NEW YORK (GenomeWeb) – A recent commentary suggests that after two decades or more of technology and method development, proteomic techniques have in recent years advanced to where they can be routinely applied to clinical questions.
Authored by executives and board members at the diagnostic firms Integrated Diagnostics and Sera Prognostics and published this week in Current Opinion in Biotechnology, the article examines the development process behind Indi's and Sera's multiplexed mass spec proteomic tests to highlight best practices for such efforts and make the case that, as they write, "identification of blood protein panels" for disease diagnosis is "now a reproducible and standard (albeit complex) process."
That said, the authors acknowledged that even as the technical challenges of proteomic test development have become more manageable, questions of regulatory approval, reimbursement, and clinical adoption remain significant hurdles to successful commercialization.
"Proteomics has long been promised to make an impact in the clinic," said Paul Kearney, president and chief scientific officer at Indi and first author on the publication. The commentary was intended, he said, "to make the community aware that that impact has happened," as well as to "make people aware of the techniques and strategies involved in bringing [the Indi and Sera] tests [to the clinic]."
Indi and Sera are the first two companies to bring multiplexed proteomic tests based on selected-reaction monitoring (SRM) mass spec to the clinic. Indi was the first, launching its Xpresys Lung test, which is intended to aid doctors in identifying whether lung nodules detected via CT scans are likely benign, in 2013. Sera launched its PreTRM test, for assessing a pregnant woman's risk of giving preterm birth, in 2015.
Despite the different indications, both tests followed similar development paths, Kearney said, noting that this suggested to the authors that their approach "is a general technique that can be used to bring other tests to the clinic using proteomics."
The authors identified six keys to successful proteomic test development. The first is use of systems biology approaches to select a large number (on the order of hundreds) of candidate markers for discovery that cover multiple cellular networks likely to be perturbed by the target condition.
"We're not talking about single gene disorders," said Gregory Critchfield, president and CEO of Sera, and an author on the paper. "We're talking about complex biology that can be dissected and understood in different ways."
As such, analysis of markers reflecting multiple different aspects of the disease process is key to ultimately achieving the necessary clinical performance.
Also important is following appropriate statistical guidelines to avoid overfitting as well as looking at a number of genetically distinct human populations to account for natural variation across populations. Additionally, the authors noted, candidate proteins should be evaluated early on not only on their diagnostic performance, but also by their analytical robustness to ensure that they can be accurately and reproducibly measured in the clinic.
The authors also put forth a peptide correlation-based technique based on the fact that peptides from the same protein will have highly correlated expression levels to allow for confident quantitation of target proteins without the use of labeled peptides or the need for measuring large numbers of unique peptides per protein, which can reduce assay sensitivity.
They also stressed the need for effective normalization approaches to account for both pre-analytic and analytic variability.
Many of these considerations were addressed in a 2012 publication from the Institutes of Medicine, said Jay Boniface, chief scientific officer at Sera, and an author on the Current Opinion paper.
"What we discovered recently is that the entities on this paper applied those rules [laid out by IOM], and showed that, in fact, that they do end in success," he said. "And what that enables now is the application of proteomics and systems approaches. And it occurred to [us] that we could provide this information to the community so that [researchers] are aware that the technology is now at a point where it's robust and can be applied clinically."
Kearney suggested that more careful adherence to the guidelines detailed in the IOM report and the Current Opinion paper could improve the translation of proteomic discoveries to the clinic — an area where the field has struggled.
"I think there's a huge gap between the early discovery work that's happening and what happens after that," he said. "Typically, the early discovery work is underpowered. I see papers all the time with 20 samples, 40 samples, what have you, and they're not validated."
"They also tend to overfit," Kearney added, "because when you have that few samples, you can always find a difference between your cases and controls. In fact, mathematically you will find something, and a lot of these studies, they're not realizing that they are destined to find something. It's probably wrong, but mathematically, they're almost sure to find something."
Here, Kearney echoed other researchers in the field who have observed that poor statistical design remains a stumbling block for many proteomics experiments.
Boniface, likewise, noted that while the goals of academic discovery projects don't always include clinical translation, even in those cases, attention to certain of the factors discussed in the Current Opinion paper is warranted.
"It's obviously quite important for results of research to be put in the public domain," he said. "You want everybody to benefit from those observations, but part of that intent is also to offer to the community the opportunity to reproduce your results."
"Imagine a scenario where you've described 50 new proteins that are important in a disease, but you've never determined which of those are analytically robust," he said. "And somebody tries to reproduce your data, but they allow the blood to clot for an hour longer, and it completely changes half of the analyses. And then those who are trying to reproduce [the original results] think that they, or you, have done something wrong. But if the original investigators had done an analytic assessment and said, 'Of these 50 proteins, here are the ones that are analytically robust,' then it allows the community to reproduce the data and science gets advanced more rapidly."
While further technical advances will likely improve proteomic biomarker development by, among other things, allowing researchers to delve more deeply into the proteome, Leroy Hood, senior author on the paper and a professor at the Institute for Systems Biology, as well as a co-founder at Indi and scientific advisor at Sera, said he believes that existing technology appropriately applied is capable of routinely identifying clinically useful protein markers and marker panels.
"We've done it two for two times with Sera Prognostics and Integrated Diagnostics," he said. "I think that suggests pretty strongly if you frame your questions properly you can get biomarkers every time."
However, the authors agreed that while the technical and scientific challenges may now be manageable, significant commercial challenges remain.
Because while Indi and Sera were the first firms to bring SRM-based proteomic tests to market, they were not the first to launch multiplexed proteomic assays. Companies including Vermillion, Crescendo Bioscience, Biodesix, Aviir, Tethys Bioscience, and Applied Proteomics have all brought multiplexed protein diagnostics to market for conditions ranging from ovarian cancer to heart disease. A number of these companies have gone bankrupt or otherwise gone out of business. Even among those like Vermillion that have managed to stay afloat, significant commercial success has remained elusive.
Indi itself has struggled since launching Xpresys Lung in 2013. The company sold only around 2,000 tests and was unable to secure coverage from Medicare before it pulled it from the market in 2016. Indi has since developed a better-performing, second-generation Xpresys Lung test, for which it has secured CMS coverage and that it plans to launch this year, but, Kearney acknowledged, it has been a long road.
"I think what we're demonstrating is, [proteomic diagnostic development] is possible and widely applicable," he said. "The other question then is where does the risk now shift. Now the risk is more towards, can you get reimbursement? It moves out of the realm of clinical science and more into the realm of the reimbursement strategy, the financing strategy for the company."
Critchfield said that by laying out the steps involved in the Current Opinion commentary, the authors hoped to demonstrate both that such an approach could lead to successful test development and also perhaps give researchers and investors a better sense of the time and resources required.
"I think many people have not been aware of how this is done," he said. "We wanted to publish this work to raise awareness [that it can be done] — that's one aspect of it. And I think the second aspect [is making people aware] that it requires significant capitalization to do it right."
"I think that the funding of these kind of companies has lagged behind the technical capabilities, and that's one of the things we wanted to highlight," he said. "Our hope was to put this out there as an example and let people know that, with the right approach, and if you're capitalized, you can actually do something that's very significant that otherwise is not possible."
Whether Indi and Sera will prove that theory out remains to be seen. Given that it has secured CMS coverage, prospects for the second-generation Xpresys Lung test are perhaps brighter than they were for the initial version, though questions of clinical adoption still remain.
As for Sera's PreTRM test, Hood suggested that the company had thus far had an easier go of it than Indi, adding, "They've done very well, and I think they're really in quite good shape with their assay."
Sera launched the test in limited release in 2016 and last year entered an agreement with Laboratory Corporation of America making that firm exclusive US distributor of the test.
Asked for details on test sales thus far, Critchfield declined to provide specific numbers, saying that the company is "just beginning to move the commercialization forward, and we're pleased with the progress that we're seeing."