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CeGaT Adds Somatic Tumor Analysis, Develops PGx Panel, Adopts Illumina Platforms

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German genetic diagnostics company CeGaT has been growing rapidly over the last year to accommodate increasing demand for next-generation sequencing-based diagnostic tests in Germany and beyond.

The firm's portfolio of gene panels for inherited disorders has expanded to 14 disease areas, including hereditary cancer. The number of panels, many of which it developed in collaboration with academic partners, has nearly doubled to about 120. CeGaT also has started offering diagnostic exome sequencing, mainly of parent-child trios.

The Tübingen-based company, whose staff doubled to about 50 over the last year, also recently launched a somatic tumor panel that includes 552 genes and a tumor exome, and is in the midst of validating a pharmacogenetic panel with more than 340 genes.

Next summer, CeGaT plans to move into a new building near its current location, construction for which started in April. The new home will provide the company with about 40,000 square feet of lab and office space and will be able to accommodate at least 100 employees.

Funding for the recent expansion of the privately-owned company comes partly from cash flow from operations and partly from debt financing.

While CeGaT originally developed and ran its NGS-based diagnostic panels exclusively on Life Technologies' SOLiD platform – last year, the company had two SOLiD 4 and two SOLiD 5500 instruments – the company largely switched to Illumina sequencers this year and is currently equipped with two HiSeq 2500, one MiSeq, two SOLiD 5500, and one Ion Torrent PGM.

The main reason for this shift in technology was the increased throughput the HiSeq offers, as well as its ability to sequence overnight if needed. "In terms of throughput and read length, I think it's the best platform on the market in a diagnostic setting," said Saskia Biskup, the company's co-founder and CEO (CSN 5/2/2012).

While the company has found that neither the HiSeq nor the SOLiD capture all genetic variants, Biskup said it is too expensive to combine both platforms, which would be her preference. Since the HiSeq offers higher coverage and read length, it offers the best chance to detect mutations at the moment, she said.

The company still uses its SOLiDs in research projects for certain applications with protocols optimized for the platform, and has found that it generally requires lower coverage than Illumina to obtain the same accuracy. "We are very happy with the SOLiD still," Biskup said.

The MiSeq allows the company to sequence a single exome overnight in urgent cases, rather than having to wait to fill up a HiSeq run. In addition, CeGaT uses the MiSeq for resequencing, as well as to validate results from the SOLiD.

It uses the Ion Torrent PGM mainly as an orthogonal platform to validate SOLiD or Illumina results, for example potential mutations – up to several hundred − in tumor genes. The coverage obtained by the PGM is high enough, Biskup pointed out, to detect mosaicism within the tumor.

For inherited disease panels, which typically yield just a couple of mutations, the company still uses Sanger sequencing for validation.

Overall, the quality of sequencing platforms has improved over the last few years, Biskup said. "When we started with exomes, sequencing protocols were not really robust, machines were not really robust," she said. "Now, we bought the HiSeq and we could immediately run it in a diagnostic setting. That has changed − the technology in general, also the SOLiD, is now very robust. In the beginning, we had lots of problems with the machines, and we always thought we bought a green banana, but now, you can buy a machine and you can immediately run it."

Very long reads with high accuracy would still be an improvement, she said, and CeGaT is keeping an eye on new sequencing technologies. "This will dramatically increase the diagnostic yield, and I would immediately go with such a platform," Biskup said. "It has huge advantages if you think about repeat regions, homology regions, or if you want to see phases," or analyze cell-free DNA from maternal blood or from liquid tumor biopsies.

Gene Panels vs. Exomes

Last year, CeGaT ran about 2,000 diagnostic gene panels and currently receives about 50 panel orders per week. It has also completed about 250 diagnostic exomes so far.

"We see huge interest from clinicians in the diagnostic panels," Biskup said. "Clinicians recognize that panel sequencing, instead of single-gene sequencing, is the way to go."

While most of its business currently comes from Germany, international orders have picked up over the last few months, especially since the company and its collaborators published results involving several of its panels in peer-reviewed journals, most recently an epilepsy study in Nature Genetics.

The increase in sample volume, combined with increased automation of DNA isolation, enrichment, sequencing, and the analysis pipeline, have allowed the firm to drop its prices from €3,500 to €4,000 per panel to €2,500 to €3,000 per panel.

Panels get updated at least every two months, depending on how fast new relevant genes are published, though the company is able to update them within a week if necessary. CeGaT continues to use Agilent's SureSelect for gene enrichment.

According to Biskup, gene panels are in many cases still preferable over exomes for disease diagnosis because they cover the target DNA more completely. CeGaT performs diagnostic exome sequencing mostly in parent-child trios where the child has a likely genetic disease but no individual gene or gene panel can be prioritized.

"With an average coverage of more than 500, a diagnostic panel has a much better sequencing quality of the target genes and is, from a medical point of view, preferred over an exome," she said, which typically has a coverage of 100-fold or so.

Prior to developing two gene panels for mitochondrial diseases, for example – one including 38 genes from mitochondrial DNA, the other 175 genes from nuclear DNA – the company considered analyzing the exome for mitochondrial disease genes instead but found that several hundred DNA regions were insufficiently covered by the exome. "That's why we decided to develop this panel," Biskup said. The company developed the panel in collaboration with MitoNet, a German research consortium.

So far, the company's diagnostic panels are only covered by private health insurance but not by Germany's non-profit sickness funds, which cover about 90 percent of the population. This is likely to change next year, however. "I am very optimistic that in 2014, they are going to implement [coverage for next-gen sequencing]," Biskup said.

Based on the results of the diagnostic panels it has run so far, CeGaT has calculated the potential cost savings that would be achieved in several disease areas if single-gene tests were replaced by NGS panels. In addition to lower costs, it has found that the diagnostic yield would increase significantly. "The difference in pricing is one thing, and what is even more important, the percentages of cases we solve, it's so dramatically higher compared to conventional diagnostics that it's so obvious this is the only way to go," Biskup said.

Tumor Diagnostics, PGx

CeGaT, which also continues to offer a variety of next-gen sequencing services for research, has traditionally focused its diagnostics business on inherited diseases. Last month, it launched a somatic tumor panel as well as a tumor exome, which both run on the HiSeq 2500 and include the analysis of a tumor/normal sample from the same patient.

The somatic tumor panel comprises 552 genes known to be involved in cancer, which CeGaT selected after performing a comprehensive review of cancer panels currently on the market. While the tumor panel is sequenced with a coverage of about 500- to 1,000-fold, the tumor exome only has a coverage of 100- to 300-fold.

CeGaT also is currently developing a pharmacogenetic panel that includes more than 340 genes, which Biskup said has been difficult to establish because of the high homology between some PGx genes. The panel has passed all requirements for coverage and completeness so far, and the company is now testing it in 150 well-characterized liver samples, which will allow it to better understand phenotype-genotype correlations related to rare variants in certain genes. In addition to offering the PGx panel as a service, CeGaT plans to license it, or parts of it, out to pharmaceutical companies.

Longer term, the company is also interested in offering non-invasive prenatal testing, but Biskup said the competition in this area is high, and there is "huge pressure" for low prices. Once health insurance starts covering NIPT, she said, CeGaT would feel comfortable getting into this market, offering testing not only for trisomies but also for other rare diseases, for example those caused by microdeletions.

For NGS-based diagnostic tests to become more pervasive, both sequencing and analysis costs will need to come down further. "The bottleneck is the analysis," Biskup said. "The most challenging part is to judge the variants and to write the report. We have to automate that, then costs will go down, and once that happens, sequencing should be available to anyone." She noted, however, that writing the medical report "cannot and should not be fully automated."

Right now, CeGaT employs two human geneticists to evaluate results, and collaborates with a number of external clinicians who helped develop the panels, who get together on a weekly basis to discuss cases before CeGaT files its medical reports.

"My vision is that once you are sick and go to the hospital, sequencing is one of the first things happening to you, like getting an ultrasound or blood testing," Biskup said.

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