ctDNA Testing Flashcards
What DNA and RNA is in the blood from cell signalling?
Exosome DNA and RNA. Very small portion of circulating nucleic acid
How long is cell free DNA and why?
~180bp as it’s still nucleosome bound
What’s the half-life of cfDNA?
10-15 minutes
How much DNA is on a nucleosome
147bp + linker.
cfDNA varies between….
People, time, tumour type
Why can tumour DNA be higher MW?
It can be 180bp from apoptosis, but 10kb from necrosis
ctDNA can be up to what percentage of cfDNA?
Up to 10%
ctDNA has a longer half life than other cfDNA, what is the half life?
About 114 minutes
What can you never be sure of with ctDNA assay?
Whether you’ve picked up any ctDNA
Whats the issue with solid biopsies?
Need skill to get sample, its invasive, patient needs to recover, patients are poorly as it is, sample is limited and can be depleted
How can ctDNA assay represent a tumour better?
If its heterogenous then it doesn’t matter what sample you take, it will be proportional
Why else is ctDNA good?
Non invasive, can do serially, rapid, no pathology assessment needed
What are the negatives of ctDNA?
Need a highly sensitive test, it’s not diagnostic? False negative risks. Variable ctDNA load. Sample handling problems.
ctDNA in plasma is unstable, so what do you do?
Separate plasma quickly, store at -80oC.
How are streck tubes useful?
They stabilise the lymphocyte membranes to protect the DNA so they don’t break open and dilute the ctDNA
NGS is sensitive to 2% VAF, what percent do we need for a ctDNA assay?
0.01-10%
What sensitive non NGS test can you use for ctDNA analysis?
ddPCR
What is the Cobas test for and what type of test?
RT-PCR test for 42 common EGFR mutations. Sensitive to 1% MAF. SQI gives indication of ctDNA load.
What does a ddPCR assay do with ctDNA?
Single molecule PCR reactions, 20,000 droplets. Sensitive to 0.1% MAF. Algorithm corrects for droplets with more than 1 DNA molecule in there.
What assay is similar to ddPCR using beads and single molecules instead of droplets?
BEAMing and Digital PCR
What do UMIs do in NGS?
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.
Why has ctDNA been applied to Lung cancer specifically?
It’s typically diagnosed late so needs improvements. Patients are very unwell so solid biopsies are tough.
LC does shed good amount of ctDNA
What’s the false negative rates of ctDNA with lung cancer patients? And why isn’t it too bad.
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.
What mutation in EGFR confers TKI resistance?
T790M
