ctDNA

Question 1

What is ctDNA?

Answer 1

Circulating tumour DNA

Represents a proportion of the total cfDNA in plasma

Question 2

How does ctDNA differ from other cfDNA?

Answer 2

Size distribution, more high molecular weight DNA

Apoptosis = 180bp
Necrosis = >10kb

Question 3

What are the advantages of testing ctDNA?

Answer 3

1. What is seen in the blood has been shed by the tumour recently and so should be representative of the current tumour genotype
2. Non-invasive - easier sampling than biopsy, better for patient
3. Can overcome tumour heterogeneity - representative of entire tumour

Question 4

What are the challenges associated with testing ctDNA?

Answer 4

1. Different likelihood of detectable ctDNA in different tumour types
2. High variability in conc of ctDNA within all tumour types
3. Highly sensitive assay needed = expensive
4. Not diagnostic
5. False negative risk
6. Samples need to be processed within 6 hrs or specialised collection tubes used

Question 5

How to qPCR, ddPCR and BEAMing compare for detecting ctDNA mutations?

Answer 5

Sensitivity:
qPCR = 1% LOD
ddPCR = 0.1% LOD
BEAMing = 0.05% LOD

Question 6

What is BEAMing?

Answer 6

Beads, emulsion, amplification, magnetics

Clonal amplification method

More sensitive than ddPCR

Question 7

How can NGS be modified to make it more sensitive?

Answer 7

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