Screening for somatic Mutations

Question 1

Most cancers are the result of what sort of mutation in what sort of cells?

Answer 1

Acquired mutations in somatic cells

Question 2

What are some technical considerations when screening for somatic mutations?

Answer 2

• Access to suitable tumour material
• Quality of DNA/RNA
• Tumour enrichment (due to background of germline sequence)
• Sensitivity of assay (% of tumour detected)
• Scope of assay (single/multiple mutations/genes)
• Reliability (will you always detect it at low level?)
• Access to relevant equipment/trained staff

Question 3

What influences the quality of the DNA in context of FFPE tissue?

Answer 3

Length of time in fixative:

• No fixation = intact DNA
• Limited Formaldehyde fixation = fragmented DNA
• Prolonged formaldehyde fixation = fragmented and cross-linked DNA

Question 4

Provide some info on tumour enrichment process in context of somatic mutation screening

Answer 4

• For solid tumours this is achieved through micro-dissection
• HandE staining of tissue sections on a slide
• Identify areas of tumour vs areas of normal tissue
• Scrape unwanted material from slide prior to extraction

Question 5

What techniques can be used for mutation enrichment?

Answer 5

• COLD-PCR (co-amplification at lower denaturation temp)
• PCR cloning
• ME-PCR (mutant enriched PCR; enzymatic digestion of WT gDNA at known mutation sites)

Question 6

What are some of the molecular markers in routine clinical use for solid tumours?

Answer 6

• KRAS
• BRAF
• EGFR
• HER2
• KIT/PDGFRa
• Sarcoma fusions

Question 7

Provide details on real time PCR in context of gene fusion detection

Answer 7

• Performed on cDNA with Primers located either side of common breakpoint
• Fluorogenic taqman probe meaning a Fluorescent signal generated in real time as PCR reaction proceeds
• Amount of fluorescence directly proportional to starting template
• high sensitivity

Question 8

What determines the sensitivity of the real time PCR assay in context of gene fusion detection?

Answer 8

• Control gene copy number (e.g. normal ABL)
• Sensitivity is crucial to ensure accurate negative result
• Aim for 1000 ABL transcripts per reaction and the result is expressed as a ratio of BCR-ABL to normal ABL

Question 9

What are some of the major issue associated with use of Sanger sequencing for somatic mutation screening?

Answer 9

• sensitivity: 10-20% of tumour present you may struggle to detect it (one reason why enrichment may be needed prior to sequencing)
• risk of artefacts

Question 10

Patients with GIST tumours tend to have mutations in either the KIT or PDGFRa genes. Provide some details on how the results are interpreted depending on the location of the mutation in the context of both tumour progression and response to treatment

Answer 10

• KIT exon 9 = more aggressive (poor response to drug: needs higher dose)
• Deletion in KIT exon 11 = more aggressive
• Duplication in KIT exon 11 = less aggressive
• Changes in exon 11 have good therapy response
• Mutation in PDGFRa exon 18 = indolent
• No response to treatment at codon 842 of ex18