Cardiovascular diagnostics company Aviir plans to launch this spring clinical tests for more than a dozen inherited cardiovascular disorders that run on both the Ion Torrent PGM and the Illumina MiSeq.
The company, which has a CLIA-certified facility in Irvine, Calif., called Aviir Diagnostics Laboratories, was founded in 2005 by cardiologists and researchers at the Stanford University School of Medicine. It focuses on diagnostic tests for the prevention and management of cardiovascular disease and metabolic syndromes.
Earlier this year, Aviir raised $10 million in the second tranche of a financing round led by Merck Global Health Innovation, which was joined by Partners & Partners, Aberdare Ventures, Bay City Capital, and New Leaf Venture Partners. Previously, the company had raised $10 million as part of the same round.
Last year, the company launched its first proprietary test, MIRISK VP, which helps doctors reclassify patients with an intermediate risk for coronary heart disease as either low-risk or high-risk. The serum-based test measures seven protein biomarkers, which it combines with clinical information to gauge a patient's risk for a severe cardiac event within five years.
In addition, Aviir offers more than 120 routine clinical tests around cardiovascular disease, including a number of molecular tests, such as for warfarin sensitivity.
More recently, the firm has developed next-generation sequencing-based tests for 18 inherited cardiovascular disorders – ranging from a single gene test to panels of more than 20 genes – that it plans to start offering this spring.
The tests cover both disorders with structural defects of the heart or its vessels, such as cardiomyopathies or Marfan syndrome, and those with defects in the electrical system of the heart, including long and short QT syndrome and Brugada syndrome.
For all of these tests, Aviir sequences the same 70 or so genes, or 1,500 exons, but the data are filtered and only those genes are analyzed that are relevant for the test a physician requested. The tests have a turnaround time of two to four weeks, depending on clinical need and volume.
"The laboratory workflow is the same for all of these tests, whether we're looking at one gene in the case of Marfan's or 20 to 25 genes in the case of the cardiomyopathies – we are going through a typical NGS library preparation process using hybrid selection," explained Bill Biggs, senior director of genetics and genomics at Aviir.
The lab originally developed and validated the 70-gene panel for the Ion Torrent PGM, which it installed in the summer of 2011. Earlier this year, it also brought in the Illumina MiSeq and is in the process of revalidating the panel on this platform.
Both sequencing platforms will be part of the commercial offering of the tests, Biggs said, enabling the lab to confirm variants identified by one platform on the other. His team is still considering confirmatory testing by Sanger sequencing – the method of choice of many clinical NGS laboratories today – but "the intention is to use two different next-gen methods to confirm variant identification."
The PGM and MiSeq agree very well on the single-nucleotide variants they call across the target genes, he said. Because the lab sequences to about 1,000-fold average depth on the MiSeq and only to about 260-fold depth on the PGM, some SNVs are called by the MiSeq but not the PGM.
Aviir found during its validation that the PGM "has issues" calling insertions and deletions accurately from SureSelect sequencing libraries, a problem the MiSeq does not have. Thus, for the time being, it is calling indels on the MiSeq. The company is monitoring Ion Torrent's progress with indel calling, though, and hopes to use the PGM for that "very soon," Biggs said.
Both platforms are currently still in an R&D environment, but the lab is finalizing their validation for the clinical tests.
"The presence of multiple [sequencing] platforms is going to be key to the success of NGS in the clinical diagnostic setting," Biggs said. "The competition between Ion and Illumina is something that, in our view, is only going to benefit the patients in the end, which is our goal."
He also welcomes that both Illumina and Life Technologies are moving their instruments into regulated environments, applying for 510(k) clearance for the MiSeq and the PGM, respectively. This, he said, is "an indication of the manufacturer's commitment to both the platform and diagnostic setting" and "gives the laboratories confidence that one is not going to have to transition to another platform, and completely revalidate all of your testing on that other platform."
To enrich the genes for sequencing, Aviir uses Agilent's SureSelect hybridization capture and finds that between 96 and 98 percent of the target DNA is covered at a sequencing depth of at least 20x, sufficient to make variant calls.
Regardless of the sample prep method or sequencing platform, the researchers found that three to five exons typically had no coverage, and another 40 to 80 exons had one or more bases with less than 20x coverage. Many of these exons, which are consistent between experiments, are GC-rich and the first exon of a gene.
Because disease-causing variants may reside in these undercovered exons, the Aviir scientists designed a custom Agilent HaloPlex target enrichment library to fill in the sequence for more than 100 exons with the lowest coverage.
Combining the SureSelect and HaloPlex enrichment enabled them to increase the target coverage from 96 percent to 99.5 percent on the PGM. Sequencing of the HaloPlex library on the MiSeq is ongoing, but the researchers expect to obtain similar results for that platform.
The lab has not looked at other target enrichment methods yet, except for the Ion AmpliSeq, which it tested more than a year ago, when it "wasn't quite ready for us yet,"
One of the biggest challenges of developing the tests was being an early adopter of the PGM. "Ion was going really fast, adding a lot of things really quickly, which was great," he said, but at the same time, "it became a little bit of a challenge for us to keep up, to translate all the great innovations that the team at Ion was making into our controlled, regulated setting."
Another challenge was constructing a pipeline for file management and data analysis that satisfied CLIA requirements, in particular being able to retain data for long periods of time and to re-access and re-evaluate data on a regular basis.
For the clinical report, Aviir focuses on variants most likely to be causative, as determined by its analytical process, which involves multiple algorithms to assess a variant's conservation, the protein region it affects, related variants, and more.
Variants of unknown significance are "admittedly the biggest challenge right now for NGS in a clinical setting," Biggs said, and no standard has emerged yet for assessing whether a novel variant is causative or not. "Everyone is wrestling with that same issue."
Aviir is in the process of building an in-house database of known disease-causing mutations, drawing on publicly available resources, which it will complement with its own variants. Many genetic testing labs keep proprietary variant databases, but Biggs said several efforts are underway to aggregate variant data and make it publicly available. "While [proprietary databases] may serve the commercial interest of a testing laboratory, in many cases [they] do not serve the interest of patients," he said.
Pricing for Aviir's NGS tests is still being determined. "We're certainly well aware of the competitive landscape in this area, as well as the rapidly evolving CPT coding and reimbursement landscape around NGS and molecular testing in general," Biggs said.
Prices can vary a lot, he added, because much of clinical testing is done under contract with specific payors. Insurance providers typically negotiate reimbursement rates with clinical laboratories, so list prices are of "limited value."
Several other laboratories – such as Partners Healthcare Center for Personalized Genetic Medicine and GeneDx – are already offering NGS-based inherited cardiovascular disease tests similar to Aviir's. "In the end, it becomes an issue of performance, pricing, and customer service," Biggs said.
As it is launching its first NGS tests, Aviir is already "actively looking" into expanding the underlying panel beyond 70 genes. "We really wanted to use this initial offering − 70 genes, 1,500 exons, 18 indications − as a model system on which to build our internal capabilities," Biggs said. File management and data processing, for example, are fully automated. In the future, Aviir may scale testing to the full exome or even genome, and may expand beyond cardiovascular diseases, he said.