ctDNA Testing

Question 1

What DNA and RNA is in the blood from cell signalling?

Answer 1

Exosome DNA and RNA. Very small portion of circulating nucleic acid

Question 2

How long is cell free DNA and why?

Answer 2

~180bp as it’s still nucleosome bound

Question 3

What’s the half-life of cfDNA?

Answer 3

10-15 minutes

Question 4

How much DNA is on a nucleosome

Answer 4

147bp + linker.

Question 5

cfDNA varies between….

Answer 5

People, time, tumour type

Question 6

Why can tumour DNA be higher MW?

Answer 6

It can be 180bp from apoptosis, but 10kb from necrosis

Question 7

ctDNA can be up to what percentage of cfDNA?

Answer 7

Up to 10%

Question 8

ctDNA has a longer half life than other cfDNA, what is the half life?

Answer 8

About 114 minutes

Question 9

What can you never be sure of with ctDNA assay?

Answer 9

Whether you’ve picked up any ctDNA

Question 10

Whats the issue with solid biopsies?

Answer 10

Need skill to get sample, its invasive, patient needs to recover, patients are poorly as it is, sample is limited and can be depleted

Question 11

How can ctDNA assay represent a tumour better?

Answer 11

If its heterogenous then it doesn’t matter what sample you take, it will be proportional

Question 12

Why else is ctDNA good?

Answer 12

Non invasive, can do serially, rapid, no pathology assessment needed

Question 13

What are the negatives of ctDNA?

Answer 13

Need a highly sensitive test, it’s not diagnostic? False negative risks. Variable ctDNA load. Sample handling problems.

Question 14

ctDNA in plasma is unstable, so what do you do?

Answer 14

Separate plasma quickly, store at -80oC.

Question 15

How are streck tubes useful?

Answer 15

They stabilise the lymphocyte membranes to protect the DNA so they don’t break open and dilute the ctDNA

Question 16

NGS is sensitive to 2% VAF, what percent do we need for a ctDNA assay?

Answer 16

0.01-10%

Question 17

What sensitive non NGS test can you use for ctDNA analysis?

Answer 17

ddPCR

Question 18

What is the Cobas test for and what type of test?

Answer 18

RT-PCR test for 42 common EGFR mutations. Sensitive to 1% MAF. SQI gives indication of ctDNA load.

Question 19

What does a ddPCR assay do with ctDNA?

Answer 19

Single molecule PCR reactions, 20,000 droplets. Sensitive to 0.1% MAF. Algorithm corrects for droplets with more than 1 DNA molecule in there.

Question 20

What assay is similar to ddPCR using beads and single molecules instead of droplets?

Answer 20

BEAMing and Digital PCR

Question 21

What do UMIs do in NGS?

Answer 21

They increase the sensitivity of NGS assays by telling you which molecules were originally there and which are there because they’ve been amplified. Good for confirming low level variants.

Question 22

Why has ctDNA been applied to Lung cancer specifically?

Answer 22

It’s typically diagnosed late so needs improvements. Patients are very unwell so solid biopsies are tough.
LC does shed good amount of ctDNA

Question 23

What’s the false negative rates of ctDNA with lung cancer patients? And why isn’t it too bad.

Answer 23

10%. Which is not awful, it’s preferred over false positives then giving an EGFR TKI when they don’t need it. A false negative with default treatment isn’t too bad.

Question 24

What mutation in EGFR confers TKI resistance?

Answer 24

T790M

Question 25

Some false positives can come from FFPE samples why?

Answer 25

Tendency to C to T transitions, for some reason

Question 26

In terms of monitoring disease, ctDNA was good for…

Answer 26

metastatic melanoma…

Question 27

The Galleri trial is doing what?

Answer 27

Multi-cancer detection via early screens. Methylation profiling is used to work out where it came from.

Question 28

Cerebral spinal fluid is also a liquid biopsy for…

Answer 28

Brain tumours and brain metastases

Question 29

What are the ethical considerations of ctDNA tests?

Answer 29

Medically anxious population from private testing.
Large downstream impact, more counselling for more diagnoses.
Some oncogenic variants are necessary but not sufficient.
How do we capture many oncogenic drivers with good sensitivity? Which panel and which hotspots do we use?