ctDNA Flashcards
What is ctDNA?
Circulating tumour DNA
Represents a proportion of the total cfDNA in plasma
How does ctDNA differ from other cfDNA?
Size distribution, more high molecular weight DNA
Apoptosis = 180bp
Necrosis = >10kb
What are the advantages of testing ctDNA?
- What is seen in the blood has been shed by the tumour recently and so should be representative of the current tumour genotype
- Non-invasive - easier sampling than biopsy, better for patient
- Can overcome tumour heterogeneity - representative of entire tumour
What are the challenges associated with testing ctDNA?
- Different likelihood of detectable ctDNA in different tumour types
- High variability in conc of ctDNA within all tumour types
- Highly sensitive assay needed = expensive
- Not diagnostic
- False negative risk
- Samples need to be processed within 6 hrs or specialised collection tubes used
How to qPCR, ddPCR and BEAMing compare for detecting ctDNA mutations?
Sensitivity:
qPCR = 1% LOD
ddPCR = 0.1% LOD
BEAMing = 0.05% LOD
What is BEAMing?
Beads, emulsion, amplification, magnetics
Clonal amplification method
More sensitive than ddPCR
How can NGS be modified to make it more sensitive?
Use of UMIs - reduce the rate of false-positive variant calls and increase sensitivity of variant detection
Accurately filter out duplicate reads and PCR error
Distinguish true variants from errors introduced by library prep/sequencing
<1% LOD achievable
