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Dutch Startup and Venture Challenge alumnus Cyclomics Developing Liquid Biopsy Assay Using Nanopore Sequencing

Dutch startup Cyclomics is developing a liquid biopsy test for tumor recurrence and therapy response monitoring that it says will be the first to use nanopore sequencing technology.

The company, a 2018 spinout from University Medical Center Utrecht, where it is currently incubated, has developed a technology that combines rolling circle amplification of target genes with sequencing on Oxford Nanopore’s MinIon sequencer.

The first application of the test, called CyclomicsSeq, is for the detection of TP53 mutations in patients with advanced head and neck cancer, both for treatment response monitoring and for recurrence detection. The company and its collaborators are currently testing this assay in a small prospective clinical trial.

Cyclomics was founded by Jeroen de Ridder, the firm’s CEO and an associate professor at the Center for Molecular Medicine at UMC Utrecht; Wigard Kloosterman, the company’s CSO and formerly an associate professor in the Department of Genetics at UMC Utrecht, who recently joined cancer vaccine firm Frame Cancer Therapeutics; and Alessio Marcozzi, a former postdoc in Kloosterman’s lab and currently principal scientist and the sole full-time employee at Cyclomics.

The cofounders had been working with Oxford Nanopore’s technology for a while — for example, in 2016, a team led by Kloosterman demonstrated the ability to identify disease-causing structural variants in the human genome using MinIon whole-genome sequencing data — and were thinking about ways of using the long-read sequencing technology in a diagnostic setting.

They came up with a protocol to target and circularize specific ctDNA fragments, which are usually about 150 bp in size, and to copy them by rolling circle amplification. This results in long DNA concatemers with multiple copies of the original fragment, which are then read by the MinIon nanopore sequencer. Using software to compare the sequences from the copies, the researchers can distinguish between sequencing errors and genuine mutations and reduce the overall error rate.

According to Marcozzi, aspects of the entire process — including how the DNA is circularized as well as details of the adapters and enzymes used — are patent-pending.

The technology could be further improved by better basecalling, for example from the raw nanopore signal, de Ridder said, which could boost accuracy and make the test more sensitive to pick up low-frequency mutations. Right now, for most mutations in the TP53 gene, for example, the false-positive rate is less than 1:1,000, which he would like to bring down to 1:10,000 or so.

The approach has several advantages over existing liquid biopsy assays for cancer mutation detection, Marcozzi said, which rely on either PCR or deep short-read sequencing.

While PCR-based tests are exquisitely sensitive, they only pick up a few predetermined mutations and require prior knowledge of these, he said, whereas sequencing-based approaches can pick up all mutations in a gene of interest. It is still helpful to know what mutations to look for, de Ridder said, but with a sequencing approach, there is no need to design a patient-specific assay.

The advantage of using a MinIon sequencer over a short-read sequencer, he said, is that the capital cost of the device is “essentially zero,” making it possible to install the test in many settings at the point of care.

While the concept of generating DNA circles from genomic targets and amplifying them through rolling circle amplification is not novel, the combination with long-read nanopore sequencing is, Marcozzi said.

PerkinElmer’s Vanadis technology, for example – which PE is currently commercializing for noninvasive prenatal testing but plans to use for cancer applications in the future – also captures cell-free DNA fragments and converts them into DNA circles, which are amplified by rolling circle replication, but reads them out using fluorescent probes instead of sequencing.

Cyclomics’ first assay analyzes the TP53 gene, which is mutated in almost 40 percent of cancers, Marcozzi said. Specifically, the company is focusing on head and neck cancer, where there is a large clinical need for better recurrence testing since radiological imaging, the current standard, is not very sensitive. About 90 percent of head and neck cancer patients carry mutations in TP53, and about half of them have their tumor recur.

In partnership with collaborators in the pathology department at UMC Utrecht, Cyclomics is currently running a clinical trial that aims to recruit 30 patients with advanced head and neck cancer undergoing treatment and 20 being monitored for recurrence. Patients under treatment receive daily doses of radiation and several rounds of chemotherapy. Along with weekly MRI scans, they get blood draws for testing with CyclomicsSeq.

The study has recruited six patients so far and will likely take a year and a half to complete. At the European Society of Human Genetics annual meeting last month, de Ridder showed early results from two patients, which he said were “extremely promising,” though it is too early to determine the sensitivity and specificity of the test.

In one of the patients, the first few MRI scans looked no different, but the blood test showed an elevated mutational burden right after treatment started, which came down after a few weeks and then increased again. This, he said, “would indicate that you get a much more fine-grained picture of the response to treatment using a blood-based test and that we can indeed achieve this with the CyclomicsSeq approach.”

The total cost of the assay is about €1,400 ($1,560) at the moment, Marcozzi said, €900 of which is related to sequencing. However, switching to the Flongle flow cell adapter, which Oxford Nanopore launched earlier this yearand which comes with small disposable flow cells, could bring sequencing costs down to about €200.

While the firm’s initial focus is on head and neck cancer, its platform can be adapted to different genes and cancer types. Its next assay will be an EGFR test, and it is also working on a genome-wide test. “Head and neck cancer is just a stepping stone for us,” de Ridder said. “Obviously, the big cancer types, in particular those for which it has been demonstrated that DNA is shed into the bloodstream, for example lung cancer, are very interesting applications for us.”

Following the completion of its first studies, the company anticipates launching its first CE-marked test for the clinical market. In the meantime, it plans to commercialize research-use-only tests for specific genes. “We have already received a number of requests and if we think they are interesting, we can definitely set up a collaboration and deliver these research-only kits, depending a bit on our capacity to produce them,” de Ridder said. In addition, it might launch a generic kit that customers can use to design their own test.

Meanwhile, Cyclomics is looking for additional funding. The firm won €25,000 from the Health~Holland Venture Challenge program in 2017 and a €50,000 SME Instrument grant from the European Commission this year, and has a small amount of private investments. In addition, the Dutch Organization for Scientific Research awarded Cyclomics and UMC Utrecht €50,000 for technical development and research and the Oncode Institute provided the two partners with a €250,000 grant for clinical testing of the technology last year.

“The next step for us is to secure some more funds exclusively for the consolidation of the company and to bring CyclomicsSeq into the research and clinical markets,” Marcozzi said. Specifically, the firm is applying to the European Union’s Eurostars program, which is partly funded by the Horizon 2020 program and could provide it with €300,000 to €500,000. Additional funding might also come from the Oncode Institute, he added.