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Cergentis Aims to Introduce TLA Kits for Cancer Companion Dx Development

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NEW YORK (GenomeWeb) – Cergentis, a Dutch technology company, is developing a new protocol for its target locus amplification (TLA) technology that for the first time will allow users to work with solid tumor biopsies.

The European Commission recently awarded Cergentis a €1.2 million grant (about $1.4 million) to support the development of its TLA technology for use in formalin-fixed, paraffin-embedded samples. The company aims to have the new kits on the market by 2020, according to CEO Max van Min.

Cergentis claims that TLA enables the targeted sequencing of complete genes, and can therefore elucidate structural changes of interest, such as gene fusions, that are missed by other techniques. To date, TLA has relied on cells and genomic DNA as a sample input, and most of the company's customers have been pharmaceutical companies that use TLA to study leukemia, for instance.

By developing a protocol that works specifically with FFPE, Cergentis hopes to also be able to reach other customers, such as pathology laboratories, that work with solid tumors. The company believes the results generated with its approach could support the development of various companion diagnostics.

"The further development of the FFPE protocol and the protocol for tumor biopsies changes the game entirely," said van Min of the company's ambitions.

Founded in Utrecht in 2012, Cergentis rolled out its target amplification method as a service the following year. In 2014, the company expanded the offering by marketing kits containing reagents for carrying out TLA in cells and genomic DNA. It also automated TLA on PerkinElmer's Janus NGS Express Workstation as well as its Sciclone G3 NGS and NGSx workstations.

TLA works by crosslinking DNA that is physically close to a gene or region of interest. The DNA is then digested with a frequently cutting restriction enzyme, and the fragments are ligated into large circles. After removing the crosslinks, the DNA is converted into smaller circles. Circles containing the gene of interest are then amplified with PCR, and the amplification products are sequenced. Target sequences are subsequently reconstructed from the reads.

Since its launch, Cergentis' technology has been adopted particularly among pharmaceutical companies, which van Min described as the company's "main customer base."

"We have been very successful in commercially introducing the protocols that will now work for the vast majority of pharma companies in cell line development, cell line quality control, animal models, and quality control for CRISPR engineering," said van Min.

Much of the work to date has been done on leukemia and similar cancers. The company together with partners at various European hospitals, including University Medical Center Rotterdam in the Netherlands and University Children's Hospital Zurich in Switzerland, has published a white paper discussing the application of TLA in minimal residual disease testing in acute and chronic leukemia, lymphoma, and myeloma patients.

Customers at the University Medical Center Groningen in the Netherlands described the use of TLA to detect translocations in acute leukemias in a Clinical Chemistry paper last month.

By expanding the application of TLA into FFPE material, Cergentis will be able offer users the opportunity to use its technology to analyze samples from solid tumors, supporting "complete gene sequencing," as the firm states in the grant's abstract, as well as breakpoint detection for use in developing minimal residual disease and circulating tumor DNA tests.

"The excitement about the opportunity that TLA presents in FFPE material is driven by the fact that the TLA protocol uses the physical proximity of sequencing in selection," noted van Min. "The consequence of that is that we are able to detect all point mutations and structural changes in any gene of interest."

Van Min added that the "fragmented nature" of DNA obtained from FFPE samples means that it has been challenging in the past for scientists to detect all categories of clinically relevant genetic alterations using other techniques. Though the DNA sequences obtained from FFPE will be "very small," van Min said they will still be cross-linked to each other, meaning that the proximity iteration of the TLA technology will be maintained.

"We will still able to use the TLA protocol to generate complete sequence information across any gene of interest and detect again all point mutations and all structural changes sensitively," he said.

"This is a real and very important sweet spot of the technology," van Min added. "It promises to enable the similar work that we are doing in leukemia at a much broader scale, and in essence for all cancer types where companion diagnostics are relevant."

Cergentis was awarded its new Horizon 2020 grant last month. The project, which is called "Targeted complete next-generation sequencing for companion diagnostics and personalized treatment of cancer," or SEQURE, is slated to run through April 2020.

Though van Min did not name any partners for the project, the company has said it will work with "leading Dutch and European cancer research institutes;" reagent, automation, and software suppliers; and pharma companies.

"We will work closely with various reagent suppliers in the NGS industry, which will help us improve the protocol itself," said van Min. He said the firm will also work together with bioinformatics companies to develop tools to detect all genetic alterations once the sequencing data has been obtained. By partnering with pathology labs and universities, Cergentis aims to generate "clinically relevant info that is used to assess its relevance in the patients being treated."

Cergentis also believes that a more flexible TLA protocol can be used in diagnostics discovery and internal research programs at companies and research institutes.

"We see an important role for the technology in the discovery of genetic alterations that will support customers in the further analysis of genes they consider to be relevant," said van Min.

Information obtained using TLA could then be migrated into companion diagnostics according to indication. Van Min specifically said that the ability of TLA to enable the detection of breakpoint sequences in the gene fusions that occur in cancers would support the development of gene fusion-specific quantitative PCR panels to quantify circulating tumor DNA in patient blood.

"That would allow us to assess how well a patient is responding to therapy, and potentially whether a patient is cured," van Min said.

In terms of competitive technologies, van Min made clear that fluorescence in situ hybridization is the approach that much of its customer base has been using to answer similar questions. Exome capture kits are also viewed as a competitive technology.

"FISH is … a complex method that is for detecting the presence or absence of gene fusions," said van Min, "while the current exome-based capture approaches will allow you to detect point mutations in the exomes, but they also miss any genetic variation that occurs in the rest of the gene and they will miss structural changes.

"Cancer is a complex disease, and the alterations that occur will be different for each patient," van Min said. "The potential of our technology to detect structural changes is a game changer."

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