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Partners in NIH Parkinson's Program Hope Combination of Proteomics Approaches Will ID Useful Markers


Under its new Parkinson's Disease Biomarker Program, the National Institute of Neurological Disorders and Stroke has funded nine research teams, most of which involve proteomic approaches, to attempt to identify biomarkers that could be used as either predictive or prognostic tools in the care of PD patients.

According to Margaret Sutherland, a program director at the NINDS, the PDBP's proteomics projects represent a range of technologies and approaches and the collaborators in program hope to synergize their efforts by sharing sample sets and in some cases replicating each others' work to confirm promising markers.

Some groups have already identified potential markers, and will work to replicate those results. Other projects are focused on expanded searches for protein markers in cerebrospinal fluid and blood plasma. Several will do both, Sutherland told ProteoMonitor this week.

She said that three of the groups — working on developing statistical methods, or specific analytical approaches — have been funded for three years, and another six groups — which will be collecting and analyzing longitudinal samples — have been funded for five years.

Several teams, including the University of Washington's Jing Zhang and Battelle's Vladislav Petyuk, are using mass spectrometry approaches, Sutherland said.

"I'm not sure I'd call it traditional, because there are a lot of new mass spec techniques coming out and these group are applying those to be more selective on the proteins you are selecting, but that is the starting point," she said.

There are also other discovery platforms being used, Sutherland said. For example, Alice Chen-Plotkin of the University of Pennsylvania is leading a group taking an aptamer-based discovery approach using technology from Somalogic. Zhang's group also plans to use the aptamer method in its larger investigation.

Dwight German and Richard Dewey of the University of Texas Southwestern Medical Center are using a peptoid library to try to identify antibodies involved in the disease's inflammatory process. "So there are more twists and turns than just going out and doing mass spec," Sutherland said.

According to Sutherland, many groups have focused in the past on plasma. But the new program is trying to build a workflow that starts with broad analysis of cerebrospinal fluid to find molecules that are unique to the PD population. Then researchers can see if any of these markers carry through to blood and serum.

Sutherland said that although some groups are coming into the program with already-identified potential markers, these possible markers are more in the replication stage than the validation stage.

"Most of the discovery so far has been on small cohorts, so we need to expand that to larger cohorts and across multiple labs so we can get something that is robust and reproducible," she said.

"Strategy-wise, we are talking about how we can pull together, for instance, a common sample people could use to calibrate their assays across labs," she added.

One group that has markers already in hand that it hopes to confirm is Chen-Plotkin's team, which received $480,000 in 2013 funding under the program. She told ProteoMonitor this week that she and her lab found two markers in previous work using multiplex immunoassays — epidermal growth factor, and apolipoprotein A1 — that they now plan to try to corroborate in larger sample sets.

According to the NIH grant abstract for the project, the group previously showed an association between plasma levels of EGF and cognitive impairment in PD — that low EGF levels might both correlate with and predate the onset of dementia in the disease. The group also found that low plasma ApoA1 levels may indicate increased PD risk.

At the same time, the team will also start fresh with a new unbiased discovery screen using Somalogic's aptamer-based technology to measure 1,000 additional molecules that might be associated with the disease.

Chen-Plotkin said the group's initial multiplex immunoassay approach was somewhat limited. "Basically there can be antibody-antibody effects that can cause false positives or false negatives when you are working with something that is immune mediated … So while that approach yielded some promising leads that we are following up, basically I was looking for a way to repeat this process, to start again with an unbiased screen that would canvass more proteins."

According to Chen-Plotkin, for the de novo screen, the team plans to look at 100 PD patients and 50 controls. For the replication of the group's two most promising markers, EGF and ApoA1, the researchers plan to use a new cohort of about 200 to 300 patients from UPenn, as well as two additional cohorts from collaborators at Columbia University and the University of Washington.

"[T]he goal is not for us to function as nine different PIs, but as a consortium, so what I anticipate happening is we'll also be able to test samples collected from other sites in the program," she said.

"We've talked to [a few other groups], and they may want to replicate our promising samples in their sets. We'll have to see what makes the most sense: should they give us samples or should we have them do the testing to make sure its not just been our magic lab's ELISA reader giving us the results?" she said.

Jing Zhang is leading the PDBP project that received the most funding this year, $1.3 million to support a broad screening effort using both mass spectrometry methods and the aptamer-based technology, as well as several techniques that mirror work going on in other program groups.

According to Zhang, his team plans to use multiple-reaction monitoring mass spec to examine patients' CSF for potential markers, as well as targeting proteins known to be involved in the disease and assessing whether they can be used as predictive or prognostic tools.

Moving forward, he said his team will couple MRM mass spec with the aptamer-based approach, as well as RNA sequencing methods being used by other groups in the program. The group will then examine its best-performing candidate markers in plasma to see if any carry through.

Zhang told ProteoMonitor that his team will also extend its research to early PD patients who are considered to be at risk for the disease but who have not developed motor symptoms in order to identify biomarkers capable of diagnosing PD at its earliest stages.

"I think if you ask the groups, the ultimate goal would be to find biomarkers for early diagnosis," Sutherland said. "But the reality with the way we set this program up with longitudinal assessments is that we're really looking for markers that tell us about disease progression."

According to Sutherland, the focus of the PDBP is currently on discovery, but the hope is that it will be able to shift as time goes on into more replication and validation, and be an ongoing effort beyond the end points of these first nine projects.