For decades, scientists have yearned for a blood test that can tell who accumulates amyloid in the brain. A simple blood draw would help fill clinical trials much more quickly and cheaply than PET scans or lumbar punctures. The dream looks set to become a reality. In the January 31 Nature, Japanese and Australian researchers unveil a sensitive and reproducible blood assay that can predict amyloid status with up to 90 percent accuracy. The test, which uses immunoprecipitation of Aβ peptides followed by mass spectrometry, works as well as existing CSF biomarkers at picking up brain amyloid. Katsuhiko Yanagisawa, National Center for Geriatrics and Gerontology in Aichi, Japan, joined with Shimadzu Corporation scientists and Colin Masters of the University of Melbourne in Australia to develop and test the serum-based method using two different research cohorts.
- New plasma Aβ assay predicts brain amyloid with 90 percent accuracy.
- The assay works in independent clinical cohorts.
- Blood tests promise to speed clinical trial recruitment, cut costs.
The report comes on the heels of work from Randall Bateman, Washington University in St. Louis, who presented and published a comparable blood test just last year (Jul 2017 conference news and Ovod et al., 2017). “It is remarkable that this group totally independently developed a very similar kind of assay, and found pretty much identical results,” Bateman told Alzforum. “This is really good news, and it tells me this approach is definitely going to work.”
Paul Aisen, University of Southern California, agreed. “The two groups’ efforts have clearly yielded plasma assays that are indicative of brain amyloidosis. That is a tremendous advance,” Aisen said.
Ups and Downs. Quantitation of plasma Aβ peptides by mass spec reveals candidate biomarkers for amyloidosis in Japanese (NCGG) and Australian cohorts (AIBL). [Nakamura et al., Nature.]
The new assays need validation, including replication and optimization in different populations, but Bateman thinks that can happen quickly. It may only take a few years for blood biomarkers to make their way into practice, he predicted.
The idea of a blood test to detect brain amyloid has been around almost as long as researchers have known about amyloid (Rumble et al., 1989). Despite many attempts over the years, no one had been able to overcome the technical challenge of precisely quantitating sticky peptides present in picomolar concentrations in a stew of thousands of other molecules. As it turns out, Koichi Tanaka, the Nobel Prize-winning chemist who helped perfect mass spec for proteins, has been quietly working on the problem. Tanaka and colleagues at Shimadzu Corporation immunoprecipitated APP-derived peptides from human plasma, then identified and quantitated them using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Kaneko et al., 2014). Although MALDI-TOF is not normally considered a quantitative technique, Tanaka and colleagues managed to develop an accurate and reproducible assay for Aβ peptides, with variances less than 10 percent for repeat measures.
They focused on three Aβ peptides: Aβ42, Aβ40, and APP669-711. The latter comprises Aβ40 with an additional three amino acids at its N terminus. Its concentration in blood does not change in people with Alzheimer’s disease, so it serves as an internal control for fluctuations in Aβ42. In a small sample of 62 people, the APP669-711/Aβ42 ratio pinpointed who had brain amyloid with more than 90 percent accuracy (Kaneko et al., 2014).
For the new study, first author Akinori Nakamura and colleagues refined and further tested the assay using binding of the amyloid PET ligand PiB as a gold standard. Initial testing in a discovery cohort of 121 Japanese, 50 of whom were amyloid-positive by PiB PET, was followed up with samples from a second group comprising 252 participants in the Australian Imaging, Biomarker, and Lifestyle Study of Aging. In AIBL, about half the participants had brain amyloid detected by a PiB, florbetapir, or flutemetamol scan. Each cohort contained a balance of people clinically classified as being cognitively normal, having mild cognitive impairment, or having AD.
Plasma Markers. Analysis reveals high sensitivity and specificity for plasma biomarkers in Japanese (left) and Australian cohorts (right). [Courtesy of Nakamura et al., Nature.]
The results were striking. In both cohorts, the researchers found a small but significant decrease in serum Aβ42 in amyloid-positive people compared with their amyloid-negative counterparts. Receiver operator characteristic (ROC), a statistical analysis that approximates accuracy, showed that by itself, Aβ42 moderately predicted PET positivity, with area under the curve (AUC) values of 87.2 percent in the Japanese and 71.8 in the Australian cohort. Ratios of Aβ40/42, APP669-711/42, or a composite biomarker that incorporated both ratios all demonstrated better predictive power, which was highest in the PiB-PET groups and slightly lower when other tracers were used. The Aβ40/42 ratio reached 90.9 percent accuracy in the Japanese cohort, 88.9 percent in the Australian PiB-only group, and 83.7 percent in the whole Australian cohort. The performance in the Australian cohort was virtually identical to what the St. Louis team achieved with their IP-MS assay in their AD cohort, Bateman told Alzforum.
In optimizing their biomarkers, the investigators broke with tradition and flipped the ratio, using Aβ40/42 as their readout, rather than the more common 42/40. They did that because putting Aβ42 in the denominator yielded data with a smoother distribution, which is important when trying to detect small differences in plasma Aβ between groups. “It’s a very small point but I think it could be a critical factor contributing to the high performance,” Masters said.
The most accurate predictor of amyloidosis turned out to be the composite, which the investigators calculated by averaging the Aβ40/42 and APP669-711/Aβ42 measures. Indeed, the composite proved equal to CSF Aβ at predicting PET positivity, showing about 80 percent accuracy in a small cohort of 46 people from AIBL who had both CSF and PET analysis.
Some commentators raised concerns about combining the two ratios in that way. “Ideally, a composite would come from two independent markers,” said Henrik Zetterberg, University of Gothenburg, Sweden. “The APP669-711/Aβ42 ratio is not independent from the Aβ40/42 ratio. In effect, the Aβ1-42 changes are counted twice."
Sid O’Bryant, University of North Texas Health Science Center, Fort Worth, shared that concern. He wrote to Alzforum that the composite score could have skewed the data given that Aβ42 is the marker that was most impactful in the first place, and the only one that was normally distributed. But he feels the method might be valuable. “While the statistical approach is not what I would have anticipated, if others can replicate the use of this approach using these analytic methods, it is possible that the authors are onto something very novel and highly important," he wrote in an email to Alzforum.
The investigators did see a systematic difference in the Aβ42 measurements between the cohorts, with values in the Australian group coming in approximately 25 percent lower. Since all the assays were done at Shimadzu, they believe the variation arises mainly during sample handing, an issue that has flummoxed CSF biomarker analysis as well (Apr 2017 conference news). Nonetheless, Nakamura was able to combine data from the two cohorts to derive a single, optimized cut-off value for the composite biomarker of 0.376. In the individual cohorts, this cut-off value still gave over 85 percent accuracy. In a separate, follow-up group of 51 Japanese subjects, the predetermined cut-off was 90 percent accurate at assigning amyloid status.
Erik Portelius, University of Gothenburg, Sweden, would like to see a comparison of the different methods being developed for serum amyloid assays. “The authors show convincingly that plasma Aβ40/Aβ42 ratio indeed reflects CNS-derived Aβ, since the plasma Aβ levels correlated with both CSF Aβ concentrations and amyloid PET. However, as the authors point out, the finding needs to be replicated. More importantly, studies comparing available methods (mass spectrometry-based and immune-based, such as ELISA) should be conducted in order to fully understand this novel promising plasma biomarker,” he wrote in an email to Alzforum (Dec 2017 conference news and Fandos et al., 2017). Portelius had some of the earliest success in measuring plasma Aβ42 by IP/mass spec (Pannee et al., 2014).
Kaj Blennow, also from UGothenburg, said he, too, would like to see comparison with ELISA and the more sensitive single-molecule array (SIMOA) approach.
If blood tests can be validated, they will pay off by reducing the number of PET screens needed to identify people with preclinical AD for prevention trials, Masters said. “At the moment, to get 100 people into a preclinical study, we have to screen nearly 700 with a PET scan. If we use a blood test first, we need to scan only about 150. That’s a huge savings in cost and efficiency,” he said. A serum biomarker might eventually be useful for differential diagnoses of dementia, and for monitoring Aβ clearance in clinical trials, though both will take a lot more study, he said.
AIBL researchers will continue to collaborate with Shimadzu, and Masters said they have many experiments planned to look at the marker longitudinally, and to ask if it correlates with cognitive impairment.
Bateman reported his group just received a grant to accelerate the development and validation of their test. “We are moving forward with a large clinical study, looking at samples from existing studies, and collecting new samples, too,” he told Alzforum. C2N Diagnostics, a St. Louis company co-founded by Bateman and his WashU colleague David Holtzman, is leading efforts to develop his IP/MS assay for commercial use.
The authors said they are now working on an automated version of the assay, to standardize the analysis and increase throughput. They are open to accepting samples from other labs outside of Japan, Tanaka wrote in an email to Alzforum.—Pat McCaffrey
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- Automated CSF Tests: Check. Blood Tests: In the Works
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No Available Further Reading
- Nakamura A, Kaneko N, Villemagne VL, Kato T, Doecke J, Doré V, Fowler C, Li QX, Martins R, Rowe C, Tomita T, Matsuzaki K, Ishii K, Ishii K, Arahata Y, Iwamoto S, Ito K, Tanaka K, Masters CL, Yanagisawa K. High performance plasma amyloid-β biomarkers for Alzheimer's disease. Nature. 2018 Feb 8;554(7691):249-254. Epub 2018 Jan 31 PubMed.