Cancer diagnostic labs have started to test next-gen sequencing-based assays from two companies, Fusion Genomics and Enzymatics, to detect gene fusions as a complement and possible alternative to existing assays.
Up until now, gene fusions in cancer, such as lung cancer, sarcomas, and hematological tumors, have mostly been detected by fluorescent in situ hybridization or RT-PCR. However, these methods cannot detect multiple gene fusions at once, determine their break points at single-base resolution, or detect fusions where one of the partners is unknown, which the new NGS-based assays from Fusion Genomics and Enzymatics promise to be able to do.
Fusion Genomics, based in Vancouver, British Columbia, has established a targeted NGS method for the detection of gene fusions in RNA that uses hybridization-based capture. The assay uses proprietary probes but has no intellectual property associated with it.
The company was founded last year as a spinout from the lab of Poul Sorensen, a senior scientist at the BC Cancer Agency and a professor of pathology and laboratory medicine at the University of British Columbia.
Last month, Sorensen's group published an assay for the detection of fusion transcripts in childhood sarcomas, called ChildSeq-RNA, in the Journal of Molecular Diagnostics. The assay looks for the gene fusions EWSR1-FLI1, EWSR1-ERG, PAX3-FOXO1, PAX7-FOXO1, EWSR1-WTI, and ETV6-NTRK3, which are found in Ewing sarcoma, alveolar rhabdomyosarcoma, desmoplastic small round cell tumors, and congenital fibrosarcomas. Along with the assay, the group developed a bioinformatic tool called ChildDecode for analyzing the data.
In their paper, the researchers tested the assay on four Ewing sarcoma cell lines and 33 clinical samples of various childhood sarcomas and found it to have high sensitivity and specificity, compared to RT-PCR.
Fusion Genomics is in the process of commercializing a version of the assay, which it calls ChildSeq-SRCT, and is currently testing it with collaborators at Texas Children's Hospital at Baylor College of Medicine and elsewhere.
The company will offer validated reagents and protocols, as well as data analysis through its FusionCloud service. While the published assay ran on the Ion Torrent platform, the commercial assay will also be available for the Illumina MiSeq, said Mohammad Qadir, Fusion Genomics' chief scientific officer and the lead author on the recent publication.
According to Qadir, the assay can detect both known gene fusions and those where one partner is unknown. "A lot of effort goes into making sure that every region is pulled down to the maximum efficiency possible," he told Clinical Sequencing News, adding that the assay's pull-down expression profiles closely match expression profiles for the same genes from total RNA-seq or qPCR.
Qadir said the assay uses 100 nanograms of starting nucleic acid but can work with as little as 10 nanograms and takes about 2.5 days to complete.
He said he believes the firm's hybridization-based capture approach "will prove to be superior in the long run" to PCR-based methods because it better reflects the diversity present in the sample. Also, it is not limited by any primers that define the beginning of a fusion. "We are pulling out the entire exons, and a fusion can be advancing anywhere, it will not matter," he said.
By the end of June, the company plans to release a version of ChildSeq-SRCT that has been optimized for formalin-fixed, paraffin-embedded samples. In addition, it has been developing a DNA-based version of the assay that it also plans to commercialize.
The firm also plans to expand the assay to include all known gene fusions in pediatric sarcomas and pediatric solid tumors, and it is working on an adult leukemia panel.
Researchers at Texas Children's Hospital have been testing ChildSeq-SRCT for possible clinical adoption. The molecular oncology service of the hospital's department of pathology currently uses both PCR-based assays and FISH to test for gene fusions in childhood sarcomas, but ChildSeq-SRCT offers several potential benefits over these, said Angshumoy Roy, an assistant professor in the departments of pathology, immunology and pediatrics at Baylor.
One advantage is that ChildSeq-SRCT can detect multiple gene fusion partner combinations at once, whereas PCR-based assays usually only detect the most common gene partners. "By targeting for pull-down several different partners in one assay, including the less common and even unknown partners, the assay claims to improve clinical sensitivity in diagnosing these sarcomas, and can potentially improve laboratory workflow by combining multiple targeted assays into one experiment," Roy told CSN.
For example, he and his colleagues ran one EWSR1-rearranged Ewing sarcoma sample which its own clinical assay showed to be negative for the two most common partner genes, FLI1 and ERG. The ChildSeq-SRCT assay, on the other hand, was able to pull down a rare partner gene. "The ability of a molecular sarcoma diagnostic assay to detect unknown partners is extremely valuable as EWSR1, ALK, and other genes are known to be promiscuous for fusing with multiple different partners," he said.
In addition to being more sensitive, the assay may also improve the lab's workflow, reduce labor, and save costs by batching several clinical samples in one run. "This is an attractive proposition for us, although this benefit is likely to be relevant for laboratories with large enough volumes [only]," Lazar said.
Going forward, the Texas researchers plan to test the assay's performance on primary tumors from its clinical sample repository, which have known translocations and gene fusions that were identified by its own clinical diagnostic tests, as well as a few unknown cases.
Beyond cancer, Fusion Genomics is eying the infectious disease market with a capture hybridization-based assay for multi-drug resistant and extensively drug-resistant tuberculosis. That test, which it is developing with collaborators, is scheduled for launch in early 2015.
In addition, the company is interested in environmental monitoring through genome sequencing and is exploring potential collaborations with other start-ups, Qadir said.
Fusion Genomics has six full-time and part-time employees at the moment. It received initial support from the National Research Council Canada and is currently funded by its co-founders and angel investors. In addition, the company is supported by VentureLabs, a business accelerator program of the University of Victoria, Simon Fraser University, the British Columbia Institute of Technology, and the Emily Carr University of Art and Design.
Fusion Genomics plans to raise additional capital in a Series A financing round within the next three months.
Enzymatics, based in Beverly, Mass., launched its first gene fusion panel last month. The assay detects fusion transcripts involving the ALK, RET, or ROS1 genes, which are important in lung cancer, and is compatible with both the Illumina MiSeq and Ion Torrent sequencing platforms.
Later this spring, Enzymatics plans to add three more gene fusion panels, for sarcomas, hematological cancers, and carcinomas, respectively.
All three assays rely on so-called anchored multiplex PCR, AMP, which was originally developed by ArcherDx, a company Enzymatics acquired for up to $50 million last year.
ArcherDx licensed the technology from Massachusetts General Hospital, where it was invented by three clinical researchers, John Iafrate, Long Phi Le, and Zongli Zheng, in the process of developing an alternative to FISH for studying gene fusions in FFPE lung cancer samples.
Last summer, MGH's Center for Integrated Diagnostics launched a clinical assay for fusion transcript detection that is based on the technology.
The AMP technology uses one target-specific primer and one common primer and allows researchers to detect both known and novel fusion partners for a gene of interest. The assays require as little as 1 nanogram of total nucleic acid and can be completed in about seven hours.
Researchers at the MD Anderson Cancer Center have been testing Enzymatics' ALK, RET, and ROS1 lung cancer gene fusion panel for potential use as part of clinical trials. In addition, they are collaborating with Enzymatics to develop gene fusion panels for sarcomas, carcinomas, melanomas, and hematological cancers.
Alexander Lazar, an associate professor of pathology at MD Anderson, told CSN that his institution has a series of ongoing lung cancer clinical trials that are driven by the presence of particular gene translocations that suggest specific therapies.
Up until now, cancer samples at MD Anderson have been tested for these translocations by FISH, either in house or through an outside laboratory. While FISH is "a very robust technique," Lazar said, the results are sometimes hard to interpret, particularly if the translocated genes are close to each other on the same chromosome, and sending samples out increases the turnaround time.
In addition to translocations, the researchers also test the samples for DNA-based mutations in genes like EGFR, KRAS, and BRAF. Extracting DNA and RNA at the same time and using the Enzymatics assay allows them to assay for DNA mutations and fusion transcripts in parallel.
"We may at a certain point sequence and analyze both the cDNA for the translocations and the genomic DNA for the mutations at the same time, but right now, we're keeping them as a separate workflow while we're evaluating this," Lazar said.
He and his colleagues are currently testing the Enzymatics assay on retrospective samples with known rearrangements that were determined by other technologies. "Once we do that, we will start moving this into our regular diagnostic workflow," he said, probably within the next few weeks to months.
Initially, they plan to perform FISH in parallel with the new assay, and "as we show that the performance characteristics of this new test are equal or superior to what we're seeing with FISH, then we will drop FISH out," Lazar said.
His team is also collaborating with Enzymatics to develop a gene fusion panel for sarcomas, carcinomas and melanomas, and hematological tumors, although "there is the potential that we can have a panel that includes all known fusion genes for all cancers," he said.
While sarcomas are rare, they involve many different translocations and a number of "promiscuous genes," such as EWSR1, that can have many fusion partners. Enzymatics' Archer technology, he said, would allow them to examine "lots of different fusion transcripts at once."
"What I'd really like to have internally is multiple methodologies to detect these [fusions]," Lazar said, such as FISH, RT-PCR, and targeted RNA sequencing, because the techniques complement each other. "My experience from running RT-PCR and FISH is, less than 3 percent of cases have shown discordance between FISH and RT-PCR results, so it's nice to have both of them."
In addition to the fusion panels, Enzymatics also plans to offer Archer assays for targeted DNA analysis this spring, as well as an assay for RNA abundance. By the end of this year, it also wants to launch assays that can analyze RNA and DNA simultaneously.