Session 4: Cancer Flashcards
What is a tumor suppressor gene
Tumor suppressors normally function to control cell growth and proliferation
LOF mutations contribute to the abnormal proliferation of cancer cells
What is an oncogene
- A gene that normally is involved in controlling cellular proliferation.
- When altered/over-activated, oncogenes can help transform normal cells into tumour cells by promoting uncontrolled cell growth.
Associated with GOF mutations
What are the main categories of TSG
Gate Keeper TSGs- control cell cycle progression e.g. TP53, cyclins and CDKs. e.g. mutation of TP53 (normally inhibits cell cycle progression) is mutated in 50% of tumours
Caretaker TSGs- maintain the fidelity of the genome by repairing damage e.g BRCA1/2 (HR), MSH2, MSH6, MLH1, PMS2 (MMR), MUTYH (BER) etc
How do TSGs restrain cell growth
- Inhibit the cell cycle (cyclins, CDKs, RB, TP53, APC)
- Apoptosis (TP53)
- Repair damage
Give 2 examples of TSGs
RB1
TP53
What is the Knudson hypothesis?
The Knudson hypothesis is derived from the genetic mechanisms underlying RB1 (first TSG discovered)
Explained the differences between hereditary (early onset bilateral retinoblastoma with risk of cancer in other tissues) and sporadic RB (usually unilateral, later onset).
2 hit hypothesis in which both alleles of a TSG must be lost to develop cancer. In hereditary cancer one copy of the gene is already KO so only a single additional acquired mutation is required resulting in earlier onset. this results in variable penetrate and apparently AD inheritance
Describe Retinoblastoma
early onset (<5yrs) aggressive childhood cancer of the eye. Characterised by whitening of the pupil.
Can be unilateral (usually sporadic) or bilateral (usually familial + increased risk of soft tissue and bone cancers)
Mutation spectrum includes SNVs, CNVs, SVs and hypermethylation of the RB1 promoter (10%). 60-70% display LOH of 1 allele with a mutation in the other allele. truncating mutations and deletions associated with almost complete penetrance whereas missense and splice site can have reduced penetrance and variable expressivity
What is the role of the RB1 gene
RB1 is found at 13q14
Key role in G1/S phase cell cycle checkpoint
nuclear phosphoprotein which is involved in cell cycle progression. When it is unphosphorlylated it bind E2F transcription factor preventing it entering the nucleus to activate transcription of target genes= cell cycle repression
When phosphorylated by cylcin D CDK 4/6 it dissociates from E2F which can then activate transcription of target genes (cylin E) and the cell can progress from G1 to S phase.
Describe p53’s role as a TSG
- second TSG to be discovered
- mutated in 50% of tumours
- essential in multiple signalling pathways associated with cell cycle, apoptosis and DNA repair
How does p53 contribute to cell cycle control
p53 can act as a transcription factor to activate the transcription of genes associated with cell cycle arrest and apoptosis.
In normal cells it is bound to MDM2 which retains it in the cytoplasm. (MDM2 required phosphorylation to migrate to the nucleus and bind to p53 and cause it to migrate to the cytoplasm) in the cytoplasm p53 is degraded by the ubiquitin/pretoeosom pathway.
in response to stress p53 is phosphorylated and acetylated = it can dissociate from MDM2 and activate transcription of genes e.g. PUMA which controls apoptosis.
Methods for loss of p53 function
- mutations to upstream genes e.g. ATM or CHEK2
- mutations in p53 (~50% of tumours)
p53 mutations can be LOF or GOF - mutations in gene that act downstream of p53 e.g. PTEN (germline mutations associated with Cowdens syndrome)
Describe the role of CDKN2A in the cell cycle.
CDKN2a encodes 2 unrelated proteins:
p16 INK4a- this inhibit CDK4/6 keeping RB1 dephosphorylated and bound to E2F = inhibits cell cycle
p14ARF- destabilises the interaction between MDM2 and p53 = p53 active resulting in cell cycle arrest
Germline mutations in CDKN2a is associated with malignant melanoma (penetrance depends on age and sun exposure)
Describe 2 examples of miRNAs that can function as TSGs
- Let-7
normally expressed in differentiated tissues but lost in NSCLC
negatively regulates cell cycle oncogenes and exogenous application to human lung caner cell reduces proliferation - miR-34 family
Lost in lung cancer
expression activated by p53 and associated with apoptosis
Name 3 additional TSGs, their molecular function and associated cancer susceptibility syndrome
- BRCA1/2- DSB repair and HR (HBOC)
- Lynch syndrome- MMR (CRC)
- APC- negative regulator of b-catenin. It is part of the B-catenin complex and targets it for ubiquitin mediated degradation. (FAP)
What are the 5 broad categories of oncogenes?
- secreted growth factors
- growth factor receptors
- Signal transducers - PIK3A
- Inhibitors of apoptosis - BCL2
- Transcription factors EWS-FLYI
Give an example of a secreted growth factor acting as an oncogene
secreted growth factors can act as an oncogene due to the constitutive activation of a growth factor gene e.g. normal wnt/b-catenin signalling is involved in embryonic development whereas over activation of the pathway is involved in many cancers including breast cancer.
Give an example of a growth factor receptor acting as an oncogene
e.g. EGFR and RET
EGFR receptor is constitutively expressed in NSCLC
- activating mutations occur in exons 18, 19 and 12.
- The gene encodes a RTK and mutation results in constitutive activation and over activation of downstream pathways
- Activating mutations can result in dependency for the cancerous cell on aberrant EGFR signalling
- Can be targeted by specific anti-EGFR therapies
- mutations can occur in the ATP binding pocket which reduce the affinity for ATP and increase the sensitivity to the RTK which competes with ATP for binding.
- Resistance mutations can occur in the catalytic domain which weaken the interaction between the inhibitor and its target
Give an example of a secreted signal transducer acting as an oncogene
PI3KA
- calls 1 signal transducer
- composed of a heterodimer of a catalytic and regulatory subunit and results in phosphorylation of phosphatidyl inositol lipids.
- plays a role in cell motabilism, motility and cell cycle regulation
- transmits signals from RTKs and GPCRs
- 13% have PI3KA mutations with a hotspot in the kinase domain
- RTK phosphorylates and activates PI3KA
- PI3KA phosphorlyates the inositol ring of PIP2 and converts it to PIP3
(PIP3 can also be directly activated by RAS by biding to the catalytic domain) - PIP3 is active and can directly bind and activated proteins with a pleckstrin homology domain e.g. PDPK1 and AKT which results in cell growth and proliferation.
PTEN tightly regulate PIP3 in normal cells by dephosphorylation to the inactive PIP2
Give an example of a an oncogene which inhibits apoptosis
BCL2
cytoplasmic protein which localises to the mitochondria and inhibits apoptosis.
overexpressed in most follicular lymphoma
t(14;18) BCL2-IGH rearangement results in BCL2 being under the control of the IGH locus
Give an example of a transcription factor acting as an oncogene
EWS-FLI1 rearrangement in Ewings sarcomma
- t(11;22)(q24;q12) most freq
- found in soft tissue and bone cancers
- results in a fusion between the FLI1 gene resulting in an aberrant transcription factor = aberrant regulation of growth and cell proliferation
What are the main mutation mechanisms for activating an oncogene (GOF)
- Point mutations- can constiutively activate a signalling pathway e.g. mutation to the regulatory domain or dimerisation domain. Results in hyperactivation of a protein that is expressed in normal amounts
- Amplification- overexpression of a non-mutated gene leading to excess protein e.g. Her-2 in BC or MYCN in NB
- Translocation to produce a novel fusion gene- aberrant or dysregulated function. e.g. BCR-ABL in CML
- Translocation into a transcriptionally active region e.g. Burkitts Lymphoma. t(8;14)(q24;q32) is seen in 75% of patients and results in juxtaposition of the MYC oncogene with an immunoglobulin (IG) locus and consequently, MYC is brought under transcriptional control of the IG locus at the same time as losing its own. Other Ig locus rearrangements are also common in cancer.
- Local DNA rearangements- fusion genes can be created by inversion or deletion of the intervening region between a gene e.g. Inv(16) in AML
- Insertional mutagenesis- Hepatitis B in hepatocellular carcinoma. Viral oncogenes insert near cellular genes such as MYC and aberrantly activate it to initiate unchecked cellular proliferation.
what is tumour mutation burden (TMB) testing?
it is a measurement of the total number of non synonymous mutations in the tumour exome
the number of somatic mutations per megabase of interrogated genomic sequence, varies across malignancies.
What is the clinical utility of TMB?
can be used as a biomarker for immunotherapy, especially for the use of immune checkpoint inhibitors.
how does TMB influence immune checkpoint inhibitors?
somatic mutations can result in the expression of neoantigens and the chance is greater the higher the TMB
The neoantigens can activate the proliferation of T-cells which act to kill the cancer cells and such tumours can be targeted with immune checkpoint inhibitors-
How do checkpoint inhibitors work
these work by blocking the binding of checkpoint proteins to their partners
prevent the off signal from being sent so T-cells kill the cancer cells.
ICIs show variable efficicy and biomarkers help stratfiy patients that will respond
- MSI high tumours also show good response to ICIs as there is a high TMB
high TMB correlates with a good response to ICIs and increased survival in some cancers including NSCLC
response is not consistent across different cancer types and there is no established threshold or methodology for TMB
What tests can be used to determine TMB?
WES/WGS panels- WES considered best but not as cheap or fast as targeted panels- targeted panels are used to exptrapolate the number of somatic coding mutations observed in the targeted genomic space to the no. that would be observed across the whole genome. tumour percentage, qualit and seq depth all influence the number of somatic mutations detected.
standardization is required to fully assess and implement TMB as a biomarker in the UK
what does ‘actionable’ mean in terms of variant interp
variant can be used a biomarker for the patients disease
what is a biomarker?
a marker of disease that can provide info that is useful for the diagnosis, prognosis or treatment of a patient
What are the current UL guidelines for somatic variant interpretation
ACMG and ACGS BPG for variant classification state that a different set of interpretation guidelines is needed for somatic variants
2017 American association for molecular pathology released guidance and a working group has been established in the UK
what are the differences between somatic and germline variant interpretation
constitutional-
homogeneous, allele fraction 0.5 or 1 (unless mosaic), low AF can generally be discounted (be wary of mosaic),
family studies are possible
Is the variant causative of phenotype?
germline- tumour heterogeneity
variable allele fraction
low AF- genuine or seq artifact
cannot use linkage
Is the variant a driver of tumourigenesis, useful for tumour classification, have prognostic implications, target-able with a drug?
describe the difference stages of the cells cycle
G1= growth 1. RNA and proteins are synthesised (not DNA). each chromosome exits as a double helix
S- DNA synthesis- each chromosome is now present as sister chromatids
G2- cell continues to grow
M- mitosis- cell stops growing and there is nuclear division (miosis) followed by cellular division (cytokinesis) to produce to daughter cells.
Go= senescence the cell has left the cell cycle and stopped dividing. the cell may re-join the cell cycle in response to specific signals.
How is the cell cycle controlled?
cell cycle checkpoints
what are the cell cycle checkpoints?
- G1/S checkpoint (restirction point) once passed the cell is commited to division and enters S phase
- G2 checkpoint ensure there is enough cytoplasmic materials available for mitosis and cyotkinesis
- Metaphase checkpoint at the end of mitosis ensure that chromosomes are correctly attached to the spindle.
What controls the cell cycle checkpoints
controlled by heterodimeric protein kinases.
Consist of:
- constituitively expressed cdk kinase- this provides the catalytic unit but is inactive without its cyclin parter
- cyclin regulatory unit which is expressed at specific times in the cell cycle.
Together they act to phosphorylate target protein and their action is reversed Cdk inhibitors (CKI’s) = phosphatases. CKI’s are frequesntly mutated in cancer.
describe the control of the G1 restriction point.
after this checkpoint the cell is irreversibly committed to cell division.
controlled by cdk4/6-cyclin D complex whihc is formed in response to growth
cdk4/6-cyclin d phosphorylation and inactivates RB1 (TSG), releasing its inhibition of E2F (transcription factor). E2F can enter the nucleus and activate transcription of genes involved in cell cycle progression.= cyclin E is produced and forms a complex with cdk2 results in in G1/s transition.
the RB1 is hypophosphorylated it is in its active form and it bound to E2F, inhibiting its TF action and preventing cell cycle progression.
Give examples of disregulation of G1/S checkpoint in cancer
In retinoblastoma RB1 is mutated so that it cannot bind E2F and control cell cycle, resulting in uncontrolled cell growth
Cyclin D is overexpressed in many cancers resulting is phosphorylation of RB1 and loss of its cell cycle control even in the absence of growth signals. Cyclin D over expression can be induce by the PI3K and RAS pathways.
how can TP53 control the cell cycle?
- can arrest the cell in G1 by the production of CKIs (reverse phsphorylation by Cdks)
- can trigger apoptosis or production of DNA repair enzymes by acting as a TF
How is TP53 regulated at the G1/S checkpoint?
in normal cells TP53 levels are kept low by binding to MDM2. TP53 induces MDM2 expression resulting in a negative feedback loop
- MDM2 is a ubiquitin ligsae that must be phosphorylated to migrate to the nucleus and interact with P53.
MDM2 binding to TP53 sequesters it in the cytoplasm where it is ubiqutinated and targeted to the proteosome for degredation.
in response to stress TP53 dissociates from MDM2. this allows it to travel to the nucleus and activate transcription of genes involved in cell cycle arrest and apoptosis e.g. PUMA
How does CDKN2A contribute to cell cycle control?
Transcribed into two unrelated genes
1. p16INK4A- inhibits cdk4/6 so RB1 is not phosphorylated and remains bound to E2F preventing the transcription of cyclins required for cell cycle progression
2. p14ARF destabilizes the MDM2-P53 interaction so p53 is released and can activated cell cycle arrest in G1
CDKN2A is mutated in many cancer we.g. multiple melanoma. cancer risk is dependent on age and sun exposure
How is the G2/M checkpoint controlled?
Cdk1 is activated by phosphorylation and dephosphorylation of specific residues and the formation of the Cdk1-cyclin B complex (AKA MPF)
How is the metaphase (spindle checkpoint) controlled?
The spindle checkpoint ensures that the chromosomes are correctly aligned on the metaphase plate and that sister chromatids are correctly attached to the spindle.
The anapahse promoting complex is activated -> this degrade cyclin B (MPF disassembly) which releases inhibition from seperase resulting in sister chromatid separation and progression to anaphase
How does disregulation of the cell cycle contribute to tumorgenesis?
failure to activate checkpoint in response to damage results in maintenance of the damage and genome instability- mutated or damaged cells remain in the cell cycle and or there is increased cell proliferation.
what are possible cell cycle therapeutic targets?
- gene silencing has been used to target MDM2- this prevents its interaction with TP53, allowing levels to rise in the cell and activation of cell cycle arrest and apoptosis.
- can inhibit the E3 ubiqutin ligase activity of MDM2 o prevent it from targeting TP53 for degredation.
how does meiosis differ from mitosis?
there are 2 rounds of division in meiosis resulting in the production of 4 haploid daughter cells.
what are the phases of meiosis?
Prophase 1
mat and pat homologues form bivalents and are held together by the synaptonemal complex. the synapsed chromosomes undergo recombination. The recombining chromosomes are physically connected at the location of the crossover (chiasmata)
chiasmata are important for correct separation in anaphase
Metaphase 1
spindle is formed and bivalents align on the metaphase plate
Anaphase 1
homologues separate. chromatids remain attached
Telophase 1
chromosome are separated at each pole and daughter cells form
Prophase/metaphase 2
cells pass directly from meiosis 1 to metaphase II with no prophase. The nuclear envelope break down, a new spindle forms and the chromosomes align (2 chromatids)
Anaphase 2
separation of centromeres and migration of chromatids to opposite poles
Telophase 2
further cell disvision occurs forming 2 haploid daughter cells.
Describe the importance of damage repair in cancer
Damage repair is important to maintain genome stability
Damage from exogenous and endogenous agents is repaired and mismatches introduced during DNA rep
the processivity of the DNA pol is the rate limiting factor in determining the mutation rate
Describe the role of NER
Nuceotide excision repair
Reparis pyrimidine dimers caused by UV damage or large chemical adducts
1) NER machinery (complex of >30 proteins) recognizes the damage as it disrupts the DNA helix.
2) nuclease excision so ther damaged DNA and surrounding bases
3) gap fill by a DNA pol using the complimentary strand as a template
4) nick ligated
there are 2 classes:
- global excision repair (repairs all DNA)
- Transcription repair (repairs DNA undergoing transcription)
defective in Xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy - increased sun sensitivity.
How does DNA damage trigger repair or apoptosis?
DNA damage activates cell cycle checkpoints resulting in cell cycle arrest and apoptosis.
failure to activate check point results in an accumulation of damage and and can result in oncogenesis.
ATM and ATR are checkpoint regulators and their dysfunction is associated with increased cancer susceptibility
Describe mismatch repair
Repairs mismatches, insertions and deletion arising during DNA replication.
- MutSa (MSH2 + MSH6) recognises mismatches and small indels
- MutSb (MHS2 + MHh3) recognises larger looped out ins and dels
MutSa recognises damaged DNA and recruits MutL (MLH1 and PMS2)- this results in excision of the mismatched bases, there is gap fill by a DNA pol and the nick is ligated by a ligase.
Mutations in MMR protains are associated with Lynch syndrome
Describe base excision repair
BER excises non-helix distorting lesions form the DNA- e.g. damage from oxidation of alkylation.
glycolases cleave the glycosidic bond between the sugar and the damaged bases to leave an AP (apurinic or apyrimidinic) site.
e.g. MUTYH (associated with AR MAP) cleaves the glycosific bond to remove 8-oxo-G caused by oxidatuve damage. If un-repaired this pairs with T instead of C resulting in a GC to AT transition.
A phosphatase then cleaves the phosphodiester bond 5’ to the AP site leaving a 5’ sugar phosphate and 3’OH. the sugar phosphate is removed to leave a single nucleotide gap which is filled by a polymerase and the DNA is ligated.
Describe NHEJ
non replicative mechanism
error prone and is responsible for many of the recurrent rearrangement seen in cancer. (50% of recurrent rearrangements have 1 breakpoint in a fragile site)
Repair is by end-joining of 2 DSbs with no homology required. Often results in small insertions or deletions at the breakpoint. The ends are edited to reveal microhomologies
1) DSB is recognised by the Ku 70/80 heterodimer which forms a scaffold that holds the DNA ends together- after formation of a synapse in whihc the broken ends overlap the KU heterodimer unwinds a small part of the DNA to reveal a region of microhomology that is present by chance
2) Artemis: DNA dependent protein kinase with 2 functions
- exonuclease to trim excess unparied overhanging ss ends
- exonuclease activity that cleaves haripins together
3) DNA pol fills remainin gaps
4) end joing by DNA ligase
NHEJ-LIGiV and XLF-SCID are 2 syndromes that are associated with dysfunction in NHEJ
Describe HR
- High fidelity repair mechanism that uses the sister chromatid to guide repair in G-phase.
- requires extensive homology (>300bp)
RAD51 is the strand exchange protein required to catalyse the invasion of the homologous DNA sequence– mediates ss invasion of the homologous sequence by the 3’ end of the ss DNA to replace the equivalent strand.
BRCA1 and BRCA2 also required to co-localise with RAD51 at the site of DNA damage to activate the repair.
NBS and BLM (helicase) are also involved in HR.
BLM is associated with Bloom syndrome (sunsensitivity, butterfly shaped red mark on face and increased risk of cancer).
NBS is associated with Nijmegen breakage syndrome (microcephaly, facial dysmorphism and increased cancer risk)
Describe translesion synthesis
Major source of mutations
Uses a low fidelity polymerase which can replicate damaged DNA and bypass a stalled replication fork but this is at the cost of a high error rate.
- low fidelity pol often incorporates mismatches even in undamaged DNA
the high error rate can be advantageous and contribute to diversity of immunoglobulins.
paradoxically translesion synthesis plays a role in suppression of cancer. It can fill passed replicative gaps and suppress the DNA damage response including cell cycle checkpoints. cellular senescence and apoptosis
- also supresses genomic rearamngements and stalled forks do not turn into DSBs.
Give examples of the cancer susceptibility syndromes
Fanconi anemia
Ataxia Telengiecstasoia
Xeroderma pigmentosum
Nimejhan breakage syndrome
Li fraumeni
HNPCC
HBOC
MAP
Give examples of the repair mechanisms and tumour susceptibility for the cancer susceptibility syndromes
FA- FA genes, corss link repair,m increased risk of AML
AT- ATM gene, DSBs and checkpoints, increased risk of acute leukemias and lymphomas
XP- XP genes, NER, increased risk of skin cancers
NBS, NBS gene, DSBs and checkpoints, increased risk of acute leukemias and lymphomas
Li Farumeni, TP53, apoptosis and checkpoints, mutated in 50% of tumours
HNPCC, MMR genes, increased risk of CRC
HBOC- BRCA1/2, HR, increased risk of breast and ovarian cancer
MAP, MUTYH (AR), involved in BER, increased risk of MAP
Describe synthetic lethality in cancer therapy?
Synthetic lethality can be used to target DNA repair pathways. It i based on the phenomenon that the presence of 2 genetic events in related pathways results in apoptosis in the presence of other stresses.
- it indicates a relatedness between 2 gene
- can be considered a feature of genetic robustness- mechanism for maintaining genome stability in the presence of stress. There is some redundancy in different pathways meaning that it one is defective an other can compensate. However loss of both will result in cell death. This can be used to selectively target cancer cells which have already mutated one of the pathways (e.g, HR in HBOC) as part of their oncogenesis. Normal cells will not be affected as they still have a fully functioning original pathway (HR)
Give an example of synthetic lethality in cancer treatment
E.g. BRCA mutations in HBOC show synthetic lethality with PARP inhibitors
-PARP inhibitors are DNA repair proteins involved in ssBs repair
- ssBs cant be repaired so are converted to DSBs at rep fork. These are also not repaired resulting in apoptosis due to the accumulation of a high level of DNA damage
- normal cells still have functioning BRCA so can repair the lesions
How is synthetic lethality identified?
you can investigate the drugability of SL targets in DNA damage response pathways using SL screens…. using genetic variability of cancerous cell or genetically engineered KO cells and investigating the functional consequences
-RNA1 screening have been used to investigate SL associated with RAS
What is stratified and personalised medicine? what are the benefits?
Using biological markers e.g. the presence of specific genetic markers to straify patients into groups based on their response to treatments
benefits:
-safer and fewer ADRs
- better response to treatment
- improved mores specific diagnostics
-only treat those that will benefit reducing costs
- improved choice
Given an example of stratified medicine in breast cancer
ERBB2/HER-2 overexpression
ERBB” encodes the growth factor receptor tyrosine kinase HER-2
- this is overexpressed in 20-30% breast cancers (Her-2 +ve) and is associated with a mores agressive cancer and high recurrence risk. (Unlikely in BRCA1 which is associated with triple -ve breast cancer)
It can be treated by the drug herceptin. Herceptins side effects include cardiac disease so only want to treat patients that will benefit.
How is HER-2 overexpression tested for?
HER-s expression is detected by IHC of tumor samples. This testing is recommended by the UK cancer network guidelines for all primary or metastatic breast cancers
staining is rated as:
0-1+ = no staining or incomplete membrane staining. Less than 10% of tumours with this level of staining will have HER-2 over expression
2+= weak to moderate membrane staining ~10% of tumours with this staining will have HER-2 over expression, re-test.
3+ = complete membrane staining. HER-2 +ve
often recommended to re-test IHC (measures protein expression) with FISH (measures gene expression)
Herceptin resistance is an issue that may result from activating PI3K mutations or inactivating PTEN mutations
Given an example of stratified medicine in hereditary breast cancer
BRCA1/2 mutations are synthetic lethal with PAR inhibitors e.g. olaparib
PAR inhibitors have been shown to stop or shrink the growth of breast, ovarian and prostrate tumours.
what are the most common genetic changes in lung cancer?
the most common mutations in lung cancer are KRAS and EGFR
ALK rearrangements resulting in aberrant fusion proteins are also common. Mostly EML4-ALK
Describe stratified medicine for EGFR in lung cancer
EGFR activating mutations are common in NSCLC and is associated with a better prognosis
EGFR is a TM tyrosine kinase receptor involved in cell growth and proliferation. Activating mutations result in constitutive downstream signalling and uncontrolled cell growth.
- mutations are found in the tyr kinase domain (exons 18-20)
EGFR positive lung cancer can be treated by Gefitinib. The drug is 100x more effective in EGFR +ve lung cancers. It works as a TKI by blocking the ATP binding site.
How are EGFR mutations detected?
EGFR mutations are detected on FFPE tumour tissue. the sample need to be carefully dissected to ensure that tumour tissue is present. Pyrosequencing of the DNA is then perfromed.
Testing for EGFR amplification by FISh in controversial as it does not actually inform on the tumour mutation status of EGFR.
How are KRAS mutations detected and treated in lung cancer?
KRAS mutations and EGFR mutations are mutually exclusive, therefore if one has been detected there is no point in testing for the other.
KRAS is a proto-oncogene. It is a G protain with intrinsic GTPase activity and acitivating mutations result in upregulation of the ERK.MAPK pathway
Mutations is found in 22% of lung cancer and is more common in smokers. KRAS mutations are difficult to target as there are multiple compensatory mechanisms
Describe the ALK fusion in lung cancer
ALK rearrangements are enriched in younger patients and never smokers
EML4-ALK is the most common fusion partner due to a paracentric inversion of chromosome 2p inv(2)(p21p23)
- can alos find ALK-KIF5B and ALK-TGF rearrangements
can be treated by the ALK inhibitors crixotinib or alectinib
generally occur independently of EGFR and KRAS mutations
Tested for by FISH or FFPE
break apart probes can identify all fusion partners
dual fusion probes can be used to look for specific rearrangement
RT-PCR is not used as it cannot detect all novel fusion partners.
What are the common mutations in sporadic CRC?
KRAS and BRAF?
What are the stratified medicine options for CRC?
KRAS and EGFR mutations are mutautlly exclusiive therefore cetuximab and panatimub are only used in WT KRAS with 5-Fu (NICE approved)
BRAF and KRAS mutations are also mutually exclusive.
- BRAF mutations are also associated with a reduced response to EGFR inhibitors.
-BRAF mutations in MSI-low of MSI-stable tumours are associated with a poorer prognosis but there is no prognostic impact in MSI-high tumours
What is the stratified medicine for BRAF activating mutations in malignant melanoma?
Malignantmelanoma is cured by surgery in the majority of case and the remainder get chemotherapy.
BRAF- ser/thr kinase downstream of M{AK/ERK and result in cell growth and proliferation.
V600E is the most common activating mutation. this result in resistance to EGFR therapy but is associated with sensitivity to MEK inhibitors.
Describe stratified treatment for CML?
CML is treated by imatinib a tur kinase inhibitor targeted to the BCR-ABL t(9;22)(q34.1;q11.2)
In blast crisis patients may also express i(17)(q10) resulting in loss of TP53- this means the cancer is refractory to treatment that works through the TP53 pathway but may respond to novel therapies shihc bypass it such as idelasib
What is the cancer UK stratified medicine programme?
partnership between the UK goevrrnmatn, CRUK and pharma companies
phase 1: pilot to demonstrate the NHS can provide routine molecular diagnosis of tumour samples
phase 2: create a national genetic pre-screening programme and advance treatment for NSCLC
define pharmacogenetics and pharmacogenomics
pharmacogenetics: individual gene drug interaction (simple relationships)
pharmacogenomics: genome wide analysis of genetic determinants of drug response (complex relationships)
GWAS, SNP array, NGS, proteomics, gene expression arrays
What are the determinants of an individual’s drug response?
ADME (pharmacokinetic factors)
Adsorption
distribution
metabolism
excretion
pharmacogenetic factors:
target proteins
downstream messengers
What are ADRs?
adverse drug reaction
adverse drug reaction
All medicines have potential side effects. e.g. NSAIDs irritate the stomach and increase the risk of stomach ulcers.
ADRs depend on age, dose, condition being treated, sex, and metaboliser type (fast or slow)
- some drugs may also interact with other medicines of food/drink e.g. statins and graprefruit juice or oral contraceptive and St. Johns wort
in 2012 the ADR1s accounted for 6.5% of hospital admissions. and 25% of patients in primary case develop an ADR ans this costs the NHS over a £1billion each year.
pharmacogenetics offers a way yo reduce ADRs as dosing can be based on the metabolism phenotype of a patient ensuring they optimum dosing.
what are the benefits of pharmacogenetic studies?
safer dosing- right dose at right time for the right patient
- reduced costs (less drug use and hospitalisation)
-improve drug development choices
- avoid toxicity and ADRs
-maximize efficacy
What is Warfarin?
Warfarin is an anti-coagulant prescribed to patients at risk of wembolism or thromboembolism
How does Warfarin act?
Warfarin act by reducing the availability of Vit K (whihc is required to activate clotting factors).
During coagulation reactions Vit K is converted to inactive Vit K epoxide. Vit K epoxide is recylced back to Vit K by the Vit K epoxide reductase (VKORC1)- this is inhibited by warfarin
Incorrect dosing risks severe bleeding.
What are the pharmacogenetic factors in Warfarin dosing?
Warfarin is metabolised by the CYP450 enzyme CYP2C9
- CYP2Cp1 = metabolisers
- CYP2C92 and CYP2C9*3 are low metaboliseras. This genotyep is associated with an increased risk of a severe bleeding episode due to reduced warfarin metabolism
the CYP2C9 alleles account for ~15% of the variation in response to Warfarin
what alleles of VKORC1 affect Warfarin dosing.
VKORC1 is the Vit K epoxide reductase and catalyses the recylcing of Vit K epoxide to Vit K
SNPs 1173C>T and -1639G>A are associated with a reduced expression of VKORC1 and is present in 37% of Caucasian Americans and 80-90% of Asians
- these patients require reduced Warfarin dosing.
What is the clinical use of testing for CYP2C9 and VKORC1 testing?
dosing is typically empirical and altered based on monitoring normalised ratios of the drug in patients.
In 2007 the FDA added a statement on Warfarin labels indicating that genotyping VKORC1 and CYP2C9 may be useful.
testing is not currently offered in the UK as it is considered to be too expensive
2 studies in 2013 found contradictory results: Pimohamed et al found a positive effect whereas Kimel et al found no benefit from testing for dosing.
What are thiopuring drugs used for?
Thiopurine drugs e.g. azothiopurin and 6-mercaptopuring are used to treat chronic inflammatory and autoimmune diseases e.g.ALL
Describe the pharmacogentics of TPMT and thiopurine drugs
TPMT is involved in thiopurine drug metabolism.
- TPMT activity is measured in clinical practice to aviod ADRs (Gi intolerance, pancreatitis and BM suppresion)
1/300 are deficient in TPMT and are likely to suffer an ADR from a standard dose of thiopurine
11% intermediate metabolisers
89% have high enzyme activity and may not reach therapeutic levels with a standard dose
What percentage of CRC is familial and what are the associated genetic syndromes?
15% is familial
3-5% is due to Lynch syndrome
1% due to FAP (APC), Juvenille polyposis syndrome (SMAD4 BMPR1A), Peutz-jeghers (STK11) and PTEN harmatoma syndrome
BMPR1A and PTEN may be deleted in 10q23 contiguous del syndrome with syndromic features dev delay and polyps
What genes are mutated in Lynch syndrome
MLH1, MSH2 (~70%)
PMS2- 1&
MSH6- 6-10%
EPCAM <1%
Lynch syndrome results in an increased risk of which cancers?
CRC, endometrial, small intestine, hepato-billiary, pancreatic, gastric, renal pelvis, bladder, prostrate, ovary, breast
what is the mean age of onset of Lynch?
45 yrs. it is older for MSH6 and PMS2 as these are the non-obligate partners onf the MutSa and MutLa heterodimers so are associated with later onset and reduced penetrance
what is the mutations spectrum of Lynch Syndrome?
- LOF mutations
- MLH1 and MSH2 are most commonly mutated and have highest penetrance
- 3% are due to deletions of the 3’ edn of the EPCAM gene, this results in loss of the stop codon and poly-A signal resulting in polymerase readthrough into downstream MSH2 and promoter hypermethylation. the EPCAM deletion can also include the MSH2 promoter or coding sequence. The result for all is LOF of MSH2
- 10Mb iinversion of chromosome 2 can disrupt MSH2 and is reported in cases of unexplained lynch syndrome
what is the clinical criteria for testing for lynch syndrome?
2 different criteria:
Amsterdam- developed to identify LS patients for research studies
Bethesda- designed to identify CRC patients who should be tested for LS. Lower specificity but higher sensitivity than Amsterdam criteria
describe IHC testing in LS
95% sensitivity
standard to test FFPE tumour tissues for the expression of LS genes by IHC
- If the obligate partner is lost e.g. MLH1 or MSH2 you will also see concurrent loss of the partner. But if the non-obligate partner is lost (MSH6 or OMS2 there is not concurrent loss of the obligate partner)
- If a patient shows loss of staining by IHC there is no need to also test for MSI as this is assumed
- IHC has lower sensitvity than MSI as it may miss LOF mutations that do not result in NMD e.g. missense mutations
- therefore if a patient is -ve for IHC should also perform MSI testing
- techinically difficult to distinguish +ve staining from artefact
if a patient shows loss of staining for MLH1/PMS2 they should have MLH1 methylation studies before proceeding to germline mutation testing
describe MSI testing in LS
MMR proteins repair mismatches and a defect in the pathway results in an increased mutation rate.
micro satellites are highly mutable making them a good target to screen for MMR deficiency
- generally screen tumour vs germline (blood samples) or regions of tumour and non-tumour on an FFPE slide.
-2/5 markers should show instability to confirm MSI
- if only 1 marker shows instability it is not enough to confirm MSI but may indicate the need for further studies
- can also be used for ovarian and endometrial cancer, but has been designed for CRC so is most snsitive for this
-if MSI is not detected an MMR defect is ruled out.
what are the most common mutations in sporadic CRC?
MLH1 promoter methylation is present in 15% and results in an absence of MLH1 by IHC. it can be detected by MS-MLPA which can methylation sites in the promoter region (can also detect MSH2 methylation due to an EPCAM deletion).
- rarely seen as a germline chage and is acquuired somatically.
V600E BRAF mutation is associated with MLH1 and indicates sporadic CRC- can be used to screen for sporadic CRC and avoid unnecessary germline mutation testing
What techniques are used for germline mutation testing in LS?
- sequencing and MLPA (although more likely to now all be performed as part of an NGS panel
- PMS2 is complicated by the presence of a highly homologous pseudogene so need to use long range PCR with primers anchored in region of divergence between the native and pseudogene to select for the native gene. nested PCR with premiers for the exons of interest for sanger seq.
what is the testing strategy?
1) test tumour for IHC or MSI- cant diagnose LS but can confirm a defect in MMR and IHC may indicate the likely affected gene
2) MLH1 promoter methylation and BRAF V600E testing to rule out spradic cases
3) test for germline MMR proteins
Describe the role of the MMR genes?
MSH2-MSH6 (MutSa) recognises single base mismatches and monoculoetide repts. MutSb recognises larger looped out errors e.g. dint rpts
MutSa inititiate repair by binding to the mismatch. Other molecules are recruited- PCNA, RCF, MutLa(MLH1-PMS2). this assembly activates the exonuclease activity of PPMS2 which makes ss breaks near the mismatch and opens EXO1 nucelase entry site. this mismatch is then excised, gap filled by a pol and nick ligated
What is associated with homozygous/compound het LS mutation?
associated with constituional mimsatch repair cancer syndrome- rare childhood cancer syndrome resulting in hematological, brain/CNS, CRC, intestinal polyps
what are the treatment and surveillance options for CRC?
full coloectomy and ilorectal anastomosis recommended
-prohylactic colectomy is not recommended as colonoscopy is effective
-removal of ovary and uterus may be considered after completion of childbearing
surveillance: colonoscopy and removal of precancerous lesions every 1-2 years from 25 years
aspirin is recommended for chemoprevention in at risk individuals
Predictive testing
Available to all at risk adults. CRC not associated with childhood cancer so testing before then is not required.
requires referral from a GC
-ve result reduces risk to that of the general population
+ve result increases risk of developing cancer- level of risk is dependent on gene, age, mutation, lifestyle, sex,
What is Familial Adenomatous polyposis (FAP)
Familial Adenomatous polyposis
most common polyps syndrome due to AD APC mutations
- characterized by the development of 100s of colonic polyps in the seconsd decade and 95% have polyps by 35
- almost 100% penetrance if left untreated- treatment is by colctomy at an early age (when 20-30 poyps are present)
- screening starts at 10-12 years and detects most CRC before onset
PST (presymp testining) in FAP
PST is offered from 10yrs so that colonoscopy can be offered due to early age of onset
- VHL and NF1 are other cancer syndromes with early onset where early PST is appropriate
APC gene
Loss of APC is an early event in colorectal adenoma.
The APC gene function in the wnt pathway by regulating phosphorylation is B-catenin and marking it for degradation, in the absence of APC B-catenin is not degraded resulting in uncontrolled cell proliferation.
APC mutation spectrum
- there is an alternatively spliced transcript of exon 9 lacking codons 312-412. This isoform is present in normal tissues and if the mutated codon is in this region it can be removed by normal splicing in the intestinal mucosa resulting in a milder phenotype.
- large final exona and nonsense muts in thus region do no always undergo NMD resulting in the presence of a partially functional protein.
-5’ mutation. There is an internal ribosomal entry site early in the transcript. therefore truncating mutation in the 1st exon can escape NMDF by initiating translation at a downstream sites- results in attenuated FAP
-Missense mutations do no confirm a diagnosis. No statistically significant increased cancer risk and PST is not offered
How is FAP diagnosed?
based on family history and CRC phenotype >100 polyps.
Genetic testing by sequencing and MLPA. Now commonly included in a CC panel
15% of APC -ve cases have a MUTYH mutation so this testing should be considered.
What is the treatment of FAP?
safest preventative strategy is surgical removal of the colon when polyps start to develop
chemotherapy includes NSAIDs, COX-2 inhibitors
What is attenuated FAP?
milder phenotype with fewer polyps (<100), likely to be underdiagnosed and associated with specific mutations.
What is MAP?
MUTYH (MYH)-associated polyposis
MUTYH associated polyposis
AR
Similar to FAP (10-100 polyps) family history can help distinguish the likely diagnosis.
FAP and MAP are example of similar phenotype different mechanism
what is the age of onset of MAP
mean age of onset is 48yrs with 40-100% lifetime risk
there is a small increased risk of adenoma in heterozygotes but screening is not warranted.
What is the role of the MUTYH gene?
Involved in BER- repair of the most common form of oxidative damage 8-oxoguanine which pairs with A instead of C resulting in a G:C to T:A transition. The glycolase excises the 8)G from the sugar phosphate backbone so the base can be repaired.
MAP mutation spectrum
almost all mutations are missense and their affect on the glycolase range from a completer to partial loss of activity
Y179C and G396D account for 90% of mutation in N EU mutations
60% of tumours also have a KRAS mutation
Testing strategy for MAP
testing is offered to patients with 10-100 polyps and no APC mutation (in reality both are likely to be tested together as part of a panel)
- can pre-screen for common N EU mutations
- failure to detect a second mut could indicate the presence of a large del, rearrangement, or another gene is responsible
what screening is offered for MAP?
colonoscopy from 18yrs and upper GI tract investigations from 25-30 therefore PST is not offered to minors
What is the incidence and risk factors of breast cancer?
Most common female cancer in the UK with 1 in 8 lifetime risk. Incidence is increasing in western countries with increased life expectancy and move to a more wetern lifetsyle
risk factors: age, genetics, HRT, smoking, obesity, alcohol. Strongest risk factor is a strong family history of BC- 15-20% of BC has a family but gene found in known familial BC gene in ~10% of cases
what factors support a suspicion of a BRCA1/2 mutation?
- BC < 50yrs
- 2x primary BC
- breast and ovarian cancer in a single individual
- breast and ovarian cancer in close relative from the same side of the family
- male BC
ovarian cancer at any age
BRCA1/2 mutation in a family member
algorithms have been developed to calculate the likelihood that an individual have a BRCA1/2 mutation (BODICEA, Myriad, BRCAPRO)
According to NICE guidelines what prior probability of BRCA1/2 mutation is required for genetic testing
10% (reduced from 20% in 2013). in some centers it may be offered to all women with triple negative BC
What is HBOC?
hereditary breast and ovarian cancer syndrome- due to mutations in BRCA1 and 2 (no sequence similarity to each other). results in increased risk of breast and ovarian cancer including Fallopian tube and peritoneal. Also increased risk of pancreatic and prostrate cancer and melanomas.
what is the penetrance of HBOC?
incomplete penetrance 38-87% lifetime risk of BC and 16-63% increased risk of OC
BRCA1
accounts for 64% of HBOC
higher overall lifetime risk of cancer than BRCA2 and cancer at much earlier age than gen pop
common in triple -ve BC
BRCA2
34% of HBOC
Associated with a wider range of cancer and higher cancer risk in males- 20% prostrate cancer and 10% male BC
BRCA1/2 protein function
involved in HR, repair of DSBs and NER
BRCA1 forms a complex with BARD1 and co-localises with BRCA2 and RAD51 at sites of DNA damage
- involved in pathways controlling cell cycle progression, check point control, gene transcription, regulation and ubiquitination
- expressed in most tissue suggesting that the expression pattern is not responsible for the restricted tissue phenotype of BC
BRCA2/RAD51 mediate HR
ss binding protein which prevents degradation of DNA at stalled replication forks. Phosphorylated when not required and dissociates from RAD51
What is the mutation spectrum in HBOC
Mainly frameshift but some missense
ENIGMA consortium focuses on determining the clinical significance of BRCA1/2 and other cancer variants
foudner mutations are present in some populations e.g. askenanzi jew (AKJ)
AKJ also have a high carrier freq
- BRCA1 c.68_69delAG
- BRCA1 c.5266dupC
- BRCA2 c.5496delT
1/1000 in UK general pop
1/40-50 in AKJ pop
some genotype phenotype correlations have been reported but are not used in clinical practice. Ovarian cancer variants cluster around exon 11 in BRCA1 and 2
What is the testing strategy for breast cancer?
sequencing and MLPA- usually part of an NGS panel nowadays
- may pre screen for AKJ mutations in appropriate individuals
PST for at risk family members (requires counselling)
NICE guidelines- unaffected family members
- may also test FFPE tumour tissue to ID familial mut if no other sample from the affected is available
Points to consider for variant interpretation in cancer syndromes
- co-segregation with disease may support pathogenicity but this is complicated by reduced penetrance and phenocopies
- tumour can be examined for LOH as support of WT allele may support pathogenicity whereas loss of mutated allele exclude pathogenicity
What are the ethics associated with genetic testing in breast cancer?
PST requires counselling- it is unethical to offer PST to minors as BC does not onset in childhood
- family member may chose not to disclose their result so their family members can’t be tested. alternatively testing may infer the result of a family member who chose not to be tested
- variable age of onset, need full understanding of risk associated with result and treatment options
What treatments are available for breast cancer?
surgery, chemo, hormone therapy
- herceptin can be offered for Her +ve tumours (tested for by IHC)
-prophylactic masectomy decreases BC risk by 90%
- bilateral prophylactic oophrectomy decreases risk by 53% for OC
- 10-24% of BRC1 and 65-99% of BRCA2 are ER+ve- these can be treated by tamoxifen (ER inhibitor) this can result in a 30-50% risk reduction and can result in menopausal symptoms and increased risk of thromboembolism and endometrial cancer so need to know tumour is ER +ve for there to be a risk benefit
knowledge of the germline mutation can influence treatment choice including surgery as risks vary for different cancer types.
Describe synthetic lethality and PARP inhibitors
PARP enzyme repairs ssBs by BER. When PARP is inhibitied these persist in the cell and develop into DSBs on replication.
HR is defective in BRCA1/2 mutated tumours therefore the DSBs can’t be repaired either and the cell accumulates lots of damage- this cant be repaired and targets the cell for apoptosis.
Normal cells are spared as they have functioning HR
Phase 3 trial of olaparib and talazoparib- 3 month progression free survival in BRCA germline carriers and metastatic disease compared to standard chemo- currenlty in use in UK for some OV cancer and in trials for BCV
surveillance techniques: self-examination, MRI and mamography. the method depends on the risk group, age and mutated gene.
Name other syndromes associated with an increased risk of BC?
Li Farumeni (TP53)- associated with increased risk of early onset BC, brain tumours, soft tissue sarcomms, osteosarcomma and adrenocortical carcinoma
Peutz-Jeghers (STK11)- associated with harmatomatous polyps and increased risk of CRC and melanin deposits in the fingers and lips
- cancer risk is also increased fro breast, lung, uterus and ovary
-93% cumulative cancer risk
Cowden syndrome (PTEN)
Multiple harmatoma syndrome - increased risk of breast cancer and non-medullary thyroid cancer (mostly pre-menopausal)
Also have cutaneous fetures: oral mucosal papillomas, tricholomas and acral kertoses
Hereditary diffuse gastric cancer/ lobular breast cancer (CHD1)
39-52% lifetime risk of lobular BC
What are the moderate penetrance BC susceptibility genes?
pathogenic variants in genes associated with damage repair may also have a moderate penetrance for BC- CHEK2, PALB2, ATM
How are polygenic risk scores used in BC
Low penetrance BC SNPs have been identified by GWAS- risk inferred by each individual loci is low and polygenic risk scores based on a number of associated SNPs has been used to stratify BC risk in different populations.
SNPs can also modify the risk of an individual with a pathogenic variant in a cancer predisposition gene and polygenic risk scores have been developed ti modify the estimated risk in patients with BRCA1/2 and CHEK2 variants
What is the genes have UKCGG consensus for inclusion in a BC NGS panel?
UK cancer genetics group published a consensus list of gene with sufficient evidence of clinical utility in testing
BRCA1/2
PALB2
PTEN
STK11
CHEK2- truncating mutations only
ATM- val242Gly only
including low penetrance genes in the panel can lead to difficulties with interpretation. if the clinical significance is unclear is can’t be used for clinical decision making and is not useful. Detection of a variant in a low penetrance gene in a family with a high incidence of BC may not account for all the risk in the family and there may be a second diagnosis present.
what are the characteristic findings in NF1?
neurofibromas and cafe au lait patches
How is NF1 diagnosed?
Usually unequivocally clinically diagnosed by the age of 8 and symptoms develop with age.
clinical diagnosis is based on:
- 5 cafe au lait macules
- NF1 in a first degree relative
- freckling in the armpit or groin
-2+ neurofibromas
- 2 + lisch nodules
- distinctive bony lesions
cognitive impairment (reduced IQ and behavioural problems) is the most common neurological manifestation
what is the life expectancy for NF1?
life expectancy usually reduced by 8 years due to malignancy and vasculopathy
How is NF1 molecularly diagnosed?
Not usually required and diagnosed clinically.
Very high new mutation rate
50% de novo and somatic mosaic is common, this can result in segmental NF1 where is only present in one or a few tissues. Therefore diagnosis using peripheral blood may not detect the mutation in sporadic cases.
tumour testing can be useful but interpretation can be complicated by the potential for a high number of acquired mutations. Testing tumour tissue can be useful for variant interp, LOH of the WY allele support pathogenicity of the variant on the mutated allele whereas loss of the variant allele excludes pathogenicity.
Testing PB is appropriate for familial cases as the mutation will be present in all tissues (non mosaic)
Genotype-phenotype correlations in NF1?
whole gene deletions are associated with earlier onset and an increased no. of neurofibromas
NF1 dup can be associated with ID and seizures
There is high clinical variability due to the presence of modifier genes
Treatment for NF1
No cure so treatment is based on treating symptoms
- vit D supplementation for fracture risk
Possible therapies could include:
gene replacement for LOF mutations
exons skipping to treat specific exons
genome editing for small muts
what is the clinical phenotype of NF1?
tumours around the brain and spinal cord can cause
pain, weakness and numbness in arms and legs (usually benign)
5% develop a malignant tumour most commonly of the peripheral nerve sheath.
children also have a 100-fold increased risk of suffering from a myeloid disorder
what is the location and product of the NF1 gene?
Found on 17q11.2 and encodes neurofibromin
NF1 acts as a TSG while -vely regulates RAS. When NF1 is mutated there is increased RAS signalling resulting in cell survival and growth through the MAPK and PI3K pathways
What are the differential diagnosis for NF1?
Noonans- share some dysmorphoc features (hypertelorism, down-slanting palepebral fissures, low set ears)
Leopard syndrome
McCune Albright syndrome (irregular cafe au lait patches)
MEN2B- tumours can be confused with neurofibromas
Describe NF2
Less common than NF1 but also AD
Characterised by the presence of vestibular schwannomas- these are benign intrcranial tumours of the myelin forming cells of the vestibular cochlea nerve and result in hearing loss, tinitis and balance problems
onset is between 18 and 24 yrs
How is NF2 diagnosed?
bilateral vestibular schwanomma or
1st degree relative with NF2 and unilateral VS
or 2 of: menigioma, schwannoma, glioma, neurofribroma
NF2 gene
encodes neurofibromin 2 AKA merlin of swchanomin
TSG in the PI2K/ Akt patheway and loss of merlin allows cells (especially schwann cells) to multiply quickly
What is the genetic testing in NF2?
sequencing and MLPA of NF2
- as with NF1 mosaicism can complicate testing
Large dels incl. NF2 which are cytogentically visible result in childhood onset and LD e.g. r(20) or chr 20 translocations.
What are the characteristic features of tuberous sclerosis?
characterised by the development of (benign) harmatomas. Symptpmsa can be mild to severe with multi organ involvement
How is TSC diagnosed?
Tuberous sclerosis
primarily diagnosed by the presence of skin lesions or secondary to epilepsy
what is the genetic cause of TSC
Due to LOF mutations in TSC1 or TSC2- these act as a TSG in the MTOR pathway. In the active state they inhibit MTOR activation
There are currently no specific drugs available but trials involveing MTOR inhibitors (e.g. rapamycin) are showing promising results
2/3 are de novo. High mutation rate means mosaicism is common and this can complicate diagnosis
TSC1 is found on 9q34 and TSC2 is on 16p13.3 (can be deleted as part of a contigous del including PKD1 resulting in TSC ans early onset PKD
What are the clinical features of TSC?
ASHLEAF
A: Ashelaf spots
S: Shagreens patches
H: Heart rhabdomyomas- can cause arrythmias
L: lung fibrosis and restircitve lung disease
E: epilepsy from cortical tuber (main cause of morbidity and mortality
A: angiomyolipoma in the kidney (benign tuours can cause end d=stage kidney disease)
F: facila angiofribromas
harmatomas in cortex and ventricles may also result in sever ID, behavioral problems and ADHD
CNS symptoms are present in 90-95% and (predominantly epilepsy)
Clinical diagnosis of TSC
can be made prenatally by the observation of cortical tubers on USS from the 2nd trimester.
most have normal life expectancy
main cause of mortality is from brain and kidney tumours
What is the role of the TSC complex as a TSG?
TSC1 (stabilises TSC1 to prevent ubiquitin degradation subunit) and TSC2 (catalytic subunit form a heterodimer with GTPase activity = TSC2 mutations are generally associated with more severe disease
- in the absence of growth signals they inhibit cell growth by converting Rheb GTP (active) to Rheb GDP (inactive). Normallly Rheb GTP activates the MTOR pathway resulting in cell growth so TSC complex inhibits this.
TSC complex is inhibited by cell growth signal
regulated post-transcriptionally by phosphorylation
MTOR is also inhibited by the drug rapamycin
Mosaicism in TSC/ Genetic testing issues
Present in a significant number of patients therefore testing of tissues other than PB may be appropriate e.g. skin, saliva, tumour, hair
detection of mosaicism is technically challenging below 2% (e.g. patients with single organ involvement)
Genetic testing is also complicated by the large size of the gene and the large number of pathogenic mutations. majority of mutations are truncating mutations, deletions or rearrangements
What is the penetrance of TSC?
AD disorder with incomplete penetrance
variable expressivity is due to the fact the TSC complex is regulated by and regulates a variation of different signalling pathways in the cell.
What treatments are available for TSC
No cure so treatment aims to treat symptoms and early identification of potentially aggressive lesions.
MTOR inhibitor (rapamycin and afinitor) can make up for deficient TSC complex by inhibiting downstream MTOR to prevent cell growth.
FDA approved in 2010 for patients with enlarging brain tumours & can also be applied topically for facial angiofibromas
MTOR inhibitors are cytostatic not cytotoxic.
MTOR inhibition may be compensated for by PDGFRB (tyr kinase). Inhibition of both in mice using rapamycin and imatinib has been shown to decrease tumour size without toxicity.
What is MEN?
Multiple endocrine neoplasia
4 subtypes
describe MEN1
due to LOF mutation in the TSG MEN1 which encodes menin at 11q13
results in tumours of the parathyroid, duodenum, pancreatic islets, pituitary gland. With the exception of gastric tumours most are not metastatic but cause signification clinical phenotype due to the secretion of endocrine hormones: IGH, insulin, gastrin, adrenocorticotropic hormone
the over production of hormones aids in the diagnosis.
Testing is by sequencing and MLPA (may be included in an NGS panel). Screening by biochemistry and imaging may be offered to family members even if a pathogenic mutation is not found
Describe MEN2 and 3
previously MEN2A and 2B
- associated with medullary thyroid carcinoma (100%), phaeochromocytoma (50%) and parathyroid tumours (50%)
caused by GOF activating mutation in RET oncogene. the location of the mutation determines the phenotype
- 1st tyr kinase domain = familial medullary thyroid carcinoma
- TM domain = MEN2
- 2nd tyr kinase domain = MEN3
any patient with medullary thyroid carcinoma should be offered testing for RET
Describe MEN4
AD LOF mutations in CDKN1B
MEN1 phenotype with no MEN1 mutation
Describe the genetics and symptoms of VHL
Caused by LOF mutations *throughout the coding seq) in the VHL gene
Associated with benign and malignant neoplasm
-renal cysts
-hemangiomas
-parangangliomas
-phaeochromocytoma (only in type 1)
-epidyidymal cysts
-pancreatic cysts
80-90% penetrance by 65yrs
Describe Peutz Jeghers
Due to LOF mutations in STK11- test my MLPA and seq
Associated with harmatomatous intestinal polyps and increased risk of CRC
- also results in dark spots on lips, armpits and groin.
surveillance is by colonoscopy, eneteroscopy, and polypectomy to remove polyps
Part of a group of disorders called lentiginomas- mostly due to mutations in the RAS/MAPK pathwya which is a targewt of MTOR signalling (leopard syndrome, PTEN, noonans)
Describe hereditary paragnaglioma/phaeochromocytoma syndromes
Due to mutations in SDHA, SDHB, SDHC, SDHD, SDHAF2 and TMEM127- therefore multigenen testing is required for diagnosis.
paraganglioma- tumour of the parganglia at the base of the skull (rare and usually benign)
phaeochromocytoma- rare usually benign tumour of the chromaffin cells- adrenal cortex
tumours occur at all ages and incidence increases at 40-50 years
The predisposing genes all appear to have different actions but despite this result in the same tumours clinically ans histologically
- highest malignancy with SDHB and PND is available if a pathogenic mutation has been identified
- genetic predisposition is indicated by family history, early onset of tumours (<45yrs) and the presence of multiple tumours
- may also get papilliary, renal and GI tumours
Describe PTEN harmatoma syndrome
AD due to LOF mutation in the PTEN TSG
PTEN acts as a tyr phosphatase - regulates PI3K/Akt/MTOR pathway and MTOR inhibitors have been approved by the FDA for treating PYEN syndromes
1) Cowden syndrome
- increased risk of breast cancer and endometiral tumours
- facial trichelommas, papilommatous papukles
-90% penetrance by 30 yrs
2) Bannayan-Riley- Ruvalcaba syndrome (BRRS)
micorcephlay, lipomatosis, hemangiomatosis, pigmented macules of the glans penis
PTEN-related proteus syndrome- progressive segmental and patchy growth of multiple tissues- most commonly connective tissue, epidermal naevi and skeleton.
Describe cutaneous malignant melanoma
ADF mutations in CDKN2A which encodes 2 unrelated proteins
P14 ARF destabilises the p53-MDM2 interaction releasing p53 to activate cell cycle arrest and apoptosis
P16INK4a- inhibits Cdk4/6 so that RB remains unphosphorylated and bound to E2F- preventing E2F from locating to the nucleus to activate transcriptions g gene required for cell cycle transition.
TSG and mutations associated with skin cancer- penetrance is dependent on age and sun exposure
