Scientists on the ONCOTECT project have found new ways to detect tumours in the blood. “DNA floats into the bloodstream and comes from all the cells of the body. Part of the DNA will also come from the tumour,” explains Alessio Marcozzi, research director at Cyclomics BV, in the Netherlands, a spin-off company from the University Medical Centre Utrecht. “The amount of circulating tumour DNA (ctDNA) in the blood, and the mutation content, are important biomarkers for tumour recurrence and response to cancer treatment,” he says. “We worked on how to collect specific mutant molecules from the bloodstream and then applied a series of biomolecular techniques to make this short molecule compatible with a DNA sequencing device such as those produced by Oxford Nanopore,” Marcozzi says, referring to the British company that specialises in the electronic DNA analysis of single molecules. “Nanopore sequencing is especially good for long molecules. But the quality of the sequencing is not very high – there’s a 5-6 % error,” he notes. “So, we start with a small molecule of DNA, we make a circle out of it and then, with molecular biology, we generate a long molecule in which the small molecule template is copied multiple times,” he explains. The Oxford Nanopore system is used to sequence this long DNA molecule. “Since the initial short DNA template is copied over and over again, any error can be cancelled out using bioinformatics. This allows very accurate detection of DNA mutations from the blood sample,” Marcozzi says.
Head and neck tumours
"We reached proof-of-principle before receiving the EU grant and we knew it was working, but we didn’t have any way of selectively picking up particular genes that are of interest in certain cancers,” Marcozzi explains. The system was tested in the laboratory on head and neck tumours which can also affect the throat and tongue. Head and neck cancers are among the top five most prevalent cancers in Europe. “We know that 90 % of a subgroup of these cancers contain a mutation in the TP53 gene. We were able to use the EU funding to tweak our technology to be able to specifically extract the information from this gene in the blood.” CT or MRI procedures, the normal way to detect such tumours, can be 'messy’ because of the presence of many different types of tissue, scars, inflammations and lymph nodes in the neck, Marcozzi says: “Unless there is a big tumour it’s quite hard to detect and with a big tumour it’s already very late for treatment.” “A CT or MRI scan cannot be done so often, so there is a real clinical need for a simple test as offered by Cyclomics.”
There were some practical challenges to reduce the time to get a result, Marcozzi explains: “We had to try different buffers, different enzymes and different components from various vendors and tweak every little detail of this long protocol to get a better result. The whole process was quite laborious and labour-intensive but we nailed it and we now have an easy and reliable test ready to hit the market.” The project is about to launch the first research-only prototype kit for use in clinical studies. “The kit is a collection of reagents that are needed to do the molecular biology in the lab, plus access the software,” Marcozzi says.
ONCOTECT, Cyclomics, nanopore, Oxford Nanopore, DNA sequencing, DNA, tumours, head and neck cancer, biomarkers