Sensitive detection of ctDNA

The business of liquid biopsy is gaining momentum as more companies strive to offer services to characterize mutations found in cell-free circulating tumour DNA (ctDNA). ctDNA are small DNA fragments that are released from dying tumour cells or present in exosomes (secretory vesicles) found circulating in the bloodstream and other bodily fluids such as urine or saliva. Using ctDNA, one can characterize certain gene-specific mutations that have a tremendous impact on treatment response, dictating personalized therapy. Monitoring mutation rates in ctDNA also serves as a potential biomarker to correlate with tumour load that can change in response to treatment. Obtaining blood is less invasive than performing tumour biopsies and the market for so-called liquid biopsies is estimated to exceed $10 billion as it can be applied to many different cancer types.

Some challenges remaining however is the sensitivity and robust detection of ctDNAs since they make up <1% of circulating DNA found in blood. Some of the currently available methods are described:

1. Digital PCR

Digital PCR is a new form of PCR that does not require reference standards. It does this by splitting up PCR reactions into several thousands of units, some of which do not contain the transcript of interest. By counting the number of positive PCR reactions, it allows for direct quantitation of the number of transcripts to begin with. The high partition rate  provides more data points leading to greater sensitivity and accuracy. Bio-Rad’s droplet digital PCR has its individual PCR units present in tiny water droplets within an oil emulsion and was successfully used to track tumour load by detecting BRAF mutations found in ctDNA.

2. Competitive allele-specific PCR (CastPCR)

CastPCR works by using mutation-specific primers to amplify only mutated alleles. At the same time, blocking primers are also used to block amplification of wildtype alleles. It works based on Taq polymerase ability to recognize mismatches at the 3′ end of the primer, proceeding with amplification only if the sequence is fully matched. Sensitivity of this detection method however is reportedley lower than digital PCR or BEAMing.

3. BEAMing

The BEAM in BEAMing stands for beads, emulsion, amplification and magnetics and relies on just those components for detecting low mutation rates. Basically both wildtype and mutation-carrying alleles are amplified by PCR, following which they are exposed to magnetic beads coated with gene-specific primers. These primers bind to the amplified DNA and get extended. The beads are then magnetically separated with the attached sequences and subject to fluorescent probes that discriminate between wildtype or mutant sequences. These are then monitored and quantified by flow cytometry. Sounds complicated but gives similar sensitivities to digital PCR.

4. PNA clamp PCR

Similar to CastPCR, PNA is a synthetically modified DNA that binds with stronger affinity to DNA due to its lack of phosphates. They therefore bind only perfectly matched sequences and are not recognized by polymerases hence cannot be amplified. PNA therefore acts as a clamp, preventing wildtype alleles from being amplified.

5. High resolution melting analysis

HRM relies on the binding of DNA dyes to double stranded DNA and giving off fluorescence which is significantly diminished when DNA is single-stranded. The binding of the dye would therefore vary as DNA strands separate at their melting temperatures. This fluorescence therefore acts as a readout to DNA melting properties which is determined by the length, GC content and sequence, and would differ  when a mutation is present.

6. Next generation sequencing

This is probably the more costly option but does not restrict one to known mutations.

As the field grows, one can expect larger trials using these detection methods. However small trials already demonstrate the utility of ctDNA more as biomarkers than for early cancer detection. FDA approval is currently only limited to measuring circulating tumour cells using CellSearch Technology by Jannsen Pharmaceuticals, however this has given different results to analysing ctDNA, with some patients having ctDNA not necessarily having detectable CTCs. More work is also needed to delineate just how and why these ctDNAs are released and how representative they are of actual cancer status. But this is not stopping the vast investment in the field with even Illumina getting into the mix.



Interesting articles from around the world for your reading pleasure:

1. Illumina’s plans on world domination

Illumina is extending its monopoly in the DNA sequencing field by suing several of its competitors including Oxford Nanopore Technologies. The latter developed an intriguing way of sequencing DNA by measuring current fluctuations as the bases (with no limit on read length) are guided through the pore by an enzyme (watch the video here). However Illumina claims ONT infringed on two exclusively licensed patents from the University of Washington with regard to the use of Mycobacterium smegmatis porinA (MspA). Illumina has also been teaming up with several diagnostic/personal consumer companies in an effort extend its genetic sequencing and interpretation capabilities to a much larger market of general consumers as opposed to research groups.

2. 3D-print your own lab equipment

It’s an interesting idea and with large potential considering how expensive lab equipment is. Furthermore the idea of everything being open-sourced allows for greater modification and improvement by the world-wide scientific community. There are currently available 3D printing designs for micropipettes, thermocyclers, centrifuges, microscopes (including two-photon), hotplates, stirrers, micromanipulators, syringe pumps and more!

3. The history of PCR

GEN did a nice article on the history of PCR and how the idea was first rejected by 15 journals!

4. Singapore’s S$19 billion five-year research budget

Singapore’s putting in a whole lot of money into developing its life science, energy, manufacturing and digital technology fields. Be interesting to see how things develop after 5-10 years!

5. Another failed Alzheimer’s drug may get a second chance

Alzheon’s drug tramiprosate which failed Phase III trials previously may get another chance after Alzheon’s re-examination of the trial data revealed significantly better responses in Apo E4 homozygous patients.

6. FDA approves genetically modified mosquitoes to halt the spread of Zika virus

Interesting article highlighting the approach to use genetically modified male Aedes Aegypti mosquitoes to mate with females producing offspring that die early. The mosquitoes are said to have limited hazardous effects to the environment as the males do not bite other animals and only survive for two days. A pilot trial in a city in San Paulo, Brazil, already proved this method was effective at reducing the Aedes aegypti population by 82%. If found effective, I bet the chances of this being applied to the control of dengue would be pretty high.

7. Finally for some stunning images

Some cool sciency images of subjects captured under the microscope in the Nikon Small World Photomicrography Competition.