DxTerity Diagnostics has developed a rapid assay for analyzing gene expression of as many as 100 genes directly from small amounts of formalin-fixed, paraffin-embedded clinical samples without prior RNA extraction. In addition, the company recently demonstrated how the assay can be used to measure HER2 gene expression levels in FFPE breast cancer samples.
The Los Angeles-based firm is now seeking clinical collaborations with partners with significant HER2 testing volume and plans to open its own CLIA-certified clinical diagnostics lab by the end of the current quarter in order to offer other assays based on its gene expression technology, DxTerity President and CEO Bob Terbrueggen told PCR Insider.
Further, DxTerity plans to launch a CLIA version of a blood-based assay that uses the same core gene expression technology to determine acute radiation exposure in individuals, and is gearing up to seek approval from the US Food and Drug Administration for the test, Terbrueggen said.
Founded in 2006 by Terbrueggen, DxTerity has looked to penetrate the genomics-based clinical testing market by leveraging its non-enzymatic amplification technology, or NEAT, which forms the basis of its diagnostic testing platform, DxDirect.
DxDirect combines NEAT with a proprietary reaction buffer called DxCollect that simultaneously lyses a sample and stabilizes RNA for ambient transport and subsequent analysis, according to the company. The technology eliminates the need for sample purification and RNA isolation, and enables direct-from-sample, single-tube, multiplexed, and highly quantitative gene expression analyses in less than four hours.
In an email to PCR Insider, Terbrueggen noted that the key to directly analyzing RNA in FFPE is that the DxDirect probes are used to make a 1:1 conversion of RNA target to a target-specific synthetic cDNA that has been engineered for robust amplification and readout.
In a typical DxDirect assay, Terbrueggen explained, the reaction buffer lyses the FFPE tissue and makes the RNA available for analysis. Then, DxDirect probes target small regions of the RNA and, as such, are relatively insensitive to RNA degradation. The DxDirect probes also have chemically reactive moieties at the 5' and 3' ends and spontaneously ligate upon hybridizing to the target RNA, creating a ligated reaction product that serves as a surrogate for the original RNA target molecules.
"The resulting ligated DxDirect probe set contains universal primer sequences for robust, quantitative, multiplex amplification, and the probes have been engineered to contain identifier sequences for easy readout by existing instrumentation," Terbrueggen said. DxDirect assays can be automated using standard liquid-handling instruments and analyzed with instrument platforms such as real-time PCR, capillary electrophoresis, and Luminex BeadArray systems.
"DxDirect has been used to simultaneously analyze up to 450 biomarkers in a single tube; however the technology is most frequently coupled with CE readout and used to analyze up to 40 genes per assay," Terbrueggen said. DxTerity also has a deal with PerkinElmer's Caliper Life Sciences to optimize its assays for the Caliper LabChip Dx instrument, although Terbrueggen declined to provide an update on that arrangement.
DxTerity is using DxDirect to develop low-cost, high-throughput clinical diagnostics in which the sample input is either a small amount of stabilized blood or FFPE tissue.
"We have worked with different sample types with equal ease," Terbrueggen said. "Our initial efforts have largely focused on our unique advantages in direct-from-blood testing. In 2011, we expanded our efforts [to look] at multiplex expression in FFPE."
In April, at the American Association for Cancer Research meeting in San Francisco, DxTerity presented a poster demonstrating the use of a DxDirect assay to analyze a 60-plex gene signature from breast and prostate tissue, finding that the obtained signature correlated closely with microarray data and results from matching freshly frozen tissue samples.
Building on that work, in October at the Association for Molecular Pathology meeting in Long Beach, Calif., company scientists presented another poster demonstrating the assay's ability to analyze HER2 gene expression directly from very small amounts (less than 1 mm2) of FFPE tissue.
More specifically, they compared the results of their assay run directly on these small FFPE samples with results obtained using their assay on RNA that was first extracted from the FFPE tissue using a Life Technologies RNA isolation kit; and with data acquired from matching freshly frozen samples. The DxDirect assay from FFPE tissue correlated highly to the other two methods.
Having conducted these in-house proof-of-principle experiments, DxTerity is now looking to commercialize its technology through partnerships and internal diagnostic test development.
"DxTerity is focused on molecular diagnostics and not the research market," Terbrueggen said. The company plans to open a CLIA lab in the current quarter in order to offer assays developed by DxTerity "as well as traditional assays that help support the clinical areas that we will be launching into," Terbrueggen said.
Regarding the HER2 test, DxTerity is seeking clinical collaborations "with a number of potential partners with significant HER2 testing volume," he added. "While there are other providers of HER2 assays, our assay has a higher sensitivity, can work directly from FFPE sample, and provides significant cost savings to the laboratory."
The company also believes that future changes in how labs are reimbursed for molecular tests like HER2 analysis will make its assay particularly attractive. "Currently labs are encouraged to utilize cumbersome, labor-intensive processes because they are reimbursed based on individual processes," Terbrueggen said. "A move to fixed-price, results-based reimbursement will make DxTerity’s HER2 test a more attractive alternative to entrenched methods" because it eliminates processing steps, he added.
In terms of in-house tests, DxTerity's biggest current project is to commercialize a blood-based gene expression assay for determining acute radiation exposure in patients. The test, called Radiation Exposure Dosage Index Diagnostic, or REDI-Dx, is a 30-plex gene expression assay that measures a panel of blood-based markers to determine the apparent level of individualized radiation exposure up to seven days post-event.
"REDI-Dx targets the emergency response and preparedness market by providing the world’s first mass-scalable solution for radiation exposure determination," Terbrueggen said. "The assay is designed for responding to national emergencies like the detonation of a nuclear bomb or a Fukushima-like event."
The company has developed the assay in collaboration with Duke University and with funding from the Biomedical Advanced Research and Development Authority.
Terbrueggen noted that REDI-Dx is a prime example of how the DxDirect assay technology can save diagnostic developers time and money.
"Most gene expression assays have a price per test of greater than $1,000 and require significant labor and testing infrastructure to achieve modest volumes of fewer than 100 samples per week," he said. "In comparison, using a single liquid-handling robot and a single CE instrument, DxTerity expects to achieve a REDI-Dx testing capacity of approximately 10,000 samples per week with a price per test of less than $100."
In addition, REDI-Dx requires only about 25 µl of whole blood collected by finger stick. "To our knowledge, this is the first example of gene signature testing from a finger stick of blood," Terbrueggen said. "The combination of these throughput and ease-of-use advantages makes DxDirect an attractive solution for large-volume diagnostics in blood without a need for special sample handling and a phlebotomist."
DxTerity plans to launch REDI-Dx in the US as a CLIA test, and is also working with undisclosed government entities outside the US to launch the test internationally, Terbrueggen said, adding that the company "also has a significant effort focused on obtaining FDA approval" for the assay.
The company also has begun several test development programs including a sepsis treatment and response test, or START; and an assay to identify cancer patients likely to suffer adverse reactions to radiation therapy.
DxTerity has also acquired from SourceDx intellectual property and know-how related to blood-based gene expression assays for cancer and autoimmune disease.