NEW YORK (GenomeWeb) – German epigenetic technology company Epiontis and collaborators this week published a seminal study demonstrating the power of Epiontis' immune cell counting platform, laying early groundwork for the potential development of clinical assays.
The study, published in Science Translational Medicine, applied Epiontis' technology — which uses epigenetic quantitative PCR to detect and count specific target cells in a sample — to dried blood spots from newborn heel pricks, showing that the approach could detect a much larger number of immune disorders than currently used methods.
Researchers also reported on the power of the technology to monitor immune cell counts in HIV infection without the need for fresh blood samples — which could be useful for testing in remote or resource-limited areas.
Sven Olek, Epiontis' managing director and now senior vice president for research at Precision for Medicine, which acquired Epiontis last fall, said in an email that he and other co-founders formed the company more than a decade ago to advance a hypothesis that DNA methylation could be harnessed to detect and characterize different cell types.
"The dominant thinking at the time was [that we] could detect cancer based on 'specific' epigenetic markers that were only found on cancer cells and not in healthy cells," he wrote. "[But] my approach … essentially argued the opposite … rather than looking at [complicated] cancer cells, let's look at specific methylation and detect/characterize healthy cells."
Olek and collages established proof of concept for their method in enumerating various cell types, including regulatory T cells, and then began what turned out to be a long process of working toward commercialization.
By 2010, the company had begun offering cell detection and quantification services to pharmaceutical customers for use in clinical trials, Olek added. "Part of what made the technology attractive to [pharma] is what we have shown in this current paper: the state of the blood sample is pretty irrelevant to our ability to successfully process it. It doesn't matter how the blood is drawn, stored, or shipped," he wrote.
The company's approach essentially provides an alternative to techniques like flow cytometry — potentially for any application where there is a need to count a particular type of immune cell.
In their study, the researchers described identification and validation of DNA methylation markers that can discriminate between target immune cells and other contents of a blood sample. In addition to analysis of T cell-associated genes CD3G/D, CD4, and CD8B, the group reported that loci in several other genes — MVD, LRP5, and LCN2 — were unmethylated only in NK cells, B cells, and neutrophils, respectively.
"Causes and consequences of methylation patterns in these regions remain unknown, but this does not affect their use for cell quantification in peripheral blood," the team wrote.
According to Olek, the Precision for Medicine acquisition was intended to help Epiontis accelerate the application of its technology, hopefully not just in research, but to address unmet clinical testing needs.
"We tried to identify areas where [our] technical advantage translates into medical benefit.," he said.
Hence the two areas explored in the firm's STM study this week: HIV monitoring and newborn screening. In both cases, Olek argued, there are "obvious shortcomings" to current approaches and a clear opportunity for Epiontis to demonstrate it could produce better results.
In the case of newborn screening, the study authors explained that among the more than 300 known primary immune deficiencies (PIDs), only one — severe combined immunodeficiencies (SCID) — can be detected at birth as a part of routine testing of heel-prick dried blood spots.
Epiontis and its collaborators showed that with the company's qPCR methodology, they could pick out cases not only of SCID, but also other disorders like X-linked agammaglobulinemia (XLA), immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, and severe congenital neutropenia (SCN).
Investigators developed real-time PCR assays for human leukocyte subpopulations and applied them to a collection of dried blood spots from 250 health newborns and 24 known cases of various immunodeficiencies.
They used assays to quantify total T, B, and NK cells, and deemed a sample suspicious if any of the three cell type-specific measurements were outside of a defined confidence region. With a confidence region locked at 99 percent, the Epiontis method could detect the cases of known immune disorders with sensitivity of about 96 percent and specificity of 98 percent. Using a cutoff of 99.9 percent, sensitivity was about 91 percent, and specificity rose to 100 percent.
In a statement, one of the study's academic authors, Stanford School of Medicine professor Rosa Bacchetta, said that while more research will be needed to validate the approach, the initial result from the trial were "very encouraging."
"Typically, children are not diagnosed [with these disorders] until they're sick and are showing symptoms [making] effective treatment much more difficult. It would be highly advantageous if we could identify these diseases at birth and begin treatment soon after birth, prior to the development of symptoms," she said.
To explore another potential clinical application, the study also examined how the Epiontis technology performed in quantifying immune cells in the context of HIV infection, using whole blood samples from 25 healthy donors and 97 HIV positive patients.
Investigators reported that they could accurately count immune cells using the epigenetic technology on a dried blood sample, with comparable results to flow cytometry.
According to Olek, monitoring blood cell counts is critical to proper treatment for HIV patients, but can be difficult to do in areas of the developing world where storing and transporting liquid blood isn't feasible.
The ability to send dried blood spots for testing in a central lab could increase the use of immune cell counts to monitor patients and improve their treatment.
Methylation PCR is not itself novel, or proprietary, but Olek said in his email that the markers the Epiontis uses to enable cell type-specific counting are patent-protected, as are other aspects of its absolute quantification methodology.
Moving forward, he said that the company is now studying larger cohorts of healthy individuals at ages up to 16 to continue to refine the ability of the technology to detect immune defects.
Other possible applications in the future include monitoring cancer patients undergoing chemotherapy or other immunosuppressive treatments with at-home finger stick sampling as a way of reducing the need for doctor's visits and repeat blood draws.
As it explores niches like this, Olek said that the firm is also working to develop a point-of-care-device that would allow the home-based measurement and data to be sent to a doctor electronically.