Session 6 - Cancer genetics Flashcards
What are the five broad categories of oncogenes? Give and example of each.
- Secreted Growth factors (Wnt1)
- Growth factor receptors (EGFR in non-small cell lung cancer)
- Signalling pathway components (PIK3CA, RAS, MAPK)
- Inhibitors of Apoptosis (BCL2)
- Transcription Factors
How can each of the classifications of oncogene contribute to malignancy?
Secreted growth factors - increase in concentration or activity can induce cell proliferation
Growth factor receptors - EGFR is a tyrosine kinase. Constitutive activation can be caused by mutations in exons 18, 19 and 20. TKIs compete for the ATP active site to block activation
Signalling pathway components cause signalling in the absence of a signal (mTOR, PI3K, RAS/MAPK)
Inhibitors of apoptosis prevent abnormal cells from programmed cell death (BCL2 t(14;18) translocations present in nearly all follicular lymphomas).
Transcription factors formed from translocation can act aberrently (EWS1/Fli1, t(11;22) in Ewing’s sarcoma.
What are the five types of gain of functions mechanisms associated with oncogenes? Give examples
- point mutations - BRAF, KRAS in CRC, melanoma
- deletions/duplications/inversions - fusion genes
- translocations - BCR-ABL1
- insertion of viral DNA to increase transcription - EBV in NHL
- gene amplification - HER2 in breast cancer.
What two ways can translocations lead to oncogenic activity?
- Translocation to form a novel fusion gene (BCR-ABL1)
- translocation of a gene into a transcriptionally active region (translocation bringing MYC under control of Ig promoter, resulting in increased MYC expression)
At which stages of the cell cycle are the control checkpoints?
Restriction point (between G1 and S) The G2/M checkpoint The Metaphase/Spindle Checkpoint
What is the cell cycle regulated by?
Cyclins and Cyclin-dependentkinases (CDKs) - these form heterodimers
What happens at the G1 restriction checkpoint?
Cell growth enables CDK/Cyclin D formation
CDK/Cyclin-D phosphorylates pRB
This releases E2F transcription factor from pRB
E2F results in expression of Cyclin E, which binds CDK2
This allow passage into S-phase
What happens at the G2/M checkpoint?
CDK1 is activated by phosphorylation and dephosphorylation of specific residues by Cyclin Activating Kinase (CAK)
This enables MPF formation
G2>M phase transition allowed
What happens at the Metphase/Spindle checkpoint?
Chromosomes assemble on metaphase plate APC activated MPF diassembled Separase inhibition released Chromatids separate and anaphase starts
At which stage of the cell cycle is the greatest oncogenic pressure exerted?
G1 restriction point
How can mutations in activation of cyclins lead to oncogenesis?
Overexpression of CDK1 by translocation or amplification can lead to increased progression through G1
Loss of CKIs
What is the role of p53?
A potent tranascription factor activated during cell stress to induce cell cycle arrest.
What interacts with TP53 in a negative feedback loop, to control expression?
MDM2 - p53 induces expression of MDM2, MDM2 causes degradation of p53.
Through which mechanisms does TP53 prevent cancer?
Activation of DNA repair
Hold cells at G2/M checkpoint to give DNA repair proteins chance to fix mutations
Induce apoptosis.
In what proportion of cancers are TP53 mutations identified?
50%
What environmental factors increase the expression of TP53?
Ionisating radiation or chemotoxic drugs
What syndrome is associated with TP53 mutations? What are the clinical features?
Li-Fraumeni.
Increased incidence of cancers - breast, colon, lung, brain, soft tissue carcinoma, osteosarcoma etc.
Which group of genes does the E2F transcription factor control?
Genes needed for S-phase
How does RB1 control activity of E2F?
unphosphorylated pRB binds E2F and prevents it acting as a TF. When pRB is phosphorylated by a CDK-Cyclin D it releases it’s inhibition of E2F and transcription of S-phase genes can progress.
What are mutation in RB1 associated with?
Retinoblastoma.
What is the mutational spectrum associated with RB1?
Frameshifts and nonsense mutations, resulting in loss of protein.
What chromosome is RB1 located on?
Chromosome 13
What is the role of CDKN2A in cell cycle control?
The gene encodes for two proteins: CDKN2A and ARF.
CDKN2A inhibits CDK activity, preventing phosphorylation of pRB, thus preventing passage to S-phase. Lack of CDKN2A results in hyperphosphorylation of pRB and activity of E2F.
ARF destabilises MDM2 and acts to maintain the levels of p53
What disease is associated with lof mutations in CDKN2A?
Multiple melanoma.
What possible therapeutic targets for cell-cycle regulators are there?
MDM2-TP53 interaction:
Block MDM2 expression
Prevent interaction between MDM2 and TP53
Prevent MDM2’s ubiquitin ligase functionality to stop degradation of TP53.
What genes are involved in HNPCC? What proportion of cases do they account for
MLH1/MSH2 - 80-90%
MSH6 - 7-10%
PMS2 - 1%
EPCAM 3’ UTR deletion - 3%
What pathway are the genes associated with HNPCC part of?
how does this pathway function
MMR
MSH2/MSH6 dimer recognise mismatched bases in DNA. MLH1/PMS2 dimer binds to the MHS2/MHS6. Exonuclease 1 is recruited into the complex and nicks the DNA. DNA pol B mends the gap
Why are mutations in PMS2 and MSH6 associated with a milder phenotype?
They are partially redundant - MLH1 and MSH2 have other partners they can pair wit (PMS1 and MSH3)
What cancers is HNPCC associated with? What are their lifetime risks?
CRC - 80% in males, lower in females Endometrial cancer - 40-60% Urinary tract cancers Stomach cancer Brain cancer
Describe the mutation spectrum associated with HNPCC
3’ deletions of EPCAM result in a EPCAM-MSH2 fusion transcript and silencing of MSH2.
Epimutation of MLH1 (somatic and germline)
10mb inversion on 2p disrupts MSH2
Largely LoF mutations: frameshift, deletion, splice.
Inversion of exons 1-7 of MSH2 is a frequenct cause of unexplained HNPCC in UK
What criteria are used to determine if an individual is at risk of having HNPCC?
Which of these guidelines were updated in 2014, why were they updated?
Amsterdam
Bethesda
Amsterdam criteria were updated to include the presence of non-CRC tumours in the family
What mutations may be indicative of a tumour being sporadic, rather than HNPCC?
MLH1 epimutation (ms-MLPA) and BRAF V600E.
Both are occasionally identified in hereditary tumours, but presence together indicates the tumour is highly likely to be sporadic.
What do MMR tumours show? Is this present in sporadic cancers?
MSI
Present in a small proportion of sporadic cancers (10%)
Describe a diagnostic strategy for CRC and suspected HNPCC
1 a) MSI using mononucleotide markers to detect instability (kit has 5 markers, 2+ = MSI-H; 1 = MSI-L; 0 = MSS)
1 b) IHC for presence/absence of MMR gene products
- MLH1 epimutation and BRAF V600E testing - if negative, less likely to be sporadic.
- Sequence target genes to identify point mutations. MLPA required to detect large del/dups. LR-PCR for PMS2 as it has pseudogenes
Why do tumours deficient in MMR genes show MSI?
The MMR genes repair mutations occurring during DNA replication. These are more likely to occur in repetitive regions to cause microsatellite instability
What are the treatment and management options for HNPCC?
Full colectomy with ileorectal anastomosis
What is the incidence of HNPCC? When do symptoms often onset?
1/300. Age 45yrs
How can primary tumours be prevented?
Colectomy - not recommended. Annual colonoscopy from age 20-25 (10 years before the onset of family cancers). Prophylactic removal of the ovaries and uterus possible in females after childbearing.
Chemoprevention - using Aspirin has shown to reduce the cancer incidence in patients with CRC - CAPP3 trial
What counselling considerations are needed for HNPCC?
AD disorder
Variable penetrance
Variabel age of cancer development
Prenatal diagnosis unusual, but available
What is the incidence of FAP?
Which gene is mutated?
What are the characteristic features?
1/8000
APC
100-1000’s polyps; 95% have polyps by age 35. Other associated cancers: fundic gland polyps, thyroid cancer, pancreatic cacner, liver cancer, osteomas, desmoids, CHRPE.
Screening starts age 10-12
Where is APC located?
5q22
What is loss of APC a known part of?
The Adenoma > Carcinoma sequence
Which pathway does the APC protein function in? How do LOF mutations cause disease?
The Wnt1/B-catenin pathway, active in early embryogenesis.
Normally APC acts by phosphorylating B-catenin to mark it for degradation. Mutant APC is unable to bind B-catenin > build up of B-catenin > increase in transcription factor expression > increase in cell growth
What two common mutations are responsible for 10% and 5% of cases of FAP, respectively?
Gln1062X and Glu1309AspfsX4
What are the genotype phenotype correlations seen in FAP?
They are dependent on the location of the mutation in the protein.
3’ mutation are associated with Gardner syndrome
5’ nonsense mutations escape NMD as there is a second ribosome entry point later in the mRNA - a shorter transcript is produced but it retains some function.
Where are most of the mutations associated with attenuated FAP found?
In the large, final exon.
Why are APC mutations in exon 9 less severe?
There is an alternatively spliced functional transcript that lacks exon 9.
In what proportion of cases is somatic mosaicism observed?
11%
Describe the testing procedure for FAP diagnosis
CRC gene panel and MLPA
What treatments are available for FAP?
Colectomy once polyps >100.
NSAIDs can help reduce risk
What other diseases are associated with APC mutation?
Attenuated FAP - later onset, less severe, fewer polyps.
Gardner syndrome - basically FAP, but with increased incidence of soft tissue tumours (desmoids).
What is the different between FAP and MAP?
MAP is AR.
MAP is similar in presentation to attenuated FAP
What is the lifetime risk of CRC associated with MAP?
40-100%
What is the disease mechanism underlying the pathogenesis of MAP?
MUTYH is a base excision repair protein
Repairs oxidative damage excising adenine bases paired with C or G. Mutations result in loss of this function.
Loss of functions causes somatic mutations to arise in other genes; these other genes include APC, BRCA1/2 and KRAS. 64% of MAP cases have a codon 12 mutation in KRAS
What is the mutation spectrum associated with MAP?
99% missense.
Two common mutations: Y179C and G369D
What is the testing strategy for MAP?
Test for APC first to rule out FAP.
Test for common two mutations.
Sequence rest of gene.
What is the screening schedule for patients with MAP?
annual colonoscopy from age 18-20. Upper GI investigations from Age 25+
What proportion of women get breast cancer in their lifetime?
What proportion of these women have a mutation in a highly penetrant susceptibility gene?
1/8
5-10%
When should hereditary BRCA be suspected?
Early onset (<50) Multiple primaries Multiple individuals in a pedigree affected BrCa and OvCa in one person One OvCa in family Male BrCa Triple negative tumour High risk population
What probability models are available to assess risk?
what does NICE recommend the cut-off is?
BRCAPRO, BOADICEA, Manchester Score, Myriad II
10% risk
What otehr cancers are BRCA1/2 associated with?
Pancreas, Prostate, ovarian, stomach, lanrygeal.
What is the lifetime risk of breast and ovarian cancer in patients with BRCA1/2 mutations?
Breast 65-80%
Ovarian ~40%
Does BRCA1 or 2 have a pseudogene?
BRCA1
Does BRCA1 or BRCA2 confer high risk to males?
BRCA2
High risk of BRCA, prostate cancer
What is the role of BRCA1 and BRCA2
Homologous recombination repair of dsDNA breaks.
BRCA1 pairs with BARD1 an BRCA2 pairs with RAD51 to repair damage in G2 of the cell cycle.
Describe the mutational spectrum in BRCA1 and BRCA2
BRCA1 - ~25% large rearrangements, 2% missense mutations
BRCA2 - ~6% large rearrangements
List some population founder mutation in BRCA1/2
Dup exon 13 in BRCA1 in UK
AJ population
BRCA1: c.68_69delAG; c.5266dupC
BRCA2: c.5946delT
What treatment options are available for BRCA1/2 mutation carriers?
Prophylactic mastectomy (90% risk reduction), bilateral salpingoophorectomy (50% risk reduction)
Tamoxifen for ER+
PARP inhibitors.
How do PARP inhibitors work?
The prevent PARP fixing ssDNA breaks. These ss breaks become dsDNA breaks and in cells with null BRCA1/2 these cannot be fixed - cells die.
List examples of other cancer predisposition syndromes
Li Fraumeni - TP53
Peutz-Jegers - STK11
NF1
Nijmegan breakage syndrome - NBN
List intermediate penetrance BRCA susceptibility genes
Ataxia Telangiectasia heterozygotes - ATM
CHEK2 c.1100delC
PALB2
RAD50
What are the diagnostic criteria for NF1?
6+ cafe-au-lait patches >5mm in diameter in prepubertal individuals Axillary freckling Lisch nodules Optic glioma 2+ neurofibromas A relative with an NF1 mutation
Additional features: Hypertension Intellectual disability Tumours JMML Scoliosis
What is the incidence of NF1?
1/3500
What does somatic mosaicism for NF1 mutations cause?
Segmental NF1
What is the inheritance pattern for NF1?
AD
What is the normal function of NF1?
Tumour suppressor gene acting in the RAS/MAPK pathway.
Loss of function causing increased RAS signalling and increased cell growth.
What are some differential diagnoses for NF1?
Noonan's Legius CFC Costello syndrome LEOPARD syndrome McCune-Albright MEN2B
What is the mutation spectrum seen in NF1?
Point mutations, small deletions.
Whole gene deletions (1.5mb common deletion) - duplication of this region causes ID and seizures.
What is the new mutation rate for NF1?
50%
Why should testing of blood be done with caution?
There is a high de novo mutation rate, and many cases are mosaic. Testing tumour material can be more useful, as blood may contain little if any evidence of the mutation. Comparing tumour vs blood can be handy.
There is a common mutation in exon 17 of NF1. What is it and what is it associated with/
2970_2972del - associated with CaL spots; not with neurofibromas.
How common is NF2?
1/25000
AD inheritance
50% de novo
Describe the clinical phenotype of NF2
Schwannomas, gliomas, vestibular schwannomas (can lead to deafness and tinnitus), meningioma, neurofibroma, cataracts, balance problems
What does loss of NF2 cause?
Schwann cell movement, growth and differentiation is affected.
What is a differential diagnosis for NF2?
Schwannomatosis - SMARCB1
Describe the testing strategy for NF2
Sequence
MLPA
Linkage analysis may be useful
Mosaicism can also be a problem - test tumour if possible
What cytgoenetic abnormalities cause NF2?
Ring 22
Large 22 deletions
chromosome 22 translocations.
What is the incidence of TSC?
What genes are associated with TSC?
1/6000
TSC1 (~30%) and TSC2 (~70%)
Name some clinical features of TSC
CNS lesions; seizures, brain tumours, SEGAs can block CSF circulation and cause hydrocephalus. Lung lesions Cardiac rhabdomyomas Renal angiomyolipomas Skin abnormalities Retinal lesions
How can TSC be diagnosed prenatally?
Presence of cortical tubers seen on 2nd trim scan.
What proportion of patients meeting the TSC diagnostic criteria have mutations in TSC1 or 2?
What proportion of cases of TSC are sporadic?
What proportion are somatic mosaic?
75-85%
2/3
6%