P3‐172: Identification and Validation of Novel Cerebrospinal Fluid Protein Biomarkers for Early Alzheimer's Disease Using Proteomics and ELISA: a Multi‐institutional Collaborative Study

For maximum effect, disease modifying therapies for Alzheimer disease (AD) must be applied during the ‘preclinical’ stage (amyloid plaques present with cognition intact) before severe neuronal loss occurs. Efficient clinical trials will require biomarkers that can identify early AD pathology, classify disease stage, monitor pathological progression, and predict cognitive decline. To discover such biomarkers, we measured AD-associated changes in cerebrospinal fluid (CSF) using two-dimensional difference-in-gel electrophoresis and liquid chromatography tandem mass spectrometry (2D-DIGE-LC-MS/MS). Because low CSF [Aβ42] correlates with amyloid deposition, we selected n = 24 CSF samples from non-demented controls (Clinical Dementia Rating [CDR0]) with high [Aβ42] and n = 24 from mild ”probable AD” (CDR1) individuals with low [Aβ42]. Samples were subjected to 2D-DIGE. Protein spots with intensity differences between groups were excised, trypsinized, and subjected to LC-MS/MS for protein identification. Findings were validated by enzyme-linked immunosorbent assays (ELISA) for eleven proteins in the original sample set, and for six proteins in a larger independent sample set (N = 292, including CDR0, 0.5 [very mild dementia], and 1 groups). CSF Aβ42, tau and phospho-tau concentrations were also measured; some CDR0 individuals had low CSF [Aβ42], indicating ‘preclinical’ AD. 119 spots differed in abundance between groups. 49 proteins were identified. Of eleven proteins measured by ELISA, six (NCAM, YKL-40, chromogranin-A, carnosinase-I, transthyretin, cystatin-C) showed differences in mean concentration between the original AD (CDR1) and control (CDR0) groups. In the independent sample set, NCAM and YKL-40 showed differences between CDR0 and CDR>0; chromogranin-A and carnosinase-I showed differences between CDR1 and CDR<1. Receiver-operator characteristic curve analyses using a stepwise logistic regression model yielded minimal biomarker panels to distinguish CDR>0 from CDR0 (tau, YKL-40, NCAM) and CDR1 from CDR<1 (tau, chromogranin-A, carnosinase-I) with areas under the curve of 0.90 and 0.88, respectively. This study identifies four novel CSF biomarkers for early AD diagnosis, and suggests that a panel of six CSF biomarkers (NCAM, YKL-40, chromogranin-A, carnosinase-I, tau, Aβ42) might aid subject categorization into four early clinicopathological stages (CDR0 without amyloid, CDR0 with amyloid [‘preclinical' AD], CDR 0.5 and CDR 1), allow monitoring of disease progression, and (in conjunction with another study: Craig-Schapiro RJ et al, submitted) predict cognitive decline.