FISH

Question 1

FISH

Answer 1

uses fluorescently labeled oligonucleotide probes, that are complementary to specific regions of the genome, to report on the copy number of these genetic loci, and their approximate position in the genome.

Question 2

Benefits to FISH

Answer 2

Able to provide this information at the level of the individual cell.

Possible to largely preserve nuclear morphology and tissue morphology during a FISH experiment, which means this technology is particularly useful for cancer investigations.

Question 3

Why is FISH ideal?

Answer 3

Many molecular genetic technologies require samples to be completely destroyed for the DNA to be extracted – as a consequence single cell information, and the location of abnormal malignant cells within a tissue – is lost.

Question 4

What is stringency?

Answer 4

Stringency governs the likelihood of two nucleic acids (oligonucleotides) being single stranded.

Stringency is a measure of how likely 2 lengths of nucleic acid will anneal, based on environmental conditions

Question 5

ANother way to think of stringency

Answer 5

Think stringency as energy available to break H-bonds

Question 6

What does FISH rely on?

Answer 6

FISH relies on the fundamental principle that complimentary sequences of nucleic acids are able to specifically anneal via the process of hybridisation in exactly the same way that PCR primers will specifically anneal to their complementary targets of a template.

Question 7

Two major parameters that govern stringency

Answer 7

temperature and the concentration of salt in the experimental system.

Question 8

What is so to allow hybridsation of FISH probe to target sequence?

Answer 8

In order to specifically hybridise a FISH probe to it’s target sequence, interphase nuclei and metaphase chromosomes are first harvested from patient samples and then adhered to microscope slides

Question 9

What must happen to enable FISH probes to bind?

Answer 9

Chromosomal DNA must be denatured in order to generate ssDNA for our FISH probes to bind. This is done by increasing the stringency of the system.
So for FISH, samples are typically denaturation at around 73 degrees.

Question 10

Why is salt important?

Answer 10

This is because salts such as NaCl and KCl, will dissociate in solution into positively and negatively charges ions.

The positive ions associate with the negatively charged DNA backbone – to cancel the electrostatic repulsion between strands. Thus – adding more salt helps to lower the stringency of the reaction because the repulsive force between ssDNA backbones of a duplex is reduced..

Question 11

Low stringency

Answer 11

Low stringency (high salt and low temp) = low energy = double stranded

Question 12

High stringency

Answer 12

High stringency (low salt, high temp) = more energy = single stranded

Question 13

What is expected at 72 degrees?

Answer 13

At this temperature we would still expect some regions to be double stranded – and so only a proportion of our probe and target sequences are expected to hybridise, once the temperature is cooled – usually to around 37 degrees.

Question 14

What should we be aware of at 37 degrees?

Answer 14

he stringency of the system is relatively low. This means probe should have hybridised nicely to our target, but at such low stringency, some of our probe will have hybridised to other regions of the genome – with imperfect complementarity. This contributes to the background signal and must be removed.

Question 15

How is background removed?

Answer 15

by washing our FISH slides in a number of different stringency buffers. It’s important to understand however that probe will still be removed from our target sites during these washes – but the goal is to maximize the true signal from these sites in comparison to the background.

Question 16

Quickly describe priciples of FISH

Answer 16

Need fluorescently labelled probe, complementary and specific to the region of target genome, also got template DNA (a nucleus) either in metaphase or interphase.

Denature probe and denature template making ssDNA.

Cool to allow probe to anneal.

When gradually cooling probe will hybridise to target site and also other parts of genome (background).

Question 17

Read

Answer 17

Nature Reviews: Genetics 2005 Oct;6(10):782-92

Question 18

What do clinical investigation using FISH use

Answer 18

Clinical investigations – only use directly labeled probes – where the fluorophore is covalently linked to the oligonucleotide sequence.

Question 19

FISH of metaphases

Answer 19

Positional information – by observing the morphology of the chromosome upon which a particular signal has colocalised

Question 20

FISH of interphases

Answer 20

Positional information - Interphase nuclei contain chromatin fibers that are far less condensed than those in metaphase – and so a much higher resolution of analysis is possible.

help to evaluate the number of copies of a particular genetic element in the genome

also report if two genetic elements have been fused together – or separated - by an abnormal rearrangement in the genome - important feature (diagnosis of certain haematological malignancies)

Question 21

What is FISH used to report?

Answer 21

FISH is used to report positional information of particular genomic loci and the copy number of these loci in an individual nucleus and nuclei of entire sections of tissue

Question 22

Gene specific probes

Answer 22

used to report on the copy number of important regions of the genome – that can have a profound impact on the prognosis of a patients’ disease.

Question 23

N-MYC probe

Answer 23

N-MYC is a transcription factor that belongs to a larger family of MYC proteins, all of which are involved in the transcriptional regulation of genes involved in cell growth, division, differentiation and apoptosis.

The genes that encode MYC proteins are considered to be proto-oncogenes – which means that these proteins have the potential to become an oncogene (i.e. a gene that has the potential to cause cancer) following a mutation event.

Question 24

What is staging?

Answer 24

. One of the first objectives of clinicians is to determine whether or not a tumour is confined to the original site – or whether it has spread – or if it is likely to spread to other parts of the body – i.e. has the caner metastisesed or is it likely to.

Question 25

What does copy number of a gene indicate (for example N-MYC)

Answer 25

anything greater than a 4 fold amplification of this gene is associated with a poor prognosis, and is observed more commonly in patients with higher stages of the disease.

Question 26

What is HER2?
What does amplification of oncogene mean?

Answer 26

Her2 is an oncogene

HER2 amplification a very useful predictive prognostic biomarker

Amplification of the HER2 oncogene is predictive of such a poor outcome, as it’s found to be amplified in around a 5th of all metastatic cases of breast cancer. It’s has also been observed in a handful of other cancers as it is involved in the transduction of proliferative and anti-apoptotic signals, in cells.

Question 27

How is amplification of HER2 identified?

Answer 27

Patient with lump
- scans to understand nature of growth
- is there evidence of abnormal growth in local and distal lymph nodes or other parts of body?
- Yes - growth will be removed and sent to histopathology
- Her2 amplification assessed by immunohistrochemistry and FISH

Question 28

Which patients would benefit with Herceptin treatment?

Answer 28

there needs to be on average twice as many genomic copies of the HER2 gene as there are copies of chromosome 17.

Question 29

In cancer, what leads to the gain and loss of chromosomes?

Answer 29

gain of whole chromosomes is a common way that oncogenes are amplified in the genome.

chromosome loss is a mechanism by which tumour suppressors are deleted.

Question 30

Therefore why is it good to characterise aneuploidy in cells?

What probe is used?

Answer 30

can provide important prognostic information about the patient

Often the more gains or losses of chromosomes, the worse the prognosis. Bladder cancer is a good example to show this to you.

Centromeric probe

Question 31

What is aneupolidy the mechanism of?

Answer 31

by which malignant cells are able to upregulate the expression of oncogenes, and down regulate tumour suppressors.

biases a cell towards enhanced proliferation and
protection against apoptosis, which drives the cancer forwards and helps to further destabilize the genome..

Question 32

What can centrometic probes also be used for?

Answer 32

Sex-mismatched bone marrow transplants

FISH is also often used to monitor the engraftment of bone marrow transplants for patients with sex-mismatched donors.

Question 33

What are bone marrow transplants used for? how? what can this data be used for?

Answer 33

often used to help cure leukaemia – this strategy relies on the immune system that differentiates from a donors bone marrow to destroy the cancerous bone marrow of the host.

So by monitoring the proportion of male and female cells in the bone marrow of a patient (i.e. the host) one is able to monitor the regression of the cancer over time.

The data is also used to help clinicians make important decisions on when to administer immuno-suppressive drugs – to limit the potency of the donors bone marrow in the host, which if allowed to proliferate out of control, could result in graft versus host disease that’ll quickly kill the patient.

Question 34

What other diagnostic information can loss/gain of chromosomes indicate?

Answer 34

what type of cancer it could be

The gain and loss of some chromosomes is also more common in certain types of cancer than others.

good is cases where its not clear type of disease patient has

Question 35

What aneuploidy chromosomes observed in myeloid leukemias

Answer 35

aneuploidy of chromosomes 5, 7, 8, 17, 20 and 21 are more commonly observed in myeloid leukaemias.

Question 36

What aneuploidy chromosomes observed in lymphoid diseases

Answer 36

chromosomes 6, 10, 12 and X.

Question 37

When is FISH used in oncology?

Answer 37

Urgent cases
Childhood leukaemia – in particular
Any new suspected case of leukaemia (child or adult) or suspected cases of relapse

Confirmation of abnormal observations

Patient monitoring, therapy response, remission status and relapse detection

Pharmacogenetic investigations

Question 38

Urgent cases

Answer 38

Identify genome that fuses two normal genes together – to form a new oncogene, which is sufficient to cause cancer.

Question 39

Confirmation of abnormal observations

Answer 39

FISH is also used to further characterise suspected abnormalities that have been identified by another technology – usually via G-banding or microarray

Question 40

What other applications are there for FISH

Answer 40

we can use this experiment to monitor patient’s over the duration of their treatment

Patient monitoring, therapy response, remission status and relapse detection

Question 41

Pharmacogenetic investigations

Answer 41

Some treatments are only prescribed when certain molecular criteria in the patient are met. – HER2 gene amplification in breast cancer and treatment with herceptin is a good example of this type of pharmacogenetic investigation.