19 Aug 2015

As amyloid fibrils hog Aβ in the brain, less ends up in the cerebrospinal fluid. Myriad studies suggest that this dearth correlates with a positive amyloid PET scan. However, the use of CSF Aβ as diagnostic tool has been stymied by lab-to-lab and day-to-day variability in the immunoassays used to quantify the peptide. Though an international consortium has tried to standardize assays and reduce this variability to acceptable levels, it still prevails to some degree. Could fully automated assay systems be the answer? At this year’s Alzheimer’s Association International Conference (AAIC), held July 18-23 in Washington, D.C., researchers reported that two of these systems, which require users to do little more than unscrew a tube and push a button, yield highly reproducible results. If given the nod by regulatory agencies, these devices could pop up in clinics and serve as both selection and monitoring tools in clinical trials. The jury is still out on whether the platforms will edge amyloid PET as a diagnostic, or academic researchers will ditch their traditional CSF assays for automated ones. Some experts suggest that much may depend on price and who controls the reimbursement purse strings. Approval of a treatment that slows or stops AD could be a game-changer, as it would unleash a flood of older adults looking for a diagnostic test, and that in turn might be enough to tip the balance toward simpler and cheaper CSF analysis, they say. 

Two kinds of variability plague the CSF measurements. On one hand, assays developed by different companies give different results. On the other, there is variability from run to run in any given assay. The Alzheimer’s Association funded the Quality Control Initiative in 2009 to attack the latter problem (see Nov 2009 news). Headed by Kaj Blennow at the University of Gothenburg in Sweden, the initiative periodically sends out CSF samples to member labs, which use their assay of choice to measure levels of Aβ42, phospho-tau (p-tau), and/or total tau. The researchers then attempt to weed out the sources of variability and standardize protocols. In 2012, the variability hovered around 25 percent (see Aug 2012 news).

At AAIC, Blennow reported that the variability hasn’t budged much in the last three years. It runs at 18 percent to 20 percent for several popular tests, including immunoassays from Meso Scale Discovery (MSD) and INNO-BIA AlzBio3 from Fujirebio (formerly Innogenetics), which was used in the Alzheimer’s Disease Neuroimagining Initiative 2 (ADNI2) and ADNI GO studies. To be fair, ADNI researchers have reduced this variability to around 10 percent by streamlining their protocols between study centers and trading human hands for pipet robots in some cases (see Figurski et al., 2012; Kang et al., 2015). However, the variability is not quite up to snuff for diagnostic standards, which ideally would fall between 2 percent and 4 percent, Blennow said. Michal Figurski of the University of Pennsylvania attributed the remaining variability to changes in lab temperature and humidity, as well as inconsistency in reagents from the manufacturer. “I do not know whether automated systems will change that. In principle they have the potential to do so; in practice, we’ll see,” Figurski told Alzforum.

Responding to this call for consistency, a handful of in vitro diagnostic companies have put forward fully automated systems that require nothing more from the user than plopping the sample tube into a machine. At AAIC, Blennow presented data from Roche Diagnostics’ Elecsys® immunoassays for Aβ42, p-tau, and tau that run on the Cobas E601 instrument. Blennow reported a variability of 0.6 to 3.5 percent for Aβ assays run at four different labs in the United States and Europe, which tested three different immunoassay lots twice a day for five days. This falls well within the realm of diagnostic acceptability, he said.

Roche Diagnostics is seeking FDA approval for the automated CSF biomarker tests as an in vitro diagnostic (IVD). The company has nearly completed the technical portion of the approval process, which ensures that the assay is consistent and reliable, explained Tobias Bittner of Roche Diagnostics in Penzberg, Germany. It now faces the more difficult hurdle of convincing the FDA that the test can be used as a clinical indicator of amyloid burden. Access to well-characterized clinical samples is key for this, and Roche is vying for its biomarker assay to be selected for the upcoming ADNI3 study.

In a poster presentation, Sandra Pereson from Fujirebio in Ghent, Belgium, summarized results from that company’s automated system, the Lumipulse. Its assay can measure CSF Aβ42 up to 4,500 pg/mL and total tau up to 2,500 pg/mL, and its in-house reproducibility for CSF Aβ42 was similar to Roche’s Cobas system. Fujirebio is also competing for the prized ADNI3 spot. The company plans to market this assay and device in Europe and the United States in addition to Japan, Pereson told Alzforum.

Assuming an automated system is approved, how will that change the landscape of biomarker testing? Bittner said the Roche Diagnostics platform is already being used under an Investigational Use Only (IUO) status for clinical trial patient selection. Roche is currently using the instrument in a Phase 3 trial of its monoclonal antibody gantenerumab in people with mild dementia due to Alzheimer’s disease. The company is also seeking the wider designation of a stand-alone IVD, which would allow physicians and other pharma companies to purchase the assays off the shelf for use in trials. As of now, they can use these Roche Diagnostics assays only through collaboration with the company.

Whether academic labs will adopt one of the automated platforms will depend on how much they will use it and why, said Henrik Zetterberg of the University of Gothenberg in Sweden. In addition to measuring Aβ, tau, and p-tau, researchers can use the instrument to quantify other potential markers, such as neuroinflammatory or cardiovascular-related proteins. Labs that process very few samples may have less incentive to automate. Zetterberg added that the current variability in tests such as AlzBio3 may still be acceptable for many research uses, but that as the cost of fully automated systems drops, more labs may jump aboard. Another factor is time. Blennow pointed out that while it only takes 20 minutes to run an assay on a fully automated machine, it takes two days before a standard ELISA returns a result.

The automated instruments could make a splash as diagnostic tools, since they are already generally used in clinical history labs at hospitals for a wide variety of diagnostic tests, Blennow said. They can run one patient sample at a time and pop out results so quickly that the clinician will have direct feedback to support the diagnostic decision. However, although CSF analysis is much cheaper than a PET scan, which costs several thousand dollars, some doctors in the United States still may prefer the latter to performing a lumbar puncture. Blennow thinks that may change once a treatment that slows the disease is approved, because then insurers will reimburse for the most cost-effective diagnostic test, especially given that CSF Aβ42 and amyloid PET give the same diagnostic accuracy. While payers currently cover either test, the Centers for Medicare and Medicaid Services (CMS) is reimbursing for PET in an exploratory study (see Apr 2015 news).

Most experts agree that CSF biomarker tests will never fully supplant amyloid PET, as the two measure two different things. Fresh data presented by Niklas Mattsson of Lund University in Sweden bore this out. While his findings strengthened the concordance between the two measures, showing that CSF Aβ, as measured by the AlzBio3 test, predicted amyloid PET positivity 98 percent of the time, he also found that a small number of patients in ADNI2 had pathological CSF Aβ42 levels but appeared in the normal range on amyloid PET. These patients subsequently tested positive in repeat amyloid-PET scans, suggesting that CSF Aβ detects earlier stages of disease.

Despite their similarities, each test has its unique benefits, Mattsson said. Unlike CSF tests, PET reflects the distribution of amyloid throughout the brain, and the scans may be particularly relevant in monitoring the effects of treatments aimed at fibrillar amyloid. They also don’t cause the headaches associated with lumbar puncture, especially in younger patients. On the other hand, CSF biomarker tests have a leg up on PET scans in that they can test multiple markers at once, and even measure the effects of treatments such as BACE inhibitors on APP processing.

While having automated systems, as well as streamlining assay techniques, will improve variability between runs of the same assay, researchers still need a way to harmonize findings between different assays. To this end, researchers led by Zetterberg and Blennow, in collaboration with the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), have whipped up a master reference material made of pooled human CSF samples that contain either low, medium, or high levels of Aβ42, confirmed by mass spectrometry (see Leinenbach et al., 2014). Once a giant batch is made by the Institute for Reference Materials and Measurements (IRMM), assay vendors will use this material to calibrate their kits.

At a pre-meeting on the eve of AAIC, members of the Alzheimer’s Association-backed Global Biomarker Standards Consortium met to discuss progress on development of the reference materials. While the Aβ42 lot is well on its way to completion, researchers are now working out strategies for similar batches of tau and p-tau. These decisions, including which isoforms of tau to use as a reference material, are set to be made at meetings in the fall, Zetterberg said, and after that researchers can start making and validating the material.—Jessica Shugart

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References

News Citations

1. Worldwide Quality Control Set to Tame Biomarker Variation 18 Nov 2009
2. CSF Markers: Goodbye, Research Use Only; Hello, Clinical 1 Aug 2012
3. $100M IDEAS: CMS Blesses Study to Evaluate Amyloid Scans in Clinical Practice 16 Apr 2015

Therapeutics Citations

1. Gantenerumab

Paper Citations

1. Figurski MJ, Waligórska T, Toledo J, Vanderstichele H, Korecka M, Lee VM, Trojanowski JQ, Shaw LM, . Improved protocol for measurement of plasma β-amyloid in longitudinal evaluation of Alzheimer's Disease Neuroimaging Initiative study patients. Alzheimers Dement. 2012 Jul;8(4):250-60. PubMed.
2. Kang JH, Korecka M, Figurski MJ, Toledo JB, Blennow K, Zetterberg H, Waligorska T, Brylska M, Fields L, Shah N, Soares H, Dean RA, Vanderstichele H, Petersen RC, Aisen PS, Saykin AJ, Weiner MW, Trojanowski JQ, Shaw LM, Alzheimer's Disease Neuroimaging Initiative. The Alzheimer's Disease Neuroimaging Initiative 2 Biomarker Core: A review of progress and plans. Alzheimers Dement. 2015 Jul;11(7):772-91. PubMed.
3. Leinenbach A, Pannee J, Dülffer T, Huber A, Bittner T, Andreasson U, Gobom J, Zetterberg H, Kobold U, Portelius E, Blennow K, IFCC Scientific Division Working Group on CSF proteins. Mass spectrometry-based candidate reference measurement procedure for quantification of amyloid-β in cerebrospinal fluid. Clin Chem. 2014 Jul;60(7):987-94. Epub 2014 May 19 PubMed.

Further Reading

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