Screening for Somatic Mutations

Question 1

Intro to somatic mutations:

Answer 1

• Most cancers are the result of acquired mutations in somatic cells = spontaneous.
• Once mut has occurred in cell get transmission of mutation into descendant cells as it divides.
• This leads clearly to a clonal production of cells.
• Within the clone all the cells will contain the mutation as a marker.
• Within any tumour system it is likely that you will get multiple somatic mutations that happen at different points within the cancer pathway - potential accumulation of different cancer mutations.
• The acquired mutations will only exist in a subset of cells.
• They exist on a background of germline sequence.
• The variant can be rare in an individual or in the human populations.
• The variant can be recurrent and therefore more common in the human population.

Question 2

What are the technical considerations that we must consider in order to assess somatic mutations?

Answer 2

1) . How are we going to access suitable tumour material in order to carry out tests?
2) . What is the quality of the DNA or RNA that you are going to look at? Cancer cells are not normal - may be accelerating towards apoptosis and the quality of DNA/RNA may be poorer that from normal cells.
3) . May wish to consider enriching for tumour cells.
4) . The sensitivity of the assay for detecting cancer cell mutations is critical - needs to be as sensitive as possible in order to detect low % of tumour.
5) . ? Scope of assay - may be just one mutation in one gene - might be that the mutation may occur anywhere within a gene - assay needs to be designed to take account of that.
6) . Reliability
7) . Access to relevant equipment and appropriately trained staff.

Question 3

What are the traditional divisions of analysis in cancer genetics?

Answer 3

1) . Haematological (leukaemia/affecting blood cells):
- CML
- AML
- MPD

2) . Solid Tumour:
- Colorectal
- Lung
- Prostate
- Breast
- Melanoma

There is commonality in the genes affecting particular pathways that drive both of these cancer types.

Question 4

How difficult it to obtain samples to test for haematological cancers?

Answer 4

• Relatively straightforward for haematological cancers to obtain specimen.
• Blood sample.
• Bone marrow sample.
• Able to process directly from blood tube.
• Repeat sampling possible at various time points in disease progression - important in monitoring treatment!
• Able to isolate specific cell types (B/T cells). Can use magnetic activated cell sorter for this (MACS).

Question 5

What might you use to separate B and T cells from a blood or BM sample?

Answer 5

• Able to isolate specific cell types (B/T cells). Can use magnetic activated cell sorter for this (MACS).

Question 6

How difficult it to obtain samples to test for solid tumours? What are the considerations in obtaining samples.

Answer 6

• Relatively difficult compared to access to haematological cancer samples.
• Invasive procedure will be required.
• Usually requires some sort of surgery - either complete removal of the tumour if it is appropriate to managing the patient, resection of site around tumour. Can also biopsy if you are just investigating growth.
• Difficult to undertake repeat sampling.
• Fresh tissue not readily available.
• Most tissue samples are usually formalin fixed.
• Size of sample will vary - will range from a biopsy to a full tumour resection.
• Cytology fluids can be used in some cases also.

Question 7

When testing tumour samples how do we go about extracting DNA or RNA? What are the different considerations when extracting DNA from blood/marrow compared to from FFET?

Answer 7

Blood/marrow:

• For blood and bone marrow extraction is quite straight forward.
• Well established tech that we would use for any blood sample to extract DNA.
• Whole process is easily automated.

FFPET:

• More complex.
• A lot of upstream sample processing is required and consequently it is less amenable to automation.
• One of the key considerations is that the paraffin wax needs to be removed.
• Length of time in fixatives influences the quality of DNA quite dramatically,

Question 8

What happens to the quality of the DNA in solid tissue the longer it is fixed in formalin?

Answer 8

• The longer the sample is help in some sort of fixative the more fragmented the DNA will become and the more cross linking will be apparent.

Question 9

Outline how we might carry out tumour enrichment for solid tumours?

Answer 9

• For solid tumours this can be achieved through micro-dissection.
• Would first H&E stain a tissue section and identify the cancer tissue vs areas of normal tissue.
• You can then scrape away the unwanted material from the slide prior to extraction meaning that the DNA is primarily coming from tumour tissue.
• Further downstream might want to think about different steps in the pathway that can help us identify whether the sequence changes are present. Looking for low levels of sequence change.
• Can use technology such as COLD-PCR (co-amplification at lower denaturation temp) to try and promote the amplification of mutations.
• Can undertake PCR cloning.
• Can use ME-PCR (mutant enriched PCR) where you digest away your wild type germline DNA at known mutation sites - where the mutation changes the restriction site due to mutation will get the mutant copies being preferentially preserved.

Question 10

How would one decide how to select an appropriate assay for the detection of somatic mutations?

Answer 10

Based on multiple considerations.

1) . Based on nature of mutations:
- single mutations within an exon
- multiple mutations within an exon
- multiple mutations in multiple exons
- gene fusion

2) . Consider available resources:
- Equipment
- Cost
- Experience and expertise

Question 11

What 2 techniques can be used to detect fusion mutations?

Answer 11

1). FISH

2) . RQ-PCR - TaqMan probes.
- Performed on cDNA generated from extracted RNA
- Primers located each side of common breakpoint
- Fluorogenic TaqMan probe
- Fluorescent signal generated in real time as PCR reaction proceeds
- Amount of fluorescence directly proportional to starting template
- High sensitivity

Question 12

Outline the use of RQ-PCR for detecting fusion mutations.

Answer 12

RQ-PCR - TaqMan probes.

• Performed on cDNA generated from extracted RNA
• Primers located each side of common breakpoint
• Fluorogenic TaqMan probe
• Fluorescent signal generated in real time as PCR reaction proceeds
• Amount of fluorescence directly proportional to starting template
• High sensitivity
• Tend to do a number of reactions for the same sample to get a picture of how much of the fusion transcript is present in the sample.
• Usually normalisation using a control gene such as ‘normal’ ABL.
• Control gene copy number reveals the sensitivity of the assay because you known what you’re expecting to see.
• Sensitivity is critical to ensure accurate negative results - when BCR-ABL transcript is not seen is it actually not there or are we just not detecting it? Need our assay to be as sensitive as it can be so we can ensure accurate negative results!
• Aim for 1000 ABL (control) transcripts per reaction as your baseline. Then express your results as a ratio of BCR-ABL to normal ABL (or whatever the control gene transcript you are using is).

Question 13

What technologies can be used to detect somatic cancer mutations other than fusions?

Answer 13

1) . Technologies where mutations can be directly identified:
- Direct Sanger sequencing
- Pyrosequencing
- Maldi/TOF
- ARMS-Scorpions (DxS)

2) . Technologies that are more pre-screen based:
- SSCP
- dHPLC
- DGGE
- HRM

ALSO NGS AND DIGITAL PCR!

Question 14

Outline the pre-screening methods that can be used to look for somatic cancer mutations that aren’t fusion based.

Answer 14

More traditional methodologies. We are looking for additional or novel peaks that indicate a sequence change in the sample.

Validation of this type of assay is critical because you need to be sure that the novel peak that you see is the result of a mutation that is going to have an effect. If the gene happens to be polymorphic might just be an example of common polymorphism - really need to understand your test to use this!

• CE-SSCA (Single Strand Confirmation Analysis).
• dHPLC (High Performance Liquid Chromatography)
• DGGE (Denaturing Gradient Gel Electrophoresis)
• HRM (High Resolution Melt analysis)

Question 15

What technologies can be used to directly detect cancer mutations other than fusions?

Answer 15

• Sanger sequencing - can get some good sequencing from a FFPE tissue sample. Sensitivity is very important here - it is reported that only if there is at least 10-20% of tumour compared to your background germline DNA will you detect the tumour mutations. Not sensitive enough below this. Good reason to consider upstream enrichment.
• Pyrosequencing - can be very sensitive because you are looking at very short fragments.
• Some commercial kits allow you to interrogate lots of different mutations in say the EGFR gene all in one go
• Can also undertake allele specific analysis - Allele Specific Oligonucleotide probes allow for the detection of point mutations - can use Southern blotting or fluorescence.

Question 16

What are some issues that you should be aware of when sequencing solid tumours?

Answer 16

• You may get some sequencing artefacts when sequencing solid tumours.
• Have to balance that sample may have been in fixative which might have altered the DNA with the fact that they are cancer cells and the DNA may already be unusual/chaotic.
• May get mutations showing on one PCR that are not then replicated on a second or third repeat? May be a true artefact? May have very low tumour levels present and the PCR might have just happened to pick up some cells depending on when you took the samples?
• Sanger sequencing may not be the most sensitive assay to use for these cases - mut may not be visible on Sanger Sequencing but may be on CSCE (Confirmation Sensitive Capillary Electrophoresis).

Question 17

What can be the value of determining specific somatic mutations in tumours?

Answer 17

Prediction of tumour progression:
- will determine how we should approach treatment.

Prediction of reaction to drug treatment:

• can use mutation information to inform on drug an dosage choice.
• Important because some of the drugs are quite unpleasant and very expensive.

Monitoring disease:

• an integral part of patient management is, where possible, to monitor disease status. Genetic analysis predicts this.
• The presence of a mutation or chromosome complement at a particular level can highlight if a patient is relapsing.
• Important to identify the disease marker.
• the test must be sensitive and accurate enough to detect the earliest sign of disease return.
• For haematological disease monitoring is achieved through regular sampling of blood and/or bone marrow.
• Comparison of low level of mutation made between consecutive samples.

Question 18

What is a GIST and what common mutations do they have?

Answer 18

• A GIST is a gastrointestinal stromal tumour.

- Mutations are commonly found in the KIT and PDGFRa genes in GISTs.

Question 19

What do different GIST mutations tell us about the likely response of the tumour to certain drugs?

Answer 19

• Exon 11 KIT mutation = good response
• Exon 9 KIT mutation = poor response
• Exon 18 of PDGFRa at codon 842 = no response.

Question 20

What do different GIST mutations tell us about the likely progression of the tumour?

Answer 20

• The specific mutations in these genes can predict how the tumour is likely to progress:
• Exon 9 mutation in KIT = more aggressive.
• Deletion in exon 11 of KIT = more aggressive
• Duplication in exon 11 of KIT = less aggressive
• Mutation in exon 18 of PDGFRa = indolent

Question 21

What is one good resource for looking at what somatic mutations have been detected in tumours?

Answer 21

• COSMIC - the Catalogue of Somatic Mutations in human cancer.
• As soon as a mutation is identified it is shared openly.

Question 22

Give an example that demonstrates the role of molecular testing in defining responses and predicting outcome.

Answer 22

• CML demonstrates the role of molecular testing in defining responses and predicting outcome.
• ELN guidelines indicate treatment milestones and monitoring strategies.
• Identify the rate of decline in BCR/ABL1 transcripts on an international scale as predictive of subsequent response.

Question 23

Outline the response changes that are likely to be observed in a CML patient who is responding well to treatment.

Answer 23

1) . Complete Haematological Response (CHR) - 10% at 6 months.
2) . Complete Cytogenetic Response (CCR) - 1% at 12 months.
3) . Major Molecular Response (MMR) - 0.1% at 18 months.
4) . 4/5 log reduction
5) . Complete Molecular Response (CMR).

Question 24

How can molecular testing be used for BMT monitoring?

Answer 24

• For haematological conditions patients may undergo stem cell transplantations (BMT).
• Success of engraftment can be monitored by FISH (sex mismatched donor/host) or microsatellite analysis (sex matched donor/host).

Question 25

Outline how the pathology services fit together in the cancer patient pathway.

Answer 25

1) . Diagnostic imaging may reveal tumour.
2) . Histopathology can asses a tumour sample, look at the morphology and classify the type of cancer.
3) . May be some molecular tests that define what type of mutation may be present.
4) . Molecular analysis may inform what type of drug may be used.
5) . If drug is effective will see evidence of the tumour disappearing in the radiology.

Question 26

What ‘new’ technologies are available that may be considered for examining somatic mutations?

Answer 26

• 2nd and 3rd generation sequencing - targeted capture array.
• Digital PCR - Raindance.

Question 27

Outline the Raindance digital PCR method and how it may be useful in detecting somatic mutations.

Answer 27

Raindance technology:

• Useful when looking at potentially low levels of DNA from low levels of tumour cells.
• Production of millions of picolitre-volume droplets.
• Each droplet performs as an individual ‘test-tube’.
• Single DNA molecule contained within each droplet.
• PCR reaction performed using fluorescence.
• Droplets pass detector with ‘counts’ positive droplets.
• Scatter plot produced - high accuracy.

Question 28

How is the diagnosis of somatic mutations in tumours going to change our approach to cancer treatment?

Answer 28

Towards Stratified Medicine:

• We can look at the molecular profile of a patients tumour and use this to decide what kind of therapy that patient would benefit from most - i.e. ‘stratify’ treatment.
• Can give patients the treatments that are most likely to work for them - benefits patients and directs resources appropriately thus saving money.

Question 29

Outline Cancer Research UK’s Stratified Medicine Programme.

Answer 29

• Recognised that there was a lot of variability in the availability of stratified diagnosis and treatment for cancers in the UK.
• Project that aims to improved stratified diagnosis and treatment of cancers in the UK.
• CRUK will fund molecular diagnostics and collect a huge number of samples to process for a number of genes in a 2 year period to prove it can be done. Alongside this they are collecting the DNA from these patients.
• CRUK also working with a company called the technology strategy board who are working to develop assays that can be used to carry out these tests.
• CRUK technology hubs receive samples from clinical hubs. Patients are consented into the programme at clinical hubs where they will have a blood sample taken and they will have their tumour sent for profiling.