Establishing an Autopsy-Based Cerebrospinal Fluid Biomarker Signature in Alzheimer Disease Patients Free

Featured Article: Shaw LM, Vanderstichele H, Knapik-Czajka M, Clark CM, Aisen PS, Petersen RC, et al. Cerebrospinal fluid biomarker signature in Alzheimer's Disease Neuroimaging Initiative subjects. Ann Neurol 2009;65:403-13.^†

At the time this work was conducted (2007 to 2008), the research use of cerebrospinal fluid (CSF) biomarker proteins linked to Alzheimer disease (AD) hallmarks—β-amyloid (Aβ) plaque load and tau pathology (Aβ1-42, t-tau, and p-tau181) —was becoming established practice amongst a number of experienced laboratories as reliable and accurate indices of brain pathology in living subjects (1–3). These studies mainly used either a widely employed ELISA methodology (4) or the newer (at the time) microbead-based biomarkers multi analyte profiling (xMAP) immunoassay format, developed by Innogenetics (now Fujirebio) (5). The data generated from more than 25 studies that used these techniques and CSF provided strong support for a more objective measurement of amyloid and tau pathology in living individuals, compared to clinical diagnosis of AD dementia (6). The clinical diagnosis of AD is only up to about 80% accurate and our 2009 study in Annals of Neurology and other studies provided the foundation for moving towards a biological basis (protein biomarker profile) for more accurate clinical diagnosis of the presence of ongoing AD brain pathology. Two populations were used in our study: (a) participants in the multicenter study, the Alzheimer's Disease Neuroimaging Initiative (ADNI; 100 patients with early AD, 196 patients with mild cognitive impairment, and 115 cognitively normal age-matched controls, all clinically diagnosed), provided CSF samples collected at 56 centers at the participants’ baseline visit, and (b) an independent population, followed at the University of Pennsylvania Alzheimer's Disease Clinical Core, of 56 autopsy-confirmed AD participants and 52 age-matched living cognitively normal controls. Besides inclusion of an ADNI-independent population for assessment of cutpoint values for each of the 3 AD biomarkers as well as for the ratios t-tau/Aβ1-42 and p-tau181/Aβ1-42, another key feature in this study was the close collaboration between an in vitro diagnostic company and a university laboratory with the aim of generating best-in-class assays for the benefit of patients, and the inclusion of autopsy diagnosis for the patients with AD, the accepted gold standard for diagnosis of this disease. This enabled (a) determination of the accuracy of prediction of AD pathology assessed by receiver operating characteristic curve (AUC) analyses, (b) determination of cutpoints for the individual proteins and their ratios, and (c) establishment of a logistic regression model that combined Aβ1-42, t-tau, and APOε4 genotype. AUC values ranged from 0.913 for CSF Aβ1-42 concentration alone up to 0.942 for the combination of Aβ1-42, t-tau, and APOε4 genotype. Importantly, the same lot of reagents was used for the analyses in both populations, thus removing lot-to-lot variability from measurement errors. At the time this work was done, it was a considerable source of variability using this “for research-use-only (RUO)” immunoassay. It was an important additional feature of this study.

These cutpoints were then applied to the ADNI participant data, for assessments of the presence or absence of AD pathology and for prediction of progression to a clinical diagnosis of AD dementia.

Despite the advances in the validation of CSF AD biomarker-based diagnosis of Alzheimer disease this study provided, using the xMAP-based immunoassay with demonstrated intra-laboratory precision performance of 5% to 10% CV across the 3 AD biomarkers, a major limitation of this RUO methodology was the limited ability to achieve inter-center agreement (CV values ranging up to 30% across centers) using a common set of CSF samples, likely due to the many steps involved in this highly manual methodology. The Annals of Neurology article featured here helped set the stage for the future development of highly automated methods developed by Roche (Elecsys platform) and Fujirebio (Lumipulse platform), each with a documented analytical precision in the 3% to 7% CV range. The Lumipulse assay was recently granted approval from the US Food and Drug Administration to market as the first in vitro diagnostic test for early detection of amyloid plaques associated with Alzheimer disease using the ratio Aβ1-42/Aβ1-40 in CSF. John Trojanowski, one of the authors of the article featured here, recently passed away and we take this opportunity to express our continued appreciation for his inspiration of relating combined measurement of biomarker analysis in biofluids to assess neuropathology in living patients.

Nonstandard Abbreviations

CSF, cerebrospinal fluid; AD, Alzheimer disease; Aβ, β-amyloid.

Author Contributions

All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved.

Authors’ Disclosures or Potential Conflicts of Interest

Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:

Employment or Leadership

K. Blennow, co-founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program.

Consultant or Advisory Role

L. Shaw, Biogen, Roche Diagnostics, Fujirebio; K. Blennow, Abcam, Axon, BioArctic, Biogen, JOMDD/Shimadzu, Lilly, MagQu, Ono Pharma, Roche Diagnostics, and Siemens Healthineers.

Stock Ownership

K. Blennow, Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program.

Honoraria

L. Shaw, Biogen, Roche Diagnostics, Fujirebio; K. Blennow, Novartis, Julius Clinical.

Research Funding

L.M. Shaw, NIH U19 AG024904 to University of Pennsylvania; in-kind support (reagents and instrumentation) from Roche Diagnostics to the laboratory for performance testing in the ADNI3 study at the University of Pennsylvania; J.Q. Trojanowski, NIH U19 AG024904 to University of Pennsylvania; H. Vanderstichele, none declared; M. Knapik-Czajka, none declared.

Expert Testimony

None declared.

Patents

None declared.

References

Author notes