Aiming to gain a foothold in Europe's emerging next-gen sequencing-based diagnostic market, Multiplicom of Belgium is offering multiplex PCR amplification kits for a range of inherited cancers and other genetic disorders.
Starting in September, the company, a spin-off from the University of Antwerp and VIB, a non-profit life sciences research institute, will offer its first CE/IVD-marked kit, the BRCA MASTR Dx assay for multiplex amplification of the BRCA 1 and 2 exons for next-gen sequencing, to genetic testing labs in Europe.
Initially, the diagnostic kit will be available for use with the Roche 454 platform, on which it was validated, but the company plans to offer versions for the Illumina MiSeq and the Ion Torrent PGM in the future. The kit is already available as a research-use-only version, which is compatible with all existing next-gen sequencing platforms.
Targeted amplification products for other indications, including genetic disorders and tumor analysis, will follow. In addition, the company is working on a non-invasive prenatal diagnostic test for chromosomal aneuploidies.
Multiplicom was founded last year to commercialize multiplex PCR amplification technology developed by the team of Jurgen Del-Favero, a researcher at the VIB Department of Molecular Genetics and a professor at the University of Antwerp. Del-Favero also serves as the firm's chief technology officer.
The company, which has about 15 employees, has a strategic partnership with the VIB Nucleomics core facility for its R&D work, which is equipped with a GS Junior, a MiSeq, and a PGM.
In April of last year, Multiplicom raised €2 million ($2.5 million) in a Series A round to develop and obtain CE-marking for diagnostic tests and to build a sales network in the European Union. €1.5 million of the funding came from venture capital firm Gimv and Gimv-managed Biotechfonds Vlaanderen, and €500,000 from VIB and the University of Antwerp.
While the current funding would allow the company to reach break-even in about two years, Multiplicom is considering raising another €5 million to be able to accelerate its test development program.
The company's Multiplexer technology, which currently allows users to pool between 10 and 200 primer sets per reaction, is based on a proprietary software algorithm to design multiplex PCR reactions in silico. The company decided not to patent the method but to keep it as a trade secret instead.
In contrast to enrichment methods, for example in-solution capture, multiplex PCR only delivers the desired target sequences and no unrelated sequences. What also sets it apart from other approaches is that every single target gets amplified. "If you want to do diagnostics on certain genes, it's important that you cover all the sequences," Del-Favero told Clinical Sequencing News at the European Society of Human Genetics annual meeting in Nuremberg, Germany, last week.
For example, when he and his colleagues amplified the BRCA 1 and 2 genes using Life Tech's AmpliSeq method, they missed 5 percent of the exon sequences, whereas their own approach amplified 100 percent of the exons.
For the last year or so, Multiplicom has been selling research-use-only Multiplex Amplification of Specific Targets for Re-Sequencing, or MASTR, kits for a range of genetic disorders: BRCA MASTR v2, covering the BRCA 1 and 2 genes; CFTR MASTR for cystic fibrosis, covering the CFTR gene; HNPCC MASTR for colorectal and endometrial cancer, covering MHL1, MSH2, parts of MSH6, PMS2, and the 3'UTR of EPCAM; FAP MASTR for familial adenomatous polyposis, covering APC and MUTYH; and ALPORT MASTR for hereditary nephritis, covering COL4A3, COL4A4, and COL4A5. In addition, it has been offering an EGFR kit, covering EGFR exons 18-21, for somatic tumor analysis.
The research assays are compatible with any existing next-gen sequencing platform and involve a two-step protocol. In the first step, the targets are amplified by multiplex PCR; in the second step, the amplification products are either barcoded using a 454-specific Molecular Identifier, or MID, kit, or further amplified using a Short Read Amplification kit that is compatible with the Illumina, PGM, and SOLiD platforms.
MID kits for the MiSeq and PGM are in the works and will be launched as soon as their respective manufacturers increase their read lengths.
For the BRCA, CFTR, FAP, and HNPCC assays, the company also offers optional PCR primer sets to look for mutations in homopolymer stretches longer than six bases, where the 454 and PGM platforms tend to have trouble. For the BRCA 1 and 2 genes, for example, the homopolymer kit includes 31 amplicons, which are analyzed by capillary electrophoresis. According to Del-Favero, labs use this additional fragment analysis assay if a mutation in a homopolymer region is suspected.
According to Luc Segers, the company's vice president of marketing and sales, the company has about 50 users throughout Europe, about 80 percent of them equipped with the 454 GS FLX or GS Junior platform, the others with the MiSeq or PGM.
Customers have already been using the RUO kits clinically, validating their tests internally. But in order to help users gain accreditation for their tests in their respective countries more easily, Multiplicom decided to obtain CE/IVD marking for its kits, starting with the BRCA assay. "We help them because we give them the data and a manufacturer-controlled kit," Segers said, "so for them, it's less work to bring that through the full accreditation process."
Also, labs using the RUO version currently need to check every reagent lot before use. "With the CE mark, they don't have to do this anymore because that is done by us," he said.
The BRCA MASTR Dx kit, which builds on the already available BRCA MASTR v2 kit for research, meets all the requirements for in vitro diagnostic kits under EU regulations. To achieve that, the company invested in an internal quality system and performed a validation study of the test with two academic partners in Belgium and one in France, using the 454 GS Junior and GS FLX systems. Once the MiSeq and PGM offer longer reads, the company also plans to validate the assay on those platforms.
Last week, Multiplicom presented results from the validation study at the ESHG conference. Using the company's protocol for amplification, sequencing, and data analysis, the partners analyzed a total of 60 samples, representing a range of BRCA mutations that had previously been analyzed by Sanger sequencing or another reference method. While the assay was 100 percent sensitive and specific for SNPs, it missed one 5-base-pair indel, which was not picked up by the software. "What we learned from that is that we have to instruct our users to be careful with indels, and we will devise some measurements they can take to reduce the chance to miss indels," Del-Favero said.
All three centers used the GS Junior for the study. In addition, one center sequenced the same amplicon library with both the GS Junior and the GS FLX and found that the results matched completely.
Moving BRCA testing to NGS
Analyzing the BRCA genes on a next-gen sequencing platform rather than by Sanger sequencing decreases the turnaround time and lowers the cost of the assay, according to Segers.
BRCA testing in Europe is highly decentralized, involving a large number of laboratories with low levels of automation and relatively small sample volumes. Sequencing the BRCA genes involves more than 90 amplicons, which can take a long time by Sanger sequencing in the absence of automation. According to a study by the French National Cancer Institute, INCA, the average turnaround time in French genetic testing labs is about nine months, Segers said.
Next-gen sequencing can bring this time down to a month to six weeks, he said, for example in a lab that performs one six-sample run per month on the GS Junior.
The cost is between 30 percent and 50 percent of that of a Sanger sequencing test, he said, including labor and consumables.
Following the launch of the BRCA MASTR Dx assay, the company plans to develop other kits under CE mark, and to expand its assay menu into other areas, for example metabolic disease, neurological disorders, cardiomyopathies, and a number of rare diseases. It also plans to update existing kits, for example its cystic fibrosis test.
Launching kits for research use only is a good way to allow laboratories to evaluate the product, Del-Favero said, while giving the company time to conduct the validation studies required for CE marking.
New tests will include not only SNP analysis but also copy number variation analysis, a feature that is currently under validation at the company. CNV analysis is achieved by adding several amplicons in genome-stable regions that can serve as a reference to calculate the copy number of other genes.
Multiplicom is also working on additional assays to detect somatic mutations in tumors. While some institutions and companies in the US, for example Foundation Medicine, are developing cancer panels with dozens of genes, "what we hear in Europe is [that] those large panels are not very interesting" except for a small number of large cancer centers, Del-Favero said. Rather, pathology and clinical diagnostics labs in Europe are asking for focused, cancer-specific sequencing panels involving no more than three or four genes, he said.
Non-invasive Prenatal Testing
Finally, Multiplicom is developing a non-invasive prenatal test for chromosomal aneuploidies, a project that is still in the research phase. Last month, the company received a €200,000 grant from the Flemish agency for Innovation by Science and Technology, IWT, to support the test development. The assay will use the company's amplification technology to generate several hundred amplicons from targets on chromosomes 13, 18, 21, X and Y in a single reaction, followed by next-gen sequencing.
The targeted approach is similar to that of Ariosa Diagnostics, which is developing a non-invasive prenatal test in the US, and has advantages over Sequenom's approach, Del-Favero said, because it is easier to set up and the data easier to analyze.
Multiplicom aims to have the technical validation of the assay completed by the end of the year, after which it plans clinical validation studies in several thousand samples.
"From a marketing point of view, we believe that there will be a very strong demand for decentralized testing" in Europe, Segers said, where legislation differs between countries and where clinicians do not like to send their samples far away. "The Sequenom approach is a centralized approach with a very optimized high-throughput system. Our assay allows [users to analyze] smaller volumes at a reasonable price" using benchtop sequencers, he said. LifeCodexx just launched a test similar to Sequenom's in Germany, which it plans to market initially in German-speaking countries.
Asked whether Multiplicom's test would violate Sequenom's patents, Segers said that the company is currently analyzing the situation. "We think we have a solution to be free to enter the market," he said.