells suppress other immune cells by producing TGF-B and IL10, expressing high levels of CD25 (IL2 receptor) to starve effector T-cells of IL2, and producing adenosine.
What is angiogenesis and its role in health and disease?
Angiogenesis is the formation of blood vessels from pre-existing vessels. It plays a crucial role in physiological processes like wound healing, the menstrual cycle, and adaptation to increased muscle activity. In pathological processes, angiogenesis contributes to cancer, inflammatory diseases, and diabetic retinopathy.
How does angiogenesis contribute to cancer pathogenesis?
Angiogenesis supports tumor growth by providing nutrients and oxygen, and it facilitates metastasis by providing a route for cancer cells to spread to other parts of the body. Hypoxia in growing tumors drives VEGF production, which promotes angiogenesis and creates a pro-angiogenic environment.
development of vessles: vasculogenesis and angiogenesis
vasculogenesis: the formation of blood vessels from mesodermal derived angioblasts
endothelial progenitors (angioblasts) differentiate from mesodermal cells
angioblasts coalesce to form first embryonic vessels - dorsal aorta and cardinal vein
angioblasts form blood islands which fuse together and remodel to form a primitive capillary plexus
angiogenesis : angiogenic remodelling of the dorsal aorta, cardinal vein and vascular plexi give rise to the arteries, veins and capillaries
angiogenic sprouting of new vessels
pericytes are recruited to stabilise capillaries
lymphatic endothelial cells sprout from veins and from the lymphatic system via lymphangiogenesis
What are the different modes of angiogenesis in cancer, and what are the stages of sprouting angiogenesis?
Modes of angiogenesis in cancer include sprouting, intussusception, vasculogenesis, vessel co-option, vascular mimicry, and endothelial differentiation. Sprouting angiogenesis involves loosening of endothelial junctions, remodeling of the basement membrane, tip cell selection and sprout extension, lumen formation, and sprout fusion.
What is HIF1, and how does it regulate VEGF production?
a transcription factor and master regulator of angiogenesis
HIF-1 is made of 2 subunits HIF-1a and HIF-1b
HIF-1a is degradation is regulated in response to O2 levels
HIF-1a and high oxygen
high levels of O2 a proline hydroxylase enzyme hydroxylates proline residues on HIF-1a
a ubiquitin ligase recognises this and binds which catalyses the poly-ubiquitination of HIF-1a
poly-ubiquitinated HIF-1a is degraded by the proteosome
HIF-1a and low oxygen
low O2 levels there is not enough O2 to drive the proline hydroxylation of HIF-1a
HIF-1a is free to bind to HIF-1b and induce transcription of genes required for the adaption to hypoxic conditions
key target = VEGF
VEGF and HIF-a and hypoxia induced expression
VEGF is most potent stimulator of angiogenesis, secreted by many tumours in response to hypoxia and oncogene signalling.
HIF-a is a TF. In normal conditions, its regulated by hydroxylation of proline residue. when hydroxylated, it can bind to pVHL. This stimulates Ubiquitination and degredation of HIF-a. Under hypoxia, pVHL becomes S-nitrosylated and cant bind to HIF-a. THen HIF-a is free to interact with HIF-b in the nucleus, leading to epxression of hypoxia related genes (VEGF)
How is VEGF involved in driving tumor vessel formation?
endothelial specific factor
VEGF-A = most potent angiogenic driver
VEGFR signals via activation of VEGFR-2 a receptor tyrosine kinase
dimerisation of VEGFR-2 activates signalling pathways which affect: proliferation/migration/survival/vascular permeability - via modulation of junctional proteins
VEGF inhibitors
prevent VEGF receptor binding so no downstream singalling.
e.g Avastin (Bevacizumab)
monoclonal ab against VEGF, used w chemo against metastatic crc, renal and nsclc. stops abberant angiogenesis, normalises tumour vasculature, promotes therapeutic delivery of chemotherapeutics- but can increase blood flow to tumour
Endogeneous angiogenesis inhibitors
Angiostatin
primary tumours produce angiogenic factors that prevent secondary tumours from being vascularised, small tumours dont produce big
How is VEGF involved in driving tumor vessel formation?
VEGF-A, the most potent VEGF family growth factor for driving angiogenesis, signals through VEGFR-2, a receptor tyrosine kinase. This activation leads to endothelial cell proliferation, survival, migration, and increased vascular permeability, which promotes tumor vessel formation.
What is the angiogenic switch?
number of pro- and anti-angiogenic factors
angiogenesis occurs when the effects of the angiogenic activators are greater than those of the inhibitors- leads to new vessels
modes of vessel formation
angiogenesis
vasculogenesis
intersusception
vessel co-option
vascular mimicry
endothelial differentiation
angiogenesis: intussusception
involves the formation of a pillar which elongates splitting the vessel in 2
- rapid inc. in capillarity
- requires minimal endothelial proliferation
angiogenesis: sprouting angiogenesis selection of a tip cell
formation of a lumen in the newly developing sprout
newly formed stalk attracts pericytes to stabilise the newly formed vessel
signalling btw tip and stalk cells maintain their different specification, expression of Delta-like 4 (DDL4) by tip cell signals to notch expressed by stalk cells
tip cells navigate in response to guidance signals and adhere to the ECM (mediated by integrins) to migrate
stalk cells behind the tip cell proliferate and extend the sprout VEGFR-2 is downregulated in stalk cells
the sprouts from adjacent vessels grow towards each other
angiogenesis: sprouting angiogenesis stabilisation of the newly formed vessel
signalling btw endothelial cells and pericytes maintain quiescence of the newly formed vessel, angiopoeitin 1 (Ang 1)
formation of tight junctions and barrier function
basement membrane deposition
pericyte maturation (PDGF, Ang1)
How is VEGF being used as a therapeutic target in cancer treatment?
inhibiting the production of angiogenic factors, using neutralizing antibodies, soluble receptors, or receptor tyrosine kinase inhibitors. Bevacizumab (Avastin) is a monoclonal antibody that targets VEGF, and other drugs like sorafenib, sunitinib, and pazopanib also target VEGF signaling.
How do tumor vessels differ from normal vessels
Tumor vessels are abnormal, leaky (increase interstitial pressure), and poorly perfused (increased VEGF), leading to persistent hypoxia.
What is vasculogenesis, and how does it contribute to vessel formation during development?
Vasculogenesis is the formation of blood vessels from mesodermal-derived angioblasts. Endothelial progenitors (angioblasts) differentiate from mesodermal cells, coalesce to form the first embryonic vessels, and create blood islands that fuse and remodel to form a primitive capillary plexus.
What is the role of HIF-1α degradation in regulating oxygen homeostasis?
HIF-1α degradation helps maintain oxygen homeostasis by responding to oxygen levels. In high oxygen conditions, HIF-1α is hydroxylated, poly-ubiquitinated, and degraded by the proteasome. In low oxygen conditions, HIF-1α stabilization promotes the expression of genes required for adaptation to hypoxic conditions, such as VEGF.
What is the most common cancer among women, and which cancer has a higher mortality rate among women?
Breast cancer is the most common cancer among women, but lung cancer has a higher mortality rate.
Why do people die from breast cancer?
People die from breast cancer due to metastatic spread to other organs such as liver, bones, and brain
epithelial proliferation -> lobular/ductal -> high/low grade
What are the four types of breast cancer?
- Histological
- In-situ carcinoma (still within the breast, e.g., 3. DCIS and lobular with no metastasis)
- Ductal
- Invasive carcinomas
What are some non-modifiable and modifiable risk factors for breast cancer?
Non-modifiable: age (peak 50-70), previous breast disease (benign), family history and BRCA, hormone-related factors. Modifiable: high socio-economic group (obesity, alcohol, high-fat diet, fewer pregnancies, and less likely to breastfeed), male gender (only 1% of cases).
What factors lead to increased exposure to estrogens and a higher risk of breast cancer?
Early menarche before 12, late menopause, no child or first child after 30, breastfeeding reduces risk, and increased BMI post-menopause.
How is oestrogen receptor (ER) positivity tested in breast cancer?
Oestrogen receptor positivity is tested in biopsies/surgically removed primary tumors and sites of metastasis spread by immunohistochemistry. A tumor is deemed positive if at least 1% of cells express ER.
Luminal A breast cancer
ER and PgR positive, HER2 negative
low proliferation (Ki67 low)
recurrence risk low
few copy number changes
few mutated genes (>5%)
responsive to endocrine therapy
less to chemotherapy
luminal B breast cancer
ER positive
either HER2 positive OR high Ki-67 and PR-ve
often aneuploid
muttions in cyclin D1/EGFR1/PIK3CA/PTEN/TP53
responsive to endocrine therapy (less than luminal A)
more sensitive to chemotherapy
Hereditary breast cancer
BRCA1/2 mutations - high penetrance (high risk of developing cancer)
- role in DNA repair
- autosomal dominant
other genes low penetrance
- TP53 - switches on DNA repair pathways
- ATM - recognised DNA damage
- CHEK2: checkpoint kinase 2 (cell cycle control)
- PALB2 -recruits RAD51 and BRCA2 to damage
- rad51 - binds at site of DNA damage
How does tamoxifen work to inhibit breast cancer?
Tamoxifen works by competitively binding to the oestrogen receptor, stopping the oestrogen receptor from dimerizing and recruiting coactivators. This alters TAF1 and TAF2 regions’ activity, keeping cells in the G1 phase of the cell cycle, preventing proliferation.
What are some common treatment-related complications of tamoxifen and aromatase inhibitors?
Menopausal symptoms (hot flushes), fatigue, painful joints, nausea, rare occurrences of deep vein thrombosis, and endometrial cancer.
What is the difference between Luminal B and Luminal A breast cancer?
Luminal B is ER positive, either HER2 positive or has high Ki-67 and PR-negative, often aneuploid, less responsive to endocrine therapy than Luminal A, more sensitive to endocrine therapy, HER2 negative and is low grade with good prognosis
What is the Oncotype DX test used for?
used to determine the most appropriate treatment for early-stage ER+ breast cancer patients. It tests a panel of 21 genes, and the results help decide whether to give chemotherapy or hormone therapy based on the number of mutations present.
What are the characteristics of HER2 overexpressing and basal-like breast cancers?
HER2 overexpressing: ER and PR absent, responsive to Herceptin. Basal-like: generally triple-negative, high metastasi
What is screening?
Screening is the investigation of asymptomatic people to classify them as likely or unlikely to have a disease. People who appear likely to have the disease are investigated further and treated if necessary
What are the prerequisites for screening according to Wilson and Junger?
Important public health problem, accepted treatment, available facilities, recognized presymptomatic or latent phase, suitable test or examination, test acceptability, understood natural history, agreed policy on treatment, economically balanced cost, and continuous case-funding.
What characteristics make a disease suitable for screening?
Relatively common, severe consequences, detectable presymptomatic phase, early treatment advantage, and evidence of net benefit.
What is lead time bias?
Lead time bias occurs when screening detects a disease in its presymptomatic phase, making the period between detection and death longer, but not necessarily increasing survival time.
length time bias
diseases that may be identified via screening are more likely to be indolent and less aggressive conditions
more aggressive disease is less likely to be detected by screening because it is likely to develop fully btw successive routine screening points
survival following screen detected disease may be lengthened by the relatively less aggressive nature of the disease process
length bias may be identified comparing the aggressiveness of disease detected clinically btw screens with that detected by screening
Define sensitivity, specificity, positive predictive value, and negative predictive value.
Sensitivity: proportion with condition who test positive.
Specificity: proportion without condition who test negative.
Positive predictive value: proportion with positive test who have the condition.
Negative predictive value: proportion with negative test who do not have the condition.
What are the three potential screening tests for prostate cancer?
1) Digital Rectal Examination (DRE)
Transrectal Ultrasound (TRUS)
Prostate Specific Antigen (PSA)
What is Severe Combined Immunodeficiency (SCID)?
SCID is a genetic disorder that weakens the immune system, making patients highly susceptible to severe recurrent infections. Treatment involves a hematopoietic stem cell transplant.
What are the first lines of defense in the immune system?
The first lines of defense include skin, saliva, stomach acid, mucous, tears, sweat, and the lymphatic system.
What are B cells and T cells, and where are they made?
types of blood immune cells that are made in the bone marrow. B cells develop in the bone marrow and make antibodies, while T cells travel to the thymus and develop before circulating in the body.
How do B cells and T cells recognize antigens?
B cells recognize antigens through a B cell receptor made up of a heavy and a light chain with a Fab antigen-binding site. When a B cell recognizes an antigen and binds, it matures into a plasma cell that releases antibodies. T cells recognize peptides on antigen-presenting cells through a T cell receptor where heavy and light chains are anchored into the cell.
What are the MHC1 and MHC2 pathways for antigen presentation?
The MHC1 pathway involves the presentation of antigens expressed inside cells, which are degraded through proteosome into small peptide fragments that are translocated to the endoplasmic reticulum and bound onto MHC1, then translocated to the surface of the cell for presentation to CD8+ T cells. The MHC2 pathway involves the presentation of peptides from extracellular proteins that are taken up into the endosome pathway, where they are degraded by proteases into small peptide fragments that are bound to MHC2 and translocated onto the surface for CD4+ T cell recognition.
What are the MHC1 and MHC2 pathways for antigen presentation?
The MHC1 pathway involves the presentation of antigens expressed inside cells, which are degraded through proteosome into small peptide fragments that are translocated to the endoplasmic reticulum and bound onto MHC1, then translocated to the surface of the cell for presentation to CD8+ T cells. The MHC2 pathway involves the presentation of peptides from extracellular proteins that are taken up into the endosome pathway, where they are degraded by proteases into small peptide fragments that are bound to MHC2 and translocated onto the surface for CD4+ T cell recognition.
How diverse are T cell receptors, and what is thymic tolerance?
There are about 10^18 possible combinations of T cell receptors, which gives the immune system the ability to recognize almost anything throughout life. Thymic tolerance is a process by which T cells that recognize self are deleted during development, which reduces the number of possible combinations to about 10^14.
Why is it a challenge for the immune system to recognize cancer?
Cancer cells look similar to normal cells in terms of immunorecognition, and T cells that recognize self are deleted during development. However, the immune system can recognize viral proteins associated with cancer, overexpressed self proteins, lineage-specific antigens, and mutated self proteins such as CDK4. Cancer cells also have mechanisms to avoid immune recognition, such as downregulation of MHC, loss of components of the antigen processing pathway, and exploiting T cell checkpoints.
What are some immunological cancer interventions?
Immunological cancer interventions include the HPV vaccine to prevent cervical cancer, checkpoint blockade to reawaken T cells by interfering with cancer ligand PDL-1 binding, and CAR T cells, which are genetically engineered T cells that can recognize cancer.
What are the three types of chemotherapy?
The three types of chemotherapy are chemotherapy for advanced disease, adjuvant chemotherapy for systemic treatment following local radiotherapy or surgery, and primary or neo-adjuvant chemotherapy for initially treating locally advanced cancer to make it more amenable to subsequent surgery and control micro metastases.
What are the principles of chemotherapy?
Chemotherapy impacts the cell cycle, and there are cell cycle-specific drugs and cell cycle non-specific drugs. Cell cycle-specific drugs act on cells in specific phases of the cell cycle and are more effective against tumors with a higher percentage of replicating cells, while cell cycle non-specific drugs act on both resting and cycling cells and are useful against tumors with low or high percentages of replicating cells. The log-kill kinetics model states that theoretically, if enough chemotherapy cycles are given, a patient can be cured.
What are the common chemotherapy agents
alkylating agents
platinum agents
antimetabolites
topoisomerase inhibitors
antimicrotubular agents
other agents
molecular targeted agents
alkylating agents
covalently transfer alkyl groups to DNA bases
1. base alkylation - monofunctional DNA adducts, subsequent processing/repair of these lesions leads to SSBs in the DNA/mispairing of nucleotides
2. 2 bases are linked together by an alkylating agent forming cross-bridges cross-linking prevents DNA from being separated for DNA synthesis/transcription
limited cell cycle specificity - binds directly to DNA
platinum agents
discovered electric current delivered to bacterial culture via platinum electrodes led to inhibition of bacterial growth
active compound found to be cisplatin
bind covalently to purine DNA bases (N7 position)
bifuncitonal intra-strand crosslinks
prevents DNA double strand from separating
not S phase specific
nephrotoxicity nad resistance
antimetabolites
acts at a level of DNA synthesis
interfere with incorporation of nucleic acid bases
class 1 purine/pyrimidine analogues
- inhibits formation of normal nucleotides
- often inhibit enzymes essential for DNA and RNA synthesis
class 2 prevent formation of reduced folate
- essential for transfer of methyl groups in DNA synthesis
usually S phase specific
acts on cancer and normal cells that are dividing rapidly
- can cause significant bone marrow and GI toxicity
no late carcinogenesis
topoisomerase inhibitors
DNA topoisomerases - ubiquitous nuclear enzymes
relax supercoiled dsDNA to allow DNA replication and RN transcription
topoisomerase I = ss nicks
- bind to and stabilise DNA topoisomerase I adducts
- inhibits re-ligation of DNA strands
- SSB in DNA
- e.g. camptothecin/irinotecan/topotecan
topoisomerase II = ds nicks
- forms a complex with topoisomerase II after cleavage of DNA
- inhibits re-ligation of DNA strands
- SSB/DSB in DNA
- e.g. etoposide/anthracyclines
swivelling of supercoiled DNA occurs at nicks followed by re-ligation to relieve torsional strain
antimicrotubular agents
prevent spindle formation
- essential for sorting and moving of chromosomes following replication at end of mitosis
vinca alkaloids
- vincristine/vinblastine/vinorelbine
- binds to tubulin, preventing polymerisation of microtubules
- inhibits progression through the cell cycle
taxanes (paclitaxel/docetaxel)
binds to tubulin (different site to vinca alkaloids)
- prevents microtubular disassembly
- disrupts normal microtubule dynamics that is required for cell division
What are the common chemotherapy toxicities on dividing cells
myelosuppression
mucositis
diarrhoes
alopecia
germinal epithelium
skin
common chemotherapy toxicities: non-replicative toxicity
nausea, vomiting
neuropathy
cardiac
renal
pulmonary
allergy
What are the criteria for assessing response to chemotherapy?
The criteria for assessing response to chemotherapy are RECIST for stable response, which is defined as <30% decrease or <20% increase in the sum of defined measurable disease (unidimensional), and disease progression, which is defined as >20% increase in the sum of all measurable lesions or new lesions. Complete response is a prerequisite for cure and improved survival, while partial response has palliative value only, offset by drug toxicity effects and a more modest impact on survival. Stable response has any impact on survival limited to continuous, and the quality of response is defined by performance status and quality of life measurements.
What are the objectives of combined chemotherapy, and how are targets selected for the development of novel cancer drugs?
provide maximum cell kill within the range of toxicity that can be tolerated for each drug, provide a broader range of coverage of resistant cells in a heterogeneous tumor population, and prevent or slow the development of drug-resistant cells. Targets are selected for the development of novel cancer drugs based on mutant oncogene products found in tumors and not normal tissue, tumor-induced alterations of the microenvironment needed for cancer progression, and genetics.
response to chemotherapy
complete response (CR)
- prerequisite (but not sufficient) for cure
- implications for improved survival
partial response (PR)
- palliative value only, offset by drug toxicity effects
- more modest impact on survival
stable response
- any impact on survival limited to continous (non-toxic) therapy
quality of response may be defined by
- performance status
- quality of life measurements
- relapse - free survival, from time all treatment discontinued
selection of targets for the development of novel cancer drugs
mutant oncogene products commonly found in tumours but not in normal tissues
tumour induced alterations of the micro environment necessary for cancer progression e.g. angiogenesis
must have a causal role in cancer development
genetics and biology must be well established
suitable for in vitro based assays
suitable for pharmacological intervention
What are the differences between targeted drugs and chemotherapy?
targeted drugs
- target abnormalities typical of cancer cells
- act selectively agains cancer cells
- limited toxicity
- often orally available
- chronic treatment may control cancer for a long time
chemotherapy
- targets common ell growth mechanisms
- equally affects normal and cancer cells
- often very toxic
- usually given IV
- given only for a limited number of cycles (6-10)
What are the four types of therapeutic modalities?
Conventional ‘small molecule’ drugs
Biopharmaceuticals
Gene therapy
Cell therapies
What is the first stage of modern drug development pathway?
drug discovery.
What is the Lipinski rule of 5?
The Lipinski rule of 5 evaluates whether a compound is likely to be orally active. A good drug candidate will have:
Oral availability > 50%
Activity such that dose ideally < 100mg per day
T1/2 clearance in blood > 3hrs
Good therapeutic index (high selectivity for target, minimal off target effects)
What are the four main adverse reactions in drug development?
Exaggerated effect of drugs
Side effects
Toxic effects unrelated to drug pharmacology
Idiosyncratic
What is drug repurposing?
Drug repurposing is using existing drugs for a new purpose, such as thalidomide for leprosy and myeloma and Viagra for high blood pressure and now for erectile dysfunction.
What is high throughput screening?
A screening program that uses a suitable assay to screen a large chemical compound library to find those with activity against the target.
What are traditional tumor markers found in body fluids?
Proteins overexpressed in tumors, tissue-specific proteins such as PSA, oncofetal antigens, and carbohydrate antigens are traditional tumor markers found in body fluids.
How is a good biomarker for a disease determined?
sensitivity = & of disease cancers correctly predicted
specificity = % of non-cancers (controls) correctly predicted
receiver operator characteristic (ROC) curve shows how sensitivity and specificity vary with the threshold used to define a positive test result
real life overall performance is influenced by prevalence of disease
positive predicted value (PPV) = % positive tests correct
negative predicted value (NPV) = % negative tests correct.
What factors determine diagnostic biomarker utility?
sensitivity and specificity
disease prevalence (NPV/PPV)
consequences of a false negative test
consequences of a false positive test
benefit to the patient - ability to detect cancer at an early stage/less invasively
cost benefit (testing costs versus costs of more expensive investigations)
circulating protein biomarkers are typically useful for monitoring disease but not screening, definitive diagnosis or treatment selection
What methods are used to validate biomarkers?
Targeted measurements such as ELISA and immunohistochemistry are used to validate biomarkers. Appropriate clinical specimens are required for validation. Prospective randomized clinical trials, thorough clinical validation, thousands of patients, properly certified assays, patients investigated long term, and definitive benefits to the NHS are also needed to validate biomarkers.
how are biomarkers assayed
ELISA (quanititative)
lateral flow device (point of care, fast but often qualitative)
dicover of new biomarkers
by OMIC tools/targeted measurements
found in tissue/bio-fluid
in only a few samples
What is a liquid biopsy?
A liquid biopsy is a non-invasive method of detecting cancer that involves analyzing blood plasma for signals from tumors. Circulating tumor cells and DNA are present in blood plasma, and their levels can be used to monitor tumor evolution and treatment response. Circulating tumor-free DNA can also be used to detect tumor-specific information such as mutations.
ctDNA challenges and solutions
challenges
- fragmented DNA
- low quality DNA
- low percentage of cell free DNA is actually ctDNA
- careful sample collection and processing to minimise genomic DNA
solutions
- next generation sequencing (NGS) of thousands of tumours identified lots of potential ctDNA biomarkers
technological advances = detection of cancer specific biomarkers in ctDNA possible
circulating tumour free DNA (ctDNA) as a biomarker
tumours shed cells into circulation (CTCs)
very low numbers
enriched using antibodies to cell surface proteins (EPCAM) or physical properties
cells stained and counted (cytokeratin +ve, CD45 -ve)
high numbers are indicative of poor outcome and need for more aggressive treatemtn
falling numbers indicate therapy is working
are prognostic in G3T1 bladder cancer - likely recurrence if present before surgery
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
a very low no. of CTCs currently excludes a role in early diagnosis
What are predictive and prognostic biomarkers?
Predictive biomarkers are used to decide which treatments are suitable for which cancer patients, while prognostic biomarkers indicate the likely course of the disease in untreated individuals. Prognostic markers can be DNA, RNA, or protein-based, and they are used to determine how aggressive the cancer is and how aggressive the treatment should be to avoid under/over treatment. Oncotype DX is an example of a breast cancer prognostic test.
How are biomarkers used for screening pre-malignant conditions?
Biomarkers such as FOBT for bowel cancer, PSA for prostate cancer, and CA-125 for ovarian cancer are used for screening pre-malignant conditions. These biomarkers detect blood or protein levels that are indicative of the presence of cancer in the body.
How are biomarkers used to assess after treatment?
Biomarkers such as CEA for colorectal cancer and PSA for prostate cancer are used to assess after treatment. These biomarkers are used to measure the recurrence of cancer after treatment and to monitor treatment response.
Compare benign and malignant tumours
Benign:
- non-invasive
- well defined border
- well differentiated
- regular nuclei
- rare mitosis
Malignant:
- invasive/metastatic
- poorly differentiated
- irregular, larger nuclei
- frequent mitosis
Define benign, malignant and metastatic
Benign: local multiplication of cells within tissue, growth confimed to specific site and no evidence of invading adjacent tissues
Malignant: growth that is locally invasive; tumour cells spread locally wihtin a tissue and are disorganised
Metastatic: tumour cells invade other tissues/ organs, a metastasis is a malignant growth formed by cells divided from a malignant primery tumour.
Define hyperplasia, dysplasia and neoplasia
Hyperplasia: increased cell number they appear normal
Dysplasia: changes in cell number and appearance, causing effecta on overall tissue archetecture. premalignant tissue
Neoplasia: bening or malignant tumour composed of cells iwth abnormal appearance and proliferation
What are the three main goals of cancer treatments?
Cancer treatments aim to kill or remove all of the cancer cells, kill most cancer cells to prolong survival, or kill some cancer cells to palliate symptoms.
What is the meaning of “remission” in cancer treatment?
“Remission” means that most of the cancer cells have been killed through treatment.
How is treatment efficacy measured in cancer patients?
measured by tumour response, which can be assessed using the Response Evaluation Criteria in Solid Tumours (RECIST).
What are the four possible outcomes of the RECIST assessment?
complete response (CR)- dissappearance of all target lesions
partial response (PR)- at least 30% decrease in sum of diameters
progression disease (PD)- at least 20% increase in sum of diameters and
stable disease (SD)- neithe sufficient shrinkage or growth
Does tumour response necessarily lead to prolonged survival?
Tumour response does not necessarily lead to prolonged survival.
What is overall survival time in cancer treatment?
Overall survival time is the time from the start of treatment to the patient’s death.
What is disease-free survival time in cancer treatment?
Disease-free survival time is the time prior to tumour relapse after radical treatment.
What is progression-free survival time in cancer treatment?
Progression-free survival time is the survival time prior to tumour progression.
Why is a large group of patients needed in a clinical trial?
A large group of patients is needed in a clinical trial to ensure that the treatment works effectively in a variety of patients from different centres.
What is a clinical trial?
A clinical trial is a planned experiment that involves patients and is designed to determine the most appropriate method of treating a medical condition in future patients.
What is the typical design for a phase III clinical trial?
randomized controlled trial (RCT), which directly compares the new treatment against the current standard of care, measuring clinically relevant outcomes.
What are the three phases of clinical trials?
phase I, which looks at safety profile and finds the maximum dosage;
phase II, which looks at the efficacy of the dosage given; and
phase III, which compares the new treatment against the standard of care to determine its true benefits.
What is a hazard ratio (HR) in cancer treatment?
measures the relative difference in survival between the new and standard treatments.
What is a waterfall plot in cancer treatment?
A waterfall plot in cancer treatment shows the amount of tumour shrinkage in each patient, with each bar representing a patient.
What are biomarkers in cancer treatment?
Biomarkers are molecules that indicate a particular biological state or condition, and can be used to predict the efficacy of treatment in cancer patients.
How is the Hazard Ratio calculated?
Hazard ratio= risk of death on NEW/ risk of death on STANDARD
What does a Hazard Ratio of 0.5 mean in cancer treatment?
Hazard Ratio of 0.5 means that the risk of death on the new treatment is half of that on the standard treatment.
How is the interpretation of the Hazard Ratio affected by the order of the comparison (new vs standard or standard vs new)?
The interpretation of the Hazard Ratio depends on the order of the comparison (new vs standard or standard vs new). For example, an HR of 0.5 is equivalent to an HR of 2, depending on the order of the comparison.
What does an HR below 1 mean in cancer treatment?
An HR below 1 means that the risk of death is reduced with the new treatment compared to the standard treatment.
the development of cancer therapy
development of chemotherapy
alkylating agents identified through toxic effect of mustard gas
folate antagonists developed from anaemia treatment
folic acid as a treatment for anaemia, given to individuals with leukaemia cause an acceleration of symptoms
folic acid antagonists developed as a chemotherapy
HPV infection and the formation of squamous neoplasia
normal cells -> HPV infection -> low grade SIL -> de-regulation of E6/7 -> high grade SIL -> invasive cancer
at each stage advances in telomerase activation, inhibition of apoptosis, genetic changes and immune response occur
air pollution components
PM
sulphur dioxide
ozone
carbon monoxide
nitrogen dioxide
polycyclic aromatic hydrocarbons (PAHs)
replicative senescence- ageing
progressive loss of telomeres [erosion] induces a DNA damage response and this triggers senescence
potent tumour suppressor
Crisis- M2 replicative senescence
2nd proliferative barrier
telomeres continue to erode
the telomeres become too short and can’t protect the chromosomes which triggers apoptosis
failure to protect the chromosomes leads to chromosome abnormalities - chromosome end to end fusions or changes in copy number
in cells with inactive pRB and p53 there is no check on proliferation - genetic instability and accumulation of mutations
extracellular matrix degrading proteases
MMPs
serine proteases
adamalysin-related membrane proteases
BMP1 type metalloprotinases
heparanases
cysteine proteases - cathepsins
cells within the tumour microenvironment
complex mix of cells that changes as the tumour develops invades and metastasises to new sites
cancer associated fibroblasts (CAFs) = important constituent of the stroma
- directly produce factors that promote angiogenesis
- indirectly attract pro-inflammatory cells such as macrophages which stimulate cancer invasion
development of vessles: angiogenesis
angiogenic remodelling of the dorsal aorta, cardinal vein and vascular plexi give rise to the arteries, veins and capillaries
angiogenic sprouting of new vessels
pericytes are recruited to stabilise capillaries
lymphatic endothelial cells sprout from veins and from the lymphatic system via lymphangiogenesis
development of vessles: angiogenesis
angiogenic remodelling of the dorsal aorta, cardinal vein and vascular plexi give rise to the arteries, veins and capillaries
angiogenic sprouting of new vessels
pericytes are recruited to stabilise capillaries
lymphatic endothelial cells sprout from veins and from the lymphatic system via lymphangiogenesis
HIF-1a and low oxygen
low O2 levels there is not enough O2 to drive the proline hydroxylation of HIF-1a
HIF-1a is free to bind to HIF-1b and induce transcription of genes required for the adaption to hypoxic conditions
key target = VEGF
von-hippel lindau disease
autosomal dominant cancer syndrome with mutation in the pVHL gene
pVHL forms the recognition components of the E3 ubiquitin ligase for HIF-1a
- pVHL provides the specificity for this E3 ubiquitin ligase
- pVHL recognises hydroxylated prolines
topoisomerase inhibitors
DNA topoisomerases - ubiquitous nuclear enzymes
relax supercoiled dsDNA to allow DNA replication and RN transcription
topoisomerase I = ss nicks
- bind to and stabilise DNA topoisomerase I adducts
- inhibits re-ligation of DNA strands
- SSB in DNA
- e.g. camptothecin/irinotecan/topotecan
topoisomerase II = ds nicks
- forms a complex with topoisomerase II after cleavage of DNA
- inhibits re-ligation of DNA strands
- SSB/DSB in DNA
- e.g. etoposide/anthracyclines
swivelling of supercoiled DNA occurs at nicks followed by re-ligation to relieve torsional strain
response to chemotherapy
complete response (CR)
- prerequisite (but not sufficient) for cure
- implications for improved survival
partial response (PR)
- palliative value only, offset by drug toxicity effects
- more modest impact on survival
stable response
- any impact on survival limited to continous (non-toxic) therapy
quality of response may be defined by
- performance status
- quality of life measurements
- relapse - free survival, from time all treatment discontinued
circulating tumour free DNA (ctDNA) as a biomarker
tumours shed cells into circulation (CTCs)
very low numbers
enriched using antibodies to cell surface proteins (EPCAM) or physical properties
cells stained and counted (cytokeratin +ve, CD45 -ve)
high numbers are indicative of poor outcome and need for more aggressive treatemtn
falling numbers indicate therapy is working
are prognostic in G3T1 bladder cancer - likely recurrence if present before surgery
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
a very low no. of CTCs currently excludes a role in early diagnosis
ctDNA challenges and solutions
challenges
- fragmented DNA
- low quality DNA
- low percentage of cell free DNA is actually ctDNA
- careful sample collection and processing to minimise genomic DNA
solutions
- next generation sequencing (NGS) of thousands of tumours identified lots of potential ctDNA biomarkers
technological advances = detection of cancer specific biomarkers in ctDNA possible
cancer genome: cancer genomics
germline mutations
- shared with relatives
- present in every cell of the body
- neutral or not
somatic mutations
- unique to patient
- present in tumour cells
- driver or passenger
cancer genome: meiosis and germline mutations
chromosome number from 46 to 23, from 2N to N
cancer genome: mitosis and somatic mutations
duplication of the 23 chromosomes: 46 or 4N
then cell division to form 2 cells with 23 chromosomes or 2N
cancer genome: BRCA1
mutation in a cancer susceptibility gene
on chromosome 17
protein has a role in genomic stability
autosomal dominant transmission
500 different mutations reported: nonsense/frameshift/missense/splice-site
cancer genome: BRCA2
mutation in a cancer susceptibility gene
on chromosome 13
protein has a role in genomic stability
autosomal dominant transmission
300 different mutations reported:
nonsense/frameshift/missense
somatic mutations: two hit hypothesis
knudson’s 2 hit hypothesis
- inheriting 1 germline mutation was not sufficient enough to cause cancer, need a second somatic hit
- more likely to get a somatic mutation if there is a germline mutation then if there was not germline mutation
if the 1st hit is a germline mutation the 2nd somatic mutation is more likely to enable cancer
somatic mutations: clonality
acquired mutations are passed on to the progeny during cell growth and division
cells with cancer linked mutations proliferate more than normal cells causing the accumulation of mutations
a cell with acquire enough mutations to become a cancer cell - subsequent cancer cells we be derived from this one cell
somatic mutations: heterogeneity
early somatic events are present in a high proportion of the cancer cells than later somatic events - present in a subset of cells
somatic mutations: next generation sequencing
clinical applications different from research - faster/cheaper/more reliable
different problem between research/clinical
portable generation of machines
WGS500 designs look at
- de novo/recessive/familial/independent cohorts/cancer pairs/metastasis/progression
somatic mutations: genotyping in tumours
doesn’t always fulfill the diploidy hypothesis
0 copy - no genotype
1 copy - A/B
2 copies - normal or homozygotes - AA/AB/BB or AA/BB
3 copies - allow up to 4 genotypes - AAA/AAB/ABB/BBB
4 copies - allow up to 5 genotypes - AAAA/AAAB/AABB/ABBB/BBBB
always appears as AA, AB or BB (orNA)
structural variations: philadelphia chromosome
abnormality of chromosome 22 - part of chromosome 9 is transferred to it
bone marrow cells containing this are found in CML
- piece of chromosome 9/chromosome 22 break off and switch places
- BCR-ABL gene is formed on chromosome 22 where the piece of chromosome 9 attaches
types of sequencing
most conventional NGS platforms use sequencing by synthesis
ion torrent uses H+ release during sequence synthesis to determine changes in sequence
illumina uses fluorophore during sequence synthesis to determine change in sequence
both methods held back by short read lengths/relatively expensive
nanopore sequencing
uses nonopores - protein pores attached to a semiconductor membrane
allows voltage detection as DNA passes through
voltage change determines base - allows detection of sequence
also theoretically allows epigenetic modifications to be studied
cancer asa chronic disease
- patient diagnosed with cancer
- cancer undergoes ‘liquid biopsy’ and sequencing to identify mutational drivers of cancer
- surgery and/or targeted therapy
- recurrence of cancer (back to 2) OR patient cured
CTCs/ctDNA
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
CRISPR/Cas9
CRISPR = clustered regularly-interspaced short palindromic repeats
CAS9 = CRISPR associated protein 9
CAS9 unwinds dsDNA and checks for matches between a guide RNA and the strand of interest
if it finds a match it induces a DSB
new sequence introduced as part of CRISPR-Cas9 complex
NHEJ/HR repairs break and inserts new DNA
Endogeneous angiogenesis inhibitors
Angiostatin
primary tumours produce angiogenic factors that prevent secondary tumours from being vascularised, small tumours dont produce big
HM: Inducing angiogenesis
some oncogenes (Myc, Ras) can upregulate expression of angiogenic factors
HM: activating invasion and metastasis
crosstalk between cancer cells and cell of the neoplastic stroma
- mesenchymal stem cells in tumour stroma secrete CCL5, stimulates invasion
- Macrophages: Metalloproteeinases, EGF
- inflammatory cells: extracellular matrix-degrating enzymes
what are the emerging hallmarks of cancer ?
1) reprogramming energy metabolism, cancder cells limit energy metabolism to glycolysis- aerobic. it allows diversion of glycolytic intermediates into biosynthesis pathways
2) evading immune system, cancer cells may paralse infiltrating CTLs and NK cells, such as TGF-b
Tumour microenviroment cells
- cancer stem cells
- endothelial cells can have tumour associated vasculature and lymphatic vessels present on peripheries of tumours
- pericytes are specialised mesenchymal cell type, stability for vascular integrity and function
- immune inflammatory cells e.g macrophages , lymphocytes, neutrophils, releases signal moleucles and can be pro and anti tumour
- cancer associated fibroblasts such as myofibroblast usually present on site of chronic inflammtion and wound healing. in tumours: proliferation, angiogenesis and invasion
Tumour microenviroment cells
- cancer stem cells
- endothelial cells can have tumour associated vasculature and lymphatic vessels present on peripheries of tumours
- pericytes are specialised mesenchymal cell type, stability for vascular integrity and function
- immune inflammatory cells e.g macrophages , lymphocytes, neutrophils, releases signal moleucles and can be pro and anti tumour
- cancer associated fibroblasts such as myofibroblast usually present on site of chronic inflammtion and wound healing. in tumours: proliferation, angiogenesis and invasion
