Genetic Changes in Cancer Part Two

Question 1

3). Describe how the loss of a gene or gene product can result in cancer.

Answer 1

• Often involves the loss of a tumour suppressor gene (TSG).
• TSGs are usually involved in maintaining the integrity of the genome.
• Both copies of the TSG will usually be compromised within a tumour.
• Alternatively, instead of being TSG related, the loss of a gene may result in haploinsufficiency for dosage sensitive gene(s).
• Loss of gene/gene product may occur due to the loss of a whole chromosome (unbalanced), deletion of part of a chromosome (unbalanced), mutation within a gene (balanced), LOH (balanced).

RB1:

• RB1 is the classic example of a TSG whereby loss can lead to cancer. Patients with bilateral retinoblastoma usually have a constitutional mutation in one copy of RB1. When the other copy is mutated or lost then retinoblastoma develops.
• Loss of RB1 is also seen in a wide variety of leukaemias.
• Deletion of this region of 13 is a poor prognosis marker in CLL and Myeloma.

TP53:

• TP53 is a TSG located on chromosome 17p. Patients with constituational mutations of TP53 have Li Fraumeni syndrome - with a very high risk of developing cancer. Loss of P53 is commonly seen in a large range of cancers - often at end stage in leukaemia.
• i(17)(q10) is a common finding in blast crisis CML. This results in the loss of 1 copy of TP53.

Deletion or loss of 5:

• 46,XX,del(5)(q22q35) or 45,XX,-5
• No TSG has been identified
• Probable dosage sensitive regions within the critical region (haploinsufficiency)
• As sole abnormality in MDS = good prognosis
• As sole abnormality in AML = poor prognosis
• As part of complex karyotype = secondary with poor prognosis

Question 2

What may be the cause for the loss of genes or gene product?

Answer 2

• loss of a whole chromosome (unbalanced)
• deletion of part of a chromosome (unbalanced)
• mutation within a gene (balanced)
• LOH (balanced) - if you get LOH and there is a mutation in the remaining homologue then the mutated remaining copy will be copied to replace the missing normal copy = effectively homozygous for the identical mutation.

Question 3

Give examples of a cancer which forms due to the loss of a gene or gene product.

Answer 3

RB1:

• RB1 is the classic example of a TSG whereby loss can lead to cancer. Patients with bilateral retinoblastoma usually have a constitutional mutation in one copy of RB1. When the other copy is mutated or lost then retinoblastoma develops.
• Loss of RB1 is also seen in a wide variety of leukaemias.
• Deletion of this region of 13 is a poor prognosis marker in CLL and Myeloma.

TP53:

• TP53 is a TSG located on chromosome 17p. Patients with constituational mutations of TP53 have Li Fraumeni syndrome - with a very high risk of developing cancer. Loss of P53 is commonly seen in a large range of cancers - often at end stage in leukaemia.
• i(17)(q10) is a common finding in blast crisis CML. This results in the loss of 1 copy of TP53.

Deletion or loss of 5:

• 46,XX,del(5)(q22q35) or 45,XX,-5
• No TSG has been identified
• Probable dosage sensitive regions within the critical region (haploinsufficiency)
• As sole abnormality in MDS = good prognosis
• As sole abnormality in AML = poor prognosis
• As part of complex karyotype = secondary with poor prognosis

Question 4

What are the general genetic characteristics that give cancer predisposition?

Answer 4

• Mutations/deletions of TSGs
• Patients with increased susceptibility to DNA damage - including the chromosome instability syndromes, e.g:
• Fanconi Anaemia
• Ataxia Telangiectasia
• Bloom Syndrome
• Nijmegen Syndrome

Question 5

What 5 methods are primarily used in detecting genetic changes in cancer?

Answer 5

1) . Conventional cytogenetics
2) . FISH
3) . PCR
4) . Microarray
5) . NGS

Question 6

What are the pros and cons of using conventional cytogenetics in cancer testing?

Answer 6

PROS:

• gives a whole genome screen for that cell
• detects balanced and unbalanced abnormalities
• provides an undirected screen
• WHO disease classifications are linked to cytogenetics entries

CONS:

• Requires fresh tissue to provide dividing cells
• Requires an experienced analyst
• Can be complex and difficult to interpret

Question 7

How can FISH be utilised in cancer testing?

Answer 7

• Usually FISH is directed to detecting a specific balanced rearrangement e.g. BCR/ABL DF - 2 colour dual fusion probes.
• Can set up probes so that they come together in a specific fusion (i.e. green on BCR, red on ABL, come together in the fusion so that you would see both red and green = fusion).
• Can also set up probes so that the two colours span the break pint and split apart when the rearrangement happens rather than coming together (MLL).

Question 8

What are the pros and cons of using FISH in cancer testing?

Answer 8

PROS:

• Can be used on metaphase preps or on non-dividing cells.
• Can be used on archive material - e.g. paraffin wax embedded tissue.
• Large numbers of cells can be scored.
• Can detect balanced and unbalanced abnormalities.
• Analyst requires less training than for conventional cytogenetic analysis.

CONS:

• Very directed - generally answers a single, specific question.
• Unexpected signal patterns can be hard to interpret.
• Low level abnormal signal patterns may be hard to distinguish from artefact - dependent on probe type.

Question 9

What are the pros and cons of using PCR in cancer testing?

Answer 9

Directed testing of the DNA for specific abnormalities - e.g. common translocations in AML or tandem duplications within the Flt-3 gene.

PROS:

• Quick and cheap
• Used to quantify low level minimal residual disease

CONS:

• Very directed
• Can’t easily detect rearrangements with many viable partners - e.g. MLL

Question 10

How can microarrays be utilised in cancer testing?

Answer 10

Classic aCGH approach:

• Test and control DNAs are co-hybridised onto a microarray target slide - often neoplastic tissue vs normal tissue from the same patient.
• The microarray carries multiple spots each containing different DNA targets.
• Testing for copy number imbalance - can’t pick up balanced rearrangements.

Expression arrays:

• Microarray with cDNA spotted onto the array.
• The test sample is either RNA or cDNA from a tumour sample.
• The control may be derived from normal tissue from the same patient.
• The microarray indicates which genes are being expressed.

SNP arrays:

• Same basic techniques as CGH array.
• Based on common hypervariable sequences - SNPs.
• Allow detection of LOH a.k.a acquired UPD (dup of one allele and loss of the other) as well as imbalance. LOH is an important mechanism, particularly for TSGs.

Question 11

What are the pros and cons of using microarrays in cancer testing?

Answer 11

PROS:

• The microarray can be tailor-made for specific uses - e.g. all known TSGs and all known oncogenes and proto-oncogenes.
• Many different loci can be screened in a single test.
• High resolution - e.g. sub-microscopic deletions etc.

CONS:

• Will only detect unbalanced abnormalities.
• Results unreliable if sample is contaminated by normal cells as many samples will be.
• High cost of both materials and equipment (not really these days).
• Any balanced abnormalities will be missed.

Question 12

How can NGS be utilised in cancer testing?

Answer 12

• New technology starting to move into the diagnostic area.
• Sequences entire DNA sequences - in theory could detect balanced and unbalanced rearrangements and LOH.
• Potential for very high throughput but set up costs are high so services are likely to be centralised.
• Like arrays requires uncontaminated target DNA.

Question 13

Conclusions:

Answer 13

• For leukaemias, a combination of cytogenetic analysis, FISH, and PCR are the techniques currently in common use in the diagnostic setting.
• Microarrays - very powerful tool for screening many loci in a single test BUT will not detect balanced rearrangements.
• NGS may come to the fore in the next 5 years.