DioGenix plans to use $1.5 million it raised last month in series B financing to support a multi-center prospective clinical trial of its targeted B-cell sequencing test for early diagnosis of multiple sclerosis.
DioGenix recently completed a mostly retrospective study of its cerebrospinal fluid targeted sequencing approach, which showed that the test performed with double the specificity and about the same sensitivity as current methods for testing CSF such as oligoclonal banding.
Larry Tiffany, DioGenix's President and CEO, said these results, if borne out in the new, fully prospective, trial, are promising enough that it believes it will be able to start offering the test, called MS Precise, as a CLIA laboratory service after the multi-center study is completed.
"We believe [that] if we see similar performance in this trial, we’d be ready to start providing this, maybe in a limited rollout, but through a CLIA lab model, starting to work with the clinical network we've already formed. They'd go from providing us patients in the clinical trial to being our core centers using the test commercially," he told Clinical Sequencing News.
DioGenix hopes MS Precise — which sequences and analyzes mutations in target regions of patients' B-cell genomes to look for patterns indicative of MS over other immune-mediated neurological diseases — will offer a more accurate tool to aid diagnosis than current CSF analysis methods.
According to Tiffany, MS diagnosis is currently based mostly on radiographic imaging. However, DioGenix estimates that about 60 percent of patients being evaluated for MS also undergo testing using oligoclonal banding as an adjunct tool, even though the method has a high false positive and false negative rate.
Tiffany said that according to the company's market research, neurologists estimate the misdiagnosis rate for MS at about 35 percent. "So imagine maybe 15 percent of the patients you're going through – who are put on $40,000 worth of medication every year – may not actually have the disease." If MS Precise can double the specificity of current CSF testing, that would be a significant improvement, he said.
Tiffany said that MS Precise is based on research by Nancy Monson, an autoimmune disease researcher at the University of Texas Southwestern. DioGenix licensed the approach from UT Southwestern and has continued to develop it toward creating a commercial test.
"It is well known that [part of ] the B-cell genome hypermutates when the immune response signaling process begins," Tiffany explained.
"In MS it is believed that somewhere on the myelin sheath begins to produce proteins. Exactly which proteins and what pattern is not well understood, but what is clear is that this starts to send a signal to the immune system to mount a response and develop an antibody repertoire. The [antibodies] that actually have good specificity with those self-antigens come back, and the B-cells focus on that clone, go through clonal expansion, and really crank up production of those antibodies," he said.
According to Tiffany, Monson's research showed that patterns of changes in the VH region of B-cells' genomes could be used to distinguish MS from other diseases that are symptomatically similar, but different in function. Monson published the initial discovery in the Journal of Neuroimmunology in 2009.
DioGenix's sequencing test, based on this initial discovery, starts with a standard CSF sample, the same as would be collected for current MS testing, according to Tiffany.
The company has not published on the method in detail, but Tiffany said that in the current iteration of the process, cells from the CSF are separated via centrifugation and then undergo targeted amplification of the VF region and sequencing on Roche's 454 platform.
Tiffany said Monson's research was initially based on Sanger sequencing, but the company saw two main advantages in adapting it to a next-gen approach.
"First, eventually the cost should be relatively very attractive," he said. "And secondly, the quality and the depth of information we get should be better."
"Also, we don't have to separate out different cell types," Tiffany explained. "We take the cell pellet [isolated from CSF by centrifugation], and we can go directly to amplifying that variable region, sequencing and looking for different mutation patterns."
Based on Monson's work, these mutational patterns, called an antibody gene signature, or AGS, are algorithmically translated into a quantitative score that indicates whether or not a patient is likely to have MS.
Tiffany said the company recently completed an initial trial that looked at about 100 subjects overall with about 80 percent analyzed retrospectively and 20 percent followed prospectively.
According to the company, in this trial, MS Precise outperformed the specificity of current standard-of-care CSF analysis in patients suspected of having MS by almost two to one with no loss of sensitivity. DioGenix is planning to publish the full results of the study soon.
Tiffany added that the sequencing data collected in this first trial and in the ongoing follow-up trial may also help the company further refine and improve the test.
"There are the hotspots [Monson] published on in the past that we use to guide our effort, but as we've dug deeper and deeper into the database we get back on each patient, we are starting to also make correlations to specific amino acid changes as it relates to these codon positions," said Tiffany.
"tThe mutational frequency method works well and is pretty compelling, but the advantage of the NGS data is that we can do that, and then also start to build more complex models and algorithms that look at specific amino acid substitutions and changes."
"It's very possible [that those models] might allow us to develop a test with greater performance across all patients, or at least in some instances," he said.
In the company's new multi-center trial, DioGenix plans to prospectively follow about 150 patients suspected of having MS through their diagnosis using standard procedures, comparing the results of MS Precise with patients' eventual diagnosis in a blinded manner.
Tiffany said the company is working with eight main centers, including Emory, Beth Israel Deaconess Medical Center in Boston, and the University of Texas Southwestern, to recruit patients.
In addition, the company is also collaborating with some large group neurology practices, including Texas Neurology in Dallas and Multicare in the Seattle area.
If the results reflect what the company saw in its previous studies, Tiffany said he expects to be able to start offering the test commercially as a CLIA lab service.
"The goal is to work through that and then start developing evidence of clinical utility for payors and other stakeholders," he said.
While DioGenix currently uses the Roche 454 for MS Precise's sequencing, Tiffany said the company is ultimately platform agnostic.
"There's a lot of debate about how other systems may be less expensive per sample," he said. "The advantages, though, are a very low error rate, and long coverage in terms of read length, since we really need to look at about 350 base pairs to make our determinations."
"But in the future, two things are going to happen. First, other systems' error rates are going down, and read lengths are going up. Also, we actually may also be able to not have to focus on these large read lengths to do our analysis."
Future decisions about sequencing platforms will come down to cost, quality of data, and read length, he said.
Tiffany also said DioGenix has not arrived on a final test price, but he said in an email that the company has contracted a preliminary pharmacoeconomic analysis that suggested that "$3,000 to $4,500 is well within the benefit provided by MSPrecise."