As the Alzheimer’s field matures, it has begun the task of moving fluid biomarkers from the discovery stage toward the clinic. For cerebrospinal fluid markers of AD, researchers have made great strides toward standardization, but the field’s more nascent blood-based biomarkers remain plagued by problems with replication and methodology. Case in point: A 2014 Nature Medicine report from Howard Federoff, then at Georgetown University Medical Center, Washington, D.C., and Mark Mapstone, then at the University of Rochester School of Medicine, New York, described a panel of 10 plasma lipids that reportedly presaged cognitive impairment within the next few years. Both Federoff and Mapstone are now at the University of California, Irvine. The paper generated excitement and headlines around the world. It also kicked off efforts by multiple groups to repeat the findings. The first of these has now been published. In the January 21 Alzheimer’s & Dementia, researchers led by Madhav Thambisetty at the National Institute on Aging in Baltimore reported they were unable to reproduce the findings in two larger, independent cohorts.
Does this mean the 10-lipid panel has no validity across populations? Not necessarily. Federoff and Mapstone note that methodological differences between the studies may explain the discrepancies. Meanwhile, replication attempts by them and others continue. More broadly, biomarker researchers point to consistent evidence that phospholipids in blood do associate with AD, and maintain that the hunt for a metabolite-based blood test will eventually bear fruit. The National Institutes of Health has put its weight behind this effort because of the advantages in cost and convenience that a blood test would have over imaging or spinal taps for routine clinical use.
Researchers lauded the current focus on replication and standardization. “This is an excellent study and a solid contribution to the field,” said Sid O’Bryant at the University of North Texas Health Science Center, Fort Worth, of Thambisetty’s work. “It does exactly what we have to do: take findings from preliminary studies into large cohorts,” O’Bryant added. Henrik Zetterberg at the University of Gothenburg, Mölndal, Sweden, noted, “These types of independent replication studies are essential. In other fields, it is extremely hard to publish these types of negative findings in high-impact journals.”
Despite much research in this area, a blood-based test for Alzheimer’s has remained elusive (see Jun 2013 webinar; AlzBiomarker database). Although numerous plasma markers differ between people with AD and healthy controls, these typically lack the sensitivity and specificity necessary for a diagnostic test (see Kivipelto et al., 2006; Doecke et al., 2012; Burnham et al., 2013). Ideally, researchers want a test that would predict who among a cognitively healthy population will go on to develop AD. A small 2007 study reported finding a panel of 18 blood proteins that might do the trick, but no one has been able to replicate that data (see Oct 2007 news).
Thus, great enthusiasm greeted the 2014 report from Federoff and Mapstone of a predictive lipid panel. The 10 plasma metabolites distinguished 28 people who went on to develop cognitive impairment within three years from 53 controls who remained healthy, and did so with 90 percent sensitivity and specificity, the authors reported (see Mar 2014 news). The authors have since refined their test to a 24-metabolite panel that they claim has somewhat better predictive power, using data from the same cohort (see Fiandaca et al., 2015).
To repeat the findings, Thambisetty and colleagues used serum samples from two different longitudinal cohorts, the Baltimore Longitudinal Study of Aging (BLSA) and the Age, Gene/Environment Susceptibility-Reykjavik Study (AGES-RS). The latter is a collaborative study between the NIA and the Icelandic Heart Association to look at risk factors for Alzheimer’s and other age-related diseases in a long-running longitudinal cohort. First author Ramon Casanova analyzed baseline and five-year serum samples using the same commercial metabolomics platform as the Federoff study did, namely the AbsoluteIDQ p180 assay from Biocrates Life Science AG, Innsbruck, Austria. In the BLSA study, the authors compared 93 people who progressed to AD with 99 others who did not. From the AGES-RS cohort, Casanova et al. analyzed 100 progressors vs. 100 who stayed stable. All participants were cognitively normal at baseline. Diagnosis was determined by clinical consensus among neuropsychologists and clinicians in the respective studies.
The 10-lipid panel performed poorly in both cohorts, with a sensitivity of around 50 percent for predicting people who would progress to AD, the authors reported. To find out if any metabolites in the AbsoluteIDQ platform correlated with disease progression, the authors then analyzed all 187 markers. They found a set of markers that had moderate predictive power in the BLSA cohort, as well as a slightly different set that distinguished people with current AD from healthy controls in the BLSA, but neither replicated in the AGES-RS cohort. While there may be a metabolite profile associated with AD, the specific panels explored so far are not clinically useful, Thambisetty told Alzforum.
Mapstone disagrees. He contends that differences in methodology, such as the use of serum instead of plasma, may account for the failure to find a signal. “We do not believe valid conclusions can be drawn from this attempt at replication,” Mapstone wrote to Alzforum (see full comment below). Speaking with Alzforum, he added, “There are certain conditions we controlled for in our study that were not taken into account in this one. If you’re doing a replication study, you need to do it prospectively and not use samples on hand.” He said he and his colleagues would detail the issues in a letter to the journal.
O’Bryant agreed that differences in the blood fraction used could play a role. “When you look across serum and plasma, the results are often different,” he told Alzforum, adding, “The jury is still out on the Federoff panel.” O’Bryant was not involved in either study.
He and others stressed that the field must standardize methodology for collection, processing, and storage of samples. In 2015, the international Blood-Based Biomarker Interest Group (BBBIG) and STandards for Alzheimer's Research in Blood biomarkers (STAR-B) published the first guidelines for processing Alzheimer’s blood protein biomarkers (see O’Bryant et al., 2015). This group comprises largely AD and assay development researchers in academia, industry, and diagnostics companies. They incorporate lessons learned from the CSF field. The group is currently preparing additional guidelines for protein biomarker validation studies, O’Bryant said.
Meanwhile, other groups are turning their attention to small molecules. The National Institute on Aging supports the Alzheimer’s Disease Metabolomics Consortium, a large initiative of different groups. It brings together eight metabolomics and informatics centers, led by Rima Kaddurah-Daouk at the Duke Institute for Brain Studies, Durham, North Carolina (see Feb 2011 news series). The consortium is applying a standardized approach to define metabolic changes in Alzheimer patients, Kaddurah-Daouk said. Numerous factors affect blood metabolites, including sex, age, diet, and medications, and they must be accounted for in analyses, she added. The consortium partners with the Alzheimer’s Disease Neuroimaging Initiative to identify new biomarkers and drug targets using ADNI samples.
As part of this effort, Kaddurah-Daouk has examined the Federoff panel in 800 participants in ADNI1, who ranged from cognitively healthy to having dementia. This particular 10-lipid panel did not predict impairment in this cohort, she told Alzforum. However, she did see a consistent correlation between Alzheimer’s disease and perturbed phospholipid metabolism, supporting other findings in the field such as those from Federoff and Thambisetty. “Many papers have come to the conclusion that there is a problem in phospholipid metabolism in AD, but it will take rigorous standardized studies to zoom in on [specific] biomarkers,” Kaddurah-Daouk said. She emphasized the need to deal with confounding factors such as medications that could have affected the findings in the reported studies. Profiling ADNI2/GO samples with use of matched plasma and serum will provide additional insight about the limitations of the data available thus far, she added.
Other biomarker experts concur that the field is slowly homing in on reliable blood markers, even if it has not found quite the right combination yet. Proteins such as clusterin and C-reactive protein, for example, consistently produce a signal in multiple studies (see Jongbloed et al., 2015; Liang et al., 2015; Yarchoan et al., 2013). “It is encouraging that different groups agree on some of the pathways/metabolites that seem to play a role in disease development and progression,” Eugenia Trushina at the Mayo Clinic in Rochester, Minnesota, wrote to Alzforum (see full comment below). Thambisetty agreed, “A large body of evidence suggests there is a blood signal associated with core pathological features of AD.”
The challenge for the field now is to refine the procedures enough to get repeatable results across different populations. This will be necessary to take these markers to the clinic. “The gap between discovery and clinical implementation is all about methods,” O’Bryant said.—Madolyn Bowman Rogers
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