Session 6 - Cancer genetics Flashcards
1
Q
What are the five broad categories of oncogenes? Give and example of each.
A
- 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
2
Q
How can each of the classifications of oncogene contribute to malignancy?
A
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.
3
Q
What are the five types of gain of functions mechanisms associated with oncogenes? Give examples
A
- 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.
4
Q
What two ways can translocations lead to oncogenic activity?
A
- 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)
5
Q
At which stages of the cell cycle are the control checkpoints?
A
Restriction point (between G1 and S) The G2/M checkpoint The Metaphase/Spindle Checkpoint
6
Q
What is the cell cycle regulated by?
A
Cyclins and Cyclin-dependentkinases (CDKs) - these form heterodimers
7
Q
What happens at the G1 restriction checkpoint?
A
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
8
Q
What happens at the G2/M checkpoint?
A
CDK1 is activated by phosphorylation and dephosphorylation of specific residues by Cyclin Activating Kinase (CAK)
This enables MPF formation
G2>M phase transition allowed
9
Q
What happens at the Metphase/Spindle checkpoint?
A
Chromosomes assemble on metaphase plate APC activated MPF diassembled Separase inhibition released Chromatids separate and anaphase starts
10
Q
At which stage of the cell cycle is the greatest oncogenic pressure exerted?
A
G1 restriction point
11
Q
How can mutations in activation of cyclins lead to oncogenesis?
A
Overexpression of CDK1 by translocation or amplification can lead to increased progression through G1
Loss of CKIs
12
Q
What is the role of p53?
A
A potent tranascription factor activated during cell stress to induce cell cycle arrest.
13
Q
What interacts with TP53 in a negative feedback loop, to control expression?
A
MDM2 - p53 induces expression of MDM2, MDM2 causes degradation of p53.
14
Q
Through which mechanisms does TP53 prevent cancer?
A
Activation of DNA repair
Hold cells at G2/M checkpoint to give DNA repair proteins chance to fix mutations
Induce apoptosis.
15
Q
In what proportion of cancers are TP53 mutations identified?
A
50%
16
Q
What environmental factors increase the expression of TP53?
A
Ionisating radiation or chemotoxic drugs
17
Q
What syndrome is associated with TP53 mutations? What are the clinical features?
A
Li-Fraumeni.
Increased incidence of cancers - breast, colon, lung, brain, soft tissue carcinoma, osteosarcoma etc.
18
Q
Which group of genes does the E2F transcription factor control?
A
Genes needed for S-phase
19
Q
How does RB1 control activity of E2F?
A
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.
20
Q
What are mutation in RB1 associated with?
A
Retinoblastoma.
21
Q
What is the mutational spectrum associated with RB1?
A
Frameshifts and nonsense mutations, resulting in loss of protein.
22
Q
What chromosome is RB1 located on?
A
Chromosome 13
23
Q
What is the role of CDKN2A in cell cycle control?
A
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
24
Q
What disease is associated with lof mutations in CDKN2A?
A
Multiple melanoma.
25
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.
26
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%
27
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
28
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)
29
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
```
30
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
31
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
32
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.
33
What do MMR tumours show? Is this present in sporadic cancers?
MSI
Present in a small proportion of sporadic cancers (10%)
34
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
2. MLH1 epimutation and BRAF V600E testing - if negative, less likely to be sporadic.
3. Sequence target genes to identify point mutations. MLPA required to detect large del/dups. LR-PCR for PMS2 as it has pseudogenes
35
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
36
What are the treatment and management options for HNPCC?
Full colectomy with ileorectal anastomosis
37
What is the incidence of HNPCC? When do symptoms often onset?
1/300. Age 45yrs
38
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
39
What counselling considerations are needed for HNPCC?
AD disorder
Variable penetrance
Variabel age of cancer development
Prenatal diagnosis unusual, but available
40
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
41
Where is APC located?
5q22
42
What is loss of APC a known part of?
The Adenoma > Carcinoma sequence
43
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
44
What two common mutations are responsible for 10% and 5% of cases of FAP, respectively?
Gln1062X and Glu1309AspfsX4
45
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.
46
Where are most of the mutations associated with attenuated FAP found?
In the large, final exon.
47
Why are APC mutations in exon 9 less severe?
There is an alternatively spliced functional transcript that lacks exon 9.
48
In what proportion of cases is somatic mosaicism observed?
11%
49
Describe the testing procedure for FAP diagnosis
CRC gene panel and MLPA
50
What treatments are available for FAP?
Colectomy once polyps >100.
| NSAIDs can help reduce risk
51
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).
52
What is the different between FAP and MAP?
MAP is AR.
MAP is similar in presentation to attenuated FAP
53
What is the lifetime risk of CRC associated with MAP?
40-100%
54
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
55
What is the mutation spectrum associated with MAP?
99% missense.
Two common mutations: Y179C and G369D
56
What is the testing strategy for MAP?
Test for APC first to rule out FAP.
Test for common two mutations.
Sequence rest of gene.
57
What is the screening schedule for patients with MAP?
annual colonoscopy from age 18-20. Upper GI investigations from Age 25+
58
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%
59
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
```
60
What probability models are available to assess risk?
what does NICE recommend the cut-off is?
BRCAPRO, BOADICEA, Manchester Score, Myriad II
10% risk
61
What otehr cancers are BRCA1/2 associated with?
Pancreas, Prostate, ovarian, stomach, lanrygeal.
62
What is the lifetime risk of breast and ovarian cancer in patients with BRCA1/2 mutations?
Breast 65-80%
Ovarian ~40%
63
Does BRCA1 or 2 have a pseudogene?
BRCA1
64
Does BRCA1 or BRCA2 confer high risk to males?
BRCA2
| High risk of BRCA, prostate cancer
65
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.
66
Describe the mutational spectrum in BRCA1 and BRCA2
BRCA1 - ~25% large rearrangements, 2% missense mutations
BRCA2 - ~6% large rearrangements
67
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
68
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.
69
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.
70
List examples of other cancer predisposition syndromes
Li Fraumeni - TP53
Peutz-Jegers - STK11
NF1
Nijmegan breakage syndrome - NBN
71
List intermediate penetrance BRCA susceptibility genes
Ataxia Telangiectasia heterozygotes - ATM
CHEK2 c.1100delC
PALB2
RAD50
72
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
```
73
What is the incidence of NF1?
1/3500
74
What does somatic mosaicism for NF1 mutations cause?
Segmental NF1
75
What is the inheritance pattern for NF1?
AD
76
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.
77
What are some differential diagnoses for NF1?
```
Noonan's
Legius
CFC
Costello syndrome
LEOPARD syndrome
McCune-Albright
MEN2B
```
78
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.
79
What is the new mutation rate for NF1?
50%
80
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.
81
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.
82
How common is NF2?
1/25000
AD inheritance
50% de novo
83
Describe the clinical phenotype of NF2
Schwannomas, gliomas, vestibular schwannomas (can lead to deafness and tinnitus), meningioma, neurofibroma, cataracts, balance problems
84
What does loss of NF2 cause?
Schwann cell movement, growth and differentiation is affected.
85
What is a differential diagnosis for NF2?
Schwannomatosis - SMARCB1
86
Describe the testing strategy for NF2
Sequence
MLPA
Linkage analysis may be useful
Mosaicism can also be a problem - test tumour if possible
87
What cytgoenetic abnormalities cause NF2?
Ring 22
Large 22 deletions
chromosome 22 translocations.
88
What is the incidence of TSC?
| What genes are associated with TSC?
1/6000
| TSC1 (~30%) and TSC2 (~70%)
89
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
```
90
How can TSC be diagnosed prenatally?
Presence of cortical tubers seen on 2nd trim scan.
91
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%
92
On which chromosomes are TSC1 and TSC2 found?
TSC1 9q34
TSC2 16p13.3
93
How does loss of TSC1/TSC2 cause disease?
Both proteins for a complex - they are individually unstable.
TSC complex restricts activation of mTORC1 (a tyrosine kinase), a regulator of protein synthesis and cell growth.
Positioned at the crossroads of multiple signalling pathways; AKT, mTOR, MAPK etc.
Loss of expression causes reduced regulation of cell growth.
94
What testing strategy can be used to detect TSC1/2 mutations?
Sequencing and MLPA.
95
What is variable expressivity in TSC caused by?
Need for 2nd hit
Environmental factors
Interactions with other signalling pathways
96
Describe the geno/pheno seen in TSC
TSC2 is more severe
There is a TSC2/PKD1 contiguous gene deletion that causes TSC with PKD
Increased risk of malignancy in TSC2, increased risk of Intellectual disability in TSC2
10% of TSC2 mutations are exon or whole gene deletions.
TSC1 mutations truncating, large rearrangements are rare.
97
What possible treatments are available for TSC?
mTOR inhibitors - rapamycin - can reduce lesion size and prevent further growth, but stopping treatment allows them to grow back.
Combining rapamycin treatment with imatinib (or other TKI) treatment suppresses the PDGFRBeta signalling pathway, that often compensates for lack of mTOR pathway. Treating with both drugs shows possible better outcomes.
98
List other cancer syndromes. Give their associated gene and a few clinical features.
MEN1 - MEN1 - Parathyroid and pancreatic tumours - AD
MEN2 - RET - Pheo's, thyroid tumours and hypothyroidism (AD)
Cowdens - PTEN - multiple harmartoma, cobblestone tongue - AD (40% mutations in exon 5)
PJ - STK11 - harmarmtomous polyposis (CRC) - AD
VHL - VHL - Renal cell carcinoma, pheo's, hemangioblastoma - AD
Gorlin - PTCH1 - Nevoid Basal Cell Carcinoma Syndrome - AD
Melanoma - CDKN2A - AD
Birt-Hogg-Dube - FLCN - lung cysts, renal tumours, skin manifestations
99
Name the 4 types of ssDNA repair and the two types of dsDNA repair
```
ssDNA:
MMR
NER
BER
Direct repair
```
dsDNA:
HRR
NHEJ (MMEJ is a low-fidelity version)
100
Which genes are in the MMR pathway?
MLH1, MSH2, MSH6, PMS2.
101
How does MMR work?
The MMR complex recognises and binds the DNA strand at the point of the lesion.
The section of the DNA including the lesion is nicked at the 5' and 3' ends and the strand removed.
DNA pol fills in the gap.
102
What does BER repair, and which hereditary cancer gene is associated with BER?
It repairs damaged bases using DNA glycosylase and AP endonucleases. followed by DNA pol and DNA ligase.
MUYTH
103
What does NER repair? What two type of NER are there?
| What cancer genes are involved?
Thymidine dimers caused by UV exposure.
Global (repairs all DNA) and Transcription-coupled (repairs DNA undergoing transcription)
XP, Cockayne syndrome, Trhichothiodystrophy
104
What mediates NHEJ?
What pathway in used if NHEJ pathway is inactive?
Diseases are associated with defects in NHEJ?
Short sections of homology present at the end of ds breaks.
MMEJ - more error prone.
XLF-SCID, LIG4 syndrome.
105
What does HRR require for dsDNA repair?
What genes are involved?
Long regions of homology (>300b) and utilises sister chromatids at G2.
BRCA1/2, RAD51, NBS (Nijmegan), BLM.
106
How can mistakes in DNA repair be advantageous?
Ig variability. Caused by translesional synthesis
107
What is synthetic lethality?
Give an example.
Two or more genetic events co-occurring resulting in impaired growth in the presence of increased stress.
PARP inhibitors in breast cancer treatment.
108
For the following familial cancer syndromes, name the functional defect (ds DNA breaks etc.) and the tumour susceptibility:
```
Fanconi's
Ataxia Telangiectasia
XP
Nijmegan
Li-Fraumeni
HNPCC
BRCA
MAP
```
Crosslink repair, AML
dsDNA breaks, checkpoints (ATM), ALL, Lymphoma
UV repair (NER), Skin cancer
dsDNA breaks, checkpoints, ALL Lymphoma
Checkpoints, cell cycle, apoptosis, multiple cancers
MMR, CRC, Endometrial cancer
HRR, BrCa, OvCa, Prostate cancer, Pancreatic cancer
BER, CRC
109
List the benefits of stratified/precision medicine.
```
Reduced ADR (2000 deaths per year)
Easier to introduce new drugs to market
Patient compliance higher with bespoke treatment
Reduced costs (6.5% hospital admissons)
Less over treatment
Right dose, right patient, better outcomes
More specific diagnoses
More informed choice of therapies
```
110
Give some examples of stratified medicine in cancer
Her2 status in breast cancer - treat with Herceptin if amplified. TKI, IHC for Her2, 0-1 = neg; 2 = borderline (FISH to check for gene amplification); 3+ = Her2 positive.
BRCA1/2 and PARP inhibitors - now a Phase 3 trial by AstraZeneca (SOLO2) for the treatment of OvCa with BRCA mutations (Olaparib)
111
Which genes should be screened in lung tumours? How should mutations be tested for, and what treatments are available?
Lung (NSCLC):
EGFR (10% tumours), screen TK domain (exons 18-21; exon 18,20,21 mutation by pyrosequencing, exon 19 deletion and exon 20 duplication by fragment analysis). Treat with Erlotininb etc.
KRAS (22% adenocarcinomas in lung), screen for activating mutations, no good treatment.
EML4-ALK (4-6% lung adenos), screen for fusion, use fusion probe to detect (2)(p21p23) inversion. Treat with Crizotinib.
112
What genes should be screen in CRC tumours?
BRAF and NRAS (10%) - Vemurafenib ineffective if no EGFR mutation. Impair response to Cetuximab.
KRAS - neutralising antibody Cetuximab. 50-70% of metastatic CRC are KRAS +ve
113
What genes should be screened in Melanoma?
BRAF - V600E in 80%, detect by RT-PCR or pyrosequencing. Treat with Vemurafenib.
cKIT mutations (also seen in GIST), treat with Imatinib but poor response.
114
What is the stratified medicine programme?
Which tumours were involved?
Which genes were screened in which tumours?
The partnership between the UK Gov, NHS and drug companies to improve diagnostics and treatment of cancers.
BrCa, OvCa, CRC, Prostate cancer, Melanoma, Lung cancer.
```
KRAS - CRC, Lung
NRAS - CRC, melanoma
BRAF - Melanoma, CRC. Lung
EGFR - Lung
TMPRSS-ERG - Prostate
EML4-ALK - Lung
PTEN - BrCa, Prostate, OvCa
PIK3CA - Lung, BrCa, OvCa, Melanoma
TP53 - BrCa, OvCa, CRC
CKIT - melanoma
```
115
What is the CRUK stratified medicine programme stage 2?
National Lung Matrix Trial. 2000 individiuals with NSCLC
Focusing on existing drugs that may be used to treat lung cancer. Genetic testing used to determine which arm of the trial individuals should enter.
116
Why is lung cancer a good candidate to perform the SMP2 trial?
1. Lung cancer is the second most common cancer in the UK
2. It is a an operationally difficult patient pathway - translating it to other cancers should be easy.
3. Poor survival.
117
What does drug response depend on?
```
Pharmacogenetics
Distribution
Absorption
Metabolism
Excretion
```
Pharmacodynamics:
Target proteins
Downstream messengers
118
What are the benefits of PGx?
```
Safe dosage
Reduced costs
Improved drug choices
Avoid ADR
Explain variable response
```
119
Which two genes are responsible for some variability in the response to Warfarin?
CYP2C9 and VKORC1
120
How does Warfarin control clotting?
It reduces vitamin K availability for clotting cascade.
121
What is VKOR's role in clotting?
It recycles vitamin K. It is inhibited by Warfarin.
122
Which are the two common SNPs associated with CYP2C9 Warfarin metabolism?
CYP2C9*2 - reduces warfarin dose requirements by 1/5.
| CYP2C9*3 - reduces warfarin dose requirements by 1/3.
123
What is the frequency of the CYP2C9 alleles in the UK population?
CYP2C9*2 - 12%
| CYP2C9*3 - 8%
124
What proportion of European/American's carry one of the common VKORC1 alleles? What dosages are required for WT/het/hom?
37%
WT: High dose
Het: Intermediate dose
Hom: Low dose
125
How is warfarin dose normally decided?
Initial does prescribed, then patient monitored using the INR. Dosage changed as required.
126
Why is genotyping of CYP2C9 and VKORC1 not routinely performed in UK?
There is conflicting evidence of the benefits of genotyping over traditional methods of monitoring.
127
Give an example of a PGx marker for cancer.
TMPT is a marker for 6-mercaptopurine, a treatment for ALL.
Activity of TPMT is measured in RBC - this is not accurate in patients with low activity and those having undergone a blood transfusion. 1/300 are TPMT deficient; patients with low activity are more likely to suffer ADR at standard levels.
Mutant alleles: TMPT*2, TMPT*3A, TMPT*3B and TMPT*3C
128
How do circulating tumour cells enter circulation?
Primary tumour cells undergo epithelial > Mesenchymal transition (EMT) to enable intravasation.
Cells enter blood stream
Cells undergo extravasation and Mesenchymal > Epithelail transition (MET) to invade a distant site.
129
What are the applications of CTC numeration?
```
Determine treatment pathways
Prognosis
Representative of primary tumour
Early stage disease - early detection
No invasive sampling
```
130
How can CTCs be enriched?
Size (ISET)
Density (FICOLL)
Cell surface markers (CD45, EpCAM binding) - CellSearch
131
How can CTCs be detected once they have been enriched?
Cytometric - Membrane proteins/Proteins secreted by viable cells (e.g. EPISPOT - antibody detection of secreted proteins - only viable cells are detected)
Nucleic acid - RT-PCR (more sensitive)
132
Give examples of clinical applications of CTC detection.
Identify driving mutations in EGFR-mutant NSCLC. Can also detect the common T790M EGFR mutation that confers drug resistance (to erlotinib) - mutation correctly identified in 12/13 cases.
133
How is AML diagnosed?
What is the incidence of AML?
What are the symptoms of AML?
Presence of 20% blasts in the PB or BM.
OR
<20% blasts, plus myeloid sarcoma, AML related chromosome abnormality or erythroid leukemia.
Adult: 2.5/100,000
Paed: 0.7/100,000
S.o.b. fatigue, bleeding, bruising, splenamegaly, tender bones, death in months if untreated.
134
How does WHO classify AML?
Cell morphology
Cell markers
Genetics
Clinical phenotype
135
List the common abnormalities seen in AML. Give cyto nomenclature, name genes involved and state prognosis.
```
t(8;21)(q22;q22) RUNX1T1-RUNX1, good prognosis
inv(16)(p13q22) CBFB-MLL good prognosis
t(15;17)(q24;q21) PML-RARA good prognosis
FLT3-ITD intermediate
FLT3-TKD intermediate
Abn 3q
-5 poor prognosis
del5p poor prognosis
-7 poor prognosis
del7q poor prognosis
complex karyotype poor prognosis
```
136
What drug is the t(15;17)(q24;q21) PML-RARA translocation sensitive to?
ATRA
137
Mutations in which gene can reduce the prognosis of t(8;21)(q22;q22) and inv(16)(p13q22) CBFB-MLL?
KIT mutations
138
What proportion of children with Down syndrome present at birth with Transient Abnormal Myelopoesis? (similar to DS-associated AML)
10%
139
What is required for AML development? Define both and give examples of each
Collaboration between 2 classes of mutation:
Class I: mutations in signalling pathways - FLT3 (ITD or TKD), PTPN11, RAS, cKIT
Class II: mutations in transcription factors - RUNX1, PML-RARA
140
Which trial is investigating the use of FLT3-inhibitors in the treatment of FLT3-mutant AML?
ALL17
141
Mutations in which novel/unclassified gene are associated with a good prognosis in AML?
Which genes do mutation in this gene commonly co-exist with?
NPM1
FLT3-ITD, FLT3-TKD, IDH.
142
Describe the diagnostic procedures used in AML.
Morphology - cell staining, blast count.
Immunophenotyping - examine cell surface markers
Cytogenetics - karyotype and FISH
Molecular testing - RNA/DNA analysis for NPM1, FLT3 and CEBPA
143
Why is RNA analysis used to in AML diagnosis?
| What is the main problem with RNA analysis?
Covers multiple transcripts
Doesn't include exons so easier to PCR
Highly sensitive
RNases are abundant in the enviroment
144
When is RT-PCR used during AML diagnosis? When is RQ-PCR used?
RT-PCR used to detect rearrangement for diagnosis
| RQ-PCR used to monitor for MRD (it is quantitative)
145
Why is a molecular diagnosis in AML important?
Allows targeting of allogenic SCT (risky) to only those most in need.
146
Is Blood or Bone marrow preferred for AML diagnosis?
Marrow
147
Describe the AML diagnostic analysis regime for a sample with:
Abn karyotype
Normal Karyotype
What technique is used for follow-up?
Abnormal karyotype - analyse 5, screen 5 more
Normal karyotype - analyse 10, screen 10 more.
FISH can be used if number of metaphases is too low.
Screen of 30 metaphases (G-banding), or 100 interphases by FISH
148
What are the TATs for cytogenetic analysis of haem-onc samples?
Provisional report on urgent - 95% at 3 days
Urgent - 95% in 14 days
Routine - 95% in 21 days
149
What is the molecular strategy for MRD monitoring of AML?
Blood samples every 6 months for inv(16)(p13.1q22), shorter intervals for t(8;21) and t(15;17) and NPM1 mutations. RQ-PCR.
150
What are the three different aspects to assessing transcript levels for MRD?
1. transcript levels at presentation
2. the extent of reduction after treatment
3. the increase following remission
151
Name three future methods of monitoring/profiling haem-onc tumours
Gene expression profiling
miRNA analysis
Epigenetic profiling - increase in methylation in AML patients at relapse.
152
What is seen in the blood sample of patients with CML?
Increased number of granulocytes and immature blasts.
153
What are the three stages of CML?
Chronic - can last many years, 90% of patients at diagnosis
Accelerated phase - accumulation of mutant cells - can last months
Blast crisis - >20% blasts in blood or marrow.
154
What is the common rearrangement seen in CML?
t(9;22)(q34q11), BCR-ABL1 in 95% of patients
Remaining 5% have other variant rearrangements involving the same region. BCR-ABL1 fusion is always present.
155
Why is characterisation of any secondary changes useful in CML?
May affect response to Imatinib.
156
What is the monitoring schedule for CML MRD?
Marrow sample at 3 and 6 months post-treatment, and every 6 months after until CCyR is achieved.
157
How is response to treatment classified in CML?
When should each milestone be reached?
Haematological Response - lack of blasts in blood/marrow, blood counts return to normal, splenomeglay subsided. (3 months)
Cytogenetic Response
Minor - >35% cells with Ph+
Partial - 1-35% cells with Ph+ (6 months)
Complete - No evidence of Ph+ cells (1 year)
Molecular Response
Major - 3 log reduction in BCR-ABL1 quantity (18 months)
Complete - no evidence of BCR-ABL1 transcripts.
158
What is the first line treatment for CML?
What are the 2 line treatments? Why are they being considered as first line treatments?
Imatinib - not curative, but leads to CCyR in 80% of patients
Nilotinib, Dasatinib - they are less sensitive to mutations occurring in the ATP-binding site of ABL1.
159
What drugs were previously available to treat CML, before the discovery of TKIs?
IFNa
| Allogenic HSCT - high risk of mortality
160
Describe the therapeutic monitoring of CML patients.
RT-PCR to determine breakpoints at diagnosis.
RQ-PCR to quantify at diagnosis and for MRD.
MMR is classed as a 3 log reduction in number of BCR-ABL1 transcripts.
Ratio between BCR-ABL1 and ABL1 generated.
This ratio is a normalised value. An international scale can be applied to make standardised interpretation possible
161
Why is ABL1 a good housekeeping gene to compare BCR-ABL1 ?
It also provides a measure of RNA quantity and quality in the test.
162
What is the detection limit of qPCR assays used?
a 5 log reduction from baseline can be detected.
163
Why are patients not routinely tested for ABL1 mutations at diagnosis?
When is ABL1 testing recommended?
New mutations can arise during disease progression to confer TKI resistance - no point testing at the start.
When patient doesn't hit their milestones or secondary resistance is observed. Patients in blast phase or in accelerated phase.
164
What are the two type of ALL?
Which is associated with genetic stratification?
B-ALL - multiple prognostic markers.
| T-ALL - no clinically useful markers to date.
165
How are B-ALL and T-ALL distinguished?
Immunophenotyping. They are morphologically identical - anaemia, thrombocytopenia, leucocytosis, neutropenia.
166
What are the most significant Adult B-ALL markers? Give their associated prognoses.
```
t(9;22)(q34;q11) BCR-ABL1 Poor prognosis
t(4;11)(q21;q23) MLL-AF1 Poor prognosis
Complex karyotype Poor prognosis
High hyperdiploidy Good prognosis
Hypodiploidy Poor prognosis
Near triploidy Poor prognosis
```
167
What are most Adult B-ALL t(9;22)(q34;q11) translocations associated with?
Deletion of IKZF1
168
What do 35% Adult and Paed T-ALL translocations involve?
Rearrangements of the TCR.
169
Activating mutations of which gene are associated with Adult T-ALL
NOTCH1
170
Describe the testing strategy for B- and T-ALL
```
Culture BM or Blood
Use TPA for B-ALL
FISH for MLL if patient <1yr
FISH for ETV6-RUNX1 if child, then iAMP21, BCR-ABL1, TCF3
FISH for BCR-ABL1 if adult, then MLL
```
171
What molecular techniques can be used in ALL diagnosis?
RT-PCR for fusions
qPCR for IGH and TCR rearrangements
MLPA for IKZF1
Best for MRD monitoring.
172
What proportion of ALL are B and T cell?
B-ALL - 85%
| T-ALL - 15%
173
What are the most significant rearrangements seen in Paediatric B-ALL?
t(9;22)(q34;q11) BCR-ABL1 Poor prognosis
MLL rearrangements most common in <1yr old. t(4;11)(q21;q23) MLL-AFF1 poor prognosis
t(12;21) ETV6-RUNX1 GOOD prognosis
iAMP21 (5+ copies of RUNX1 on one chr21) - poor prognosis
HeH - GOOD prognosis
Near haploidy - poor prognosis
TCF3 rearrangements t(1;19) TCF3-PBX1 - Intermediate prognosis
IGH rearrangments (t(5;14) IL3-IGH)
174
How is CLL diagnosed?
Monomorphic small B-cells.
| Anaemia, cytopaenia
175
What is the incidence of CLL related to?
Age
2-6/100,000 overall
12.8/100,000 by age 65 years. Most common adult leukaemia.
176
What proportion of families appear to have a genetic component to their CLL?
5-10%
177
What is the life expectancy of an individual with CLL?
Variable. Can present as MBL > CLL > Richter's syndrome (most severe - aks DLBCL)
178
Why is FISH recommended for CLL testing?
The B cells don't grow well in culture, even after TPA stimulation.
179
What are the 4 common rearrangements associated with CLL? give their associated prognoses.
Trisomy 12 - Intermediate prognosis (114 month survival)
del(13)(q14.3) - Good prognosis (133 month survival)
Del(17)(p13) TP53 deletion Very poor prog (32 mo)
del(11)(q23) ATM deletion Poo prog (72 mo)
180
What other translocations are associated with CLL? What are their prognoses?
IGH rearrangements:
t(8;14) IGH-MYC
t(14;18) IGH-BCL2
All IGH rearrangements associated with a poor prognosis.
181
Which genes are commonly mutated in CLL?
```
TP52
ATM
NOTCH1 - nonsense mutation is most common recurrent CLL mutation
BIRC3
SF3B1
```
182
How did Rossi, 2013, recommend CLL mutations were subgrouped according to prognosis?
1 High risk TP53 deletion, BIRC3 deletion
2 Intermediate risk NOTCH1/SF3B1 mutation or del(11)(q23)/ATM mutation
3 Low risk Trisomy 12 or normal karyo
4 V low risk del(13)(q14.3)
183
What tests are recommended for the diagnosis/prognosis of CLL?
FISH and sequencing.
| Blood smear, blood count, immunophenotyping
184
What genetic tests do the British Commitee for Standards in Haematology recommend are performed for CLL patients?
Screen for TP53 loss and TP53 mutation
185
Which cell markers are also indicators of poor prognosis in CLL?
CD38+ and ZAP70+
186
What are the first line therapies for CLL?
FR and FCR
| Alemtuzumab for TP53 mutation (FDA-approved)
187
What are the features associated with Myeloma/Plasma cell neoplasms?
C - hypercalcaemia
R - renal failure
A - anaemia
B - bone disease
188
What can be detected in the urine and serum of patients with myeloma?
Paraprotein
189
What rearrangements are common in Myeloma?
Hyperdiploidy - 50% of patients - good prognosis
Others - poor prognosis - most IGH rearrangements (14q32)
```
5 most common:
t(4;14)(p16.3;q32) Poor prog, FGFR3/MMSET
t(6;14) Good prog. CCND3/IGH
t(11;14) Good prog. CCND1/IGH
t(14;16) Poor prog. MAF/IGH
t(14;20) V. poor prog MAFB/IGH
```
190
What changes are associated with disease progression in Myeloma?
del13
del17p
del1p, dup1q
MYC++
191
What do EMN guidelines recommend are FISH'd for for Myeloma diagnosis/prognosis?
What additional regions can be screened?
t(4;14)
t(14;16)
17p13 del
1p+ / 1q-
Extended:
t(11;14)
t(14;20)
Ploidy
192
How can myeloma be treated?
```
Chemo
Steroids
Thalidomide
SCT
Newer drugs showing promise in patients with v poor prognosis.
```
193
Name the two different types of Lymphoma
Hodgkins lymphoma
Non-Hodgkins lymphoma
Can be B or T cell
194
What is Hodgkins Lymphoma characterised by?
Reed-Sternberg cells.
| EBV genome identified in 50% of cases
195
What sub-types of NHL are there?
| Name a mutation found in each
DLBCL (Richert's syndrome) - t(14;18) IGH-BCL2; t(3;14) IGH-BCL6
Mantle Cell lymphoma - t(11;14) IGH-BCL1; SOX11 transcription factor only present in MCL.
Burkitt's - t(8;14)(q24;q32) IGH-MYC; t(2;8) IGK-MYC; t(8;22) IGL-MYC
Follicular - t(14;18)(q32;q21) IGH-BCL2; 60% have 6q21 deletion
Marginal Zone - MALT and IGH rearrangements
Anaplastic Large Cell Lymphoma (T-cell) - ALK rearrangements (2p23); t(1;2) ALK-TPM3; t(2;5) ALK-NPM
196
List testing methods for Lymphoma cases
Karyotype to detect complex, need dividing cells
FISH for break-aparts - useful when genes have multiple translocation partners, can test FFPE
aCGH - only detects balanced, won't detect low level mosaicism, don't need dividing cells
SNP array - can detect UPD and LOH. Tumour vs Normal
Array gene expression
PCR for common translocations, MRD
IHC for cell surface marker expression
197
What can be a significant problem in Lymphoma testing?
How can this problem be overcome?
High grade lymphomas have problems with cell apoptosis; lack of dividing cells for karyotype.
Harvest same day cultures, culture with B-cell mitogens (PMA).
198
How should karyotyping be performed on LN biopsies and infiltrated bone marrow for Lymphoma?
LN biopsy: 20 mets screened for common abn - if clone found, analyse 5, and screen further 5.
Marrow: screen min of 20 mets if normal - if abn analyse 5, screen 5.
199
What can MDS develop into?
AML
200
How can MDS arise?
De novo: virus, smoking, hereditary disorders, Down syndrome, Fanconi's, benzene exposure.
Secondary to cytotoxic chemotherapy
201
What scoring system can be used to offer prognosis of MDS?
IPSS-R - only useful at presentation
| WPSS - WHO score that can take into account treatment cycles.
202
What is the most important prognostic marker for MDS?
Karyotype
203
What abnormalities are seen on MDS karyotype?
```
5q- Good prognosis
-7 Poor prognosis
7q- Poor prognosis
Trisomy 8 Intermediate prognosis
3q- Poor prognosis
17p- (TP53) poor prognosis
Complex - very poor prognosis
Others (rare)
```
204
How is 5q- thought to cause MDS?
What do these patients respond to?
Haploinsufficiency of genes in the region
RPS14, SPARC, CTNNA1, EGR1
Lenolidomide
205
What genes are implicated in MDS?
What are many of them involved in?
```
TET2
EZH2
TP53
SF3B1
ASXL1
UTX
DNMT3A
IDH1/2
```
Epigenetic modification and methylation.
206
What is the diagnostic strategy for MDS?
Karyotype.
207
What three therapeutic target agents have been approved for use in MDS by FDA?
Lenolidomide (low risk MDS)
azacitidine (high risk MDS)
decitabine (high risk MDS)
208
What are the purposes of MRD monitoring?
To detect early relapse
Allow target driven dosage and duration of treatment
Detect reponse to treatment
To avoid toxicity
209
What methods are available for monitoring MRD?
FISH - low sensitivity
RQ-PCR - determine breakpoints of translocations at diagnosis, compare against ABL, used for BCR-ABL (CML), PML-RARA (APL) and Ig-TCR (T-ALL)
qPCR - t(14;18) IGH-BCL2 rearrangement in 80% Follicular Lymphoma
Tandem duplication PCR (IS-PCR) - FLT3-ITD (AML)
Flow cytometry for cell markers.
210
Describe MRD monitoring in ALL
Largely by flow cytometry.
Start monitoring 2-3 weeks post remission induction therapy
Detect B-ALL and T-ALL
Ig-TCR rearrangement testing if identified at diagnosis - need unique primers for each patient.
Gene fusion detection (40% of B-ALL patients) - ETV6-RUNX1, TCF3-PBX1, MLL-AFF1, BCR-ABL
Flow - 10e-4
RQPCR - 10e-5
211
Describe MRD monitoring in AML
Haematology
Cytogenetics
FISH
Fusion gene monitoring - t(8;21), inv(16) and t(15;17)
FLT3-ITD status - PCR, compare shorter allele to longer allele. only 10e-2 sensitivity due to PCR bias
WT1 monitoring - overexpressed in 80-90% AML
Flow cytometry for abnormal cell markers - 94% suitable
212
Describe monitoring in CML
Ph+ in cytogenetics
Fusion gene monitoring with interphase FISH
qRT-PCR
213
Describe monitoring on Mantel Cell lymphoma
qPCR for IGH rearrangments - early lack of IGH means good prognosis
214
Describe monitoring for Follicular lymphoma
PCR for t(14;18) IGH-BCL2 (in 80% of patients)
215
How can BMT be monitored?
By sex mismatched and detection of SRY/ AMELX/Y etc. by QF-PCR
By sex matched QF-PCR for polymorphic markers
216
What are the three phases of clinical trials? Briefly describe each.
Phase I - early stage, very small numbers of patients, determine safety and efficacy. Dose scaling (determine tolarance to different drug dosage)
Phase II - Larger scale trial, sub-group of patients with a specific type of cancer. Sometimes placebo vs drug - does the drug work. ID patients who would most benefit, optimise dose. Sometimes randomised
Phase III - randomised, large scale trial, multiple patients, new drug vs current best. Is survival improved.
217
What trial is currently in progress for Lung Cancer?
Small Cell Lung Cancer treatment with Olaparib. Randomised, phase II trial of Olaparib vs placebo.
218
What is the RIO study for Breast Cancer?
Window study of the effect of treating TNT and BRCA1/2 carriers with PARP inhibitor - determine the percentage of tumours that are sensitive to PARP treatment.
219
Name a clinical trial for CRC
```
FOCUS-4 - aims to stratify patients into 4 groups for targeted treatment:
KRAS/NRAS +
BRAF+
PIK3CA+ or PTEN loss
WT
```
220
Name an ALL trial.
UKALL14 - adults - stratify into T1/T2 (T-cell) and B1/B2 (B-cell) for treatment
UKALL 2011 - children and young adults - ALL and lymphoma
UKALL 60+
221
Name an AML trial
AML17 - two aims
1. In patients with APL, test ATRA + AIDA vs ATRA.
2. In patients with AML - stratify into 5 risk groups and trial different treatments - e.g. FLT3 inhibitor if mutated.
222
What is the SPIRIT2 trial?
Testing the efficacy of Imatinib vs nilotinib and dasatinib in CML. Compare event free survival.
223
What is the DESTINY trial?
Investigate the feasibility of reducing/stopping TKI treatment in CML patients that are excellent responders.
Need to be <0.1% BCR-ABL (MMR) for at least 12 months.
Need to have been treated with imatinib, dasatinib or nilotinib for a minimum of 3 years.
224
List some myeloproliferative neoplasms
```
CML
CMML
JMML
CNL
PV
ET
Myelofibrosis
```
225
What are the presenting features of MPNs?
```
Increased number of mature cells in the BM. Can lead to bone marrow failure.
Organomegaly
Peak incidence 50-70 years old
6-10/100,000
Good prognosis
```
226
What molecular changes are commonly found in MPN?
JAK2 mutations - V617F most common, followed by rare mutations in exon 12. Present in nearly all PV cases.
MPL exon 10 mutations seen in ET and myelofibrosis
CALR -common mutations in exon 9, all frameshift
PDGFRA - respond to TKI treatment
PDGFRB - respond to TKI treatment
FGFR1 - doesn't respond TKI treatment
227
What disorder is transient myeloproliferative disorder seen in?
Down syndrome.
228
Describe the diagnostic strategy for MPN.
Cytogenetics
FISH for PDGFRA fusion and BCR-ABL
Sequence for mutations in specific genes (JAK2 V617F)
Molecular diagnostics will become more important as the number of personalised medicines increases.
