Bianca Van Broeck

Diagnostic Accuracy of Cerebrospinal Fluid Amyloid-beta Isoforms for Early and Differential Dementia Diagnosis

BACKGROUNDnOverlapping cerebrospinal fluid biomarkers (CSF) levels between Alzheimers disease (AD) and non-AD patients decrease differential diagnostic accuracy of the AD core CSF biomarkers. Amyloid-β (Aβ) isoforms might improve the AD versus non-AD differential diagnosis.nnnOBJECTIVEnTo determine the added diagnostic value of Aβ isoforms, Aβ(1-37), Aβ(1-38), and Aβ(1-40), as compared to the AD CSF biomarkers Aβ(1-42), T-tau, and P-tau(181P).nnnMETHODSnCSF from patients with dementia due to AD (n = 50), non-AD dementias (n = 50), mild cognitive impairment due to AD (n = 50) and non-demented controls (n = 50) was analyzed with a prototype multiplex assay using MSD detection technology. The non-AD group consisted of frontotemporal dementia (FTD; n = 17), dementia with Lewy bodies (DLB; n = 17), and vascular dementia (n = 16).nnnRESULTSnAβ(1-37) and Aβ(1-38) increased accuracy to differentiate AD from FTD or DLB. Aβ(1-37), Aβ(1-38), and Aβ(1-40) levels correlated with Mini-Mental State Examination scores and disease duration in dementia due to AD. The Aβ(1-42)/Aβ(1-40) ratio improved diagnostic performance of Aβ(1-42) in most differential diagnostic situations. Aβ(1-42) levels were lower in APOE ε4 carriers compared to non-carriers.nnnCONCLUSIONSnAβ isoforms help to differentiate AD from FTD and DLB. Aβ isoforms increase diagnostic performance of Aβ(1-42). In contrast to Aβ1-42, Aβ isoforms seem to be correlated with disease severity in AD. Adding the Aβ isoforms to the current biomarker panel could enhance diagnostic accuracy.

Synthesis and Evaluation of a Zr-89-Labeled Monoclonal Antibody for Immuno-PET Imaging of Amyloid-β Deposition in the Brain

PurposeThe aim of this study was to evaluate the in vitro and in vivo characteristics of [89Zr]JRF/AβN/25, a radiolabeled monoclonal antibody directed against amyloid-β (Aβ).ProceduresJRF/AβN/25 was labeled with 89Zr following modification with desferal. The affinity of the tracer for Aβ1–40 was determined in a saturation binding assay. In vitro stability was evaluated, and in vivo plasma stability and biodistribution of [89Zr]Df-Bz-JRF/AβN/25 were determined in wild-type mice. To evaluate whether the antibody can cross the blood-brain barrier, brain uptake in wild-type mice was additionally assessed by ex vivo autoradiography.Results[89Zr]Df-Bz-JRF/AβN/25 was obtained in an average radiochemical yield of 50 % and a radiochemical purity of >97 %. A saturation binding assay demonstrated specific binding of [89Zr]Df-Bz-JRF/AβN/25 to Aβ1–40 with nanomolar affinity. The tracer was stable in buffer and proved to be stable in vivo with >92 % intact monoclonal antibody (mAb) remaining in the plasma at 48 h post injection. A biodistribution study showed a slow blood clearance with no significant accumulation of activity in any of the organs. Furthermore, [89Zr]Df-Bz-JRF/AβN/25 demonstrated modest brain penetration, which slowly decreased in time. This cerebral uptake was confirmed by ex vivo autoradiography.Conclusions[89Zr]Df-Bz-JRF/AβN/25 binds with high affinity to Aβ1–40. The tracer displays an acceptable in vivo stability and is able to cross the blood-brain barrier. [89Zr]Df-Bz-JRF/AβN/25 might therefore be a potential candidate for in vivo imaging of Aβ deposition in the brain.

Impact of frequent cerebrospinal fluid sampling on Aβ levels: systematic approach to elucidate influencing factors

BackgroundCerebrospinal fluid (CSF) amyloid-beta (Aβ) peptides are predictive biomarkers for Alzheimer’s disease and are proposed as pharmacodynamic markers for amyloid-lowering therapies. However, frequent sampling results in fluctuating CSF Aβ levels that have a tendency to increase compared with baseline. The impact of sampling frequency, volume, catheterization procedure, and ibuprofen pretreatment on CSF Aβ levels using continuous sampling over 36xa0h was assessed.MethodsIn this open-label biomarker study, healthy participants (nu2009=u200918; either sex, age 55u2009−u200985xa0years) were randomized into one of three cohorts (nu2009=u20096/cohort; high-frequency sampling). In all cohorts except cohort 2 (sampling started 6xa0h post catheterization), sampling through lumbar catheterization started immediately post catheterization. Cohort 3 received ibuprofen (800xa0mg) before catheterization. Following interim data review, an additional cohort 4 (nu2009=u20096) with an optimized sampling scheme (low-frequency and lower volume) was included. CSF Aβ1–37, Aβ1–38, Aβ1–40, and Aβ1–42 levels were analyzed.ResultsIncreases and fluctuations in mean CSF Aβ levels occurred in cohorts 1–3 at times of high-frequency sampling. Some outliers were observed (cohorts 2 and 3) with an extreme pronunciation of this effect. Cohort 4 demonstrated minimal fluctuation of CSF Aβ both on a group and an individual level. Intersubject variability in CSF Aβ profiles over time was observed in all cohorts.ConclusionsCSF Aβ level fluctuation upon catheterization primarily depends on the sampling frequency and volume, but not on the catheterization procedure or inflammatory reaction. An optimized low-frequency sampling protocol minimizes or eliminates fluctuation of CSF Aβ levels, which will improve the capability of accurately measuring the pharmacodynamic read-out for amyloid-lowering therapies.Trial registrationClinicalTrials.gov NCT01436188. Registered 15 September 2011.

Profiling the dynamics of CSF and plasma Aβ reduction after treatment with JNJ-54861911, a potent oral BACE inhibitor

Safety, tolerability, pharmacokinetics, and pharmacodynamics of a novel β‐site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitor, JNJ‐54861911, were assessed after single and multiple dosing in healthy participants.

Diffusion kurtosis imaging allows the early detection and longitudinal follow-up of amyloid-β-induced pathology

BackgroundAlzheimer’s disease (AD) is a progressive neurodegenerative disorder and the most common cause of dementia in the elderly population. In this study, we used the APP/PS1 transgenic mouse model to explore the feasibility of using diffusion kurtosis imaging (DKI) as a tool for the early detection of microstructural changes in the brain due to amyloid-β (Aβ) plaque deposition.MethodsWe longitudinally acquired DKI data of wild-type (WT) and APP/PS1 mice at 2, 4, 6 and 8xa0months of age, after which these mice were sacrificed for histological examination. Three additional cohorts of mice were also included at 2, 4 and 6xa0months of age to allow voxel-based co-registration between diffusion tensor and diffusion kurtosis xa0metrics and immunohistochemistry.ResultsChanges were observed in diffusion tensorxa0(DT) and diffusion kurtosisxa0(DK) metrics in many of the 23 regions of interest that were analysed. Mean and axialxa0kurtosis were greatly increased owing to Aβ-induced pathological changes in the motor cortex of APP/PS1 mice at 4, 6 and 8xa0months of age. Additionally, fractional anisotropy (FA) was decreased in APP/PS1 mice at these respective ages. Linear discriminant analysis of the motor cortex data indicated that combining diffusion tensorxa0and diffusion kurtosis metrics permits improved separation of WT from APP/PS1 mice compared with either diffusion tensor or diffusion kurtosis metrics alone. We observed that mean kurtosis and FA are the critical metrics for a correct genotype classification. Furthermore, using a newly developed platform to co-register the in vivo diffusion-weighted magnetic resonance imaging with multiple 3D histological stacks, we found high correlations between DK metrics and anti-Aβ (clone 4G8) antibody, glial fibrillary acidic protein, ionised calcium-binding adapter molecule 1 and myelin basic protein immunohistochemistry. Finally, we observed reduced FA in the septal nuclei of APP/PS1 mice at all ages investigated. The latter was at least partially also observed by voxel-based statistical parametric mapping, which showed significantly reduced FA in the septal nuclei, as well as in the corpus callosum, of 8-month-old APP/PS1 mice compared with WT mice.ConclusionsOur results indicate that DKI metrics hold tremendous potential for the early detection and longitudinal follow-up of Aβ-induced pathology.

Evaluation of μPET outcome measures to detect disease modification induced by BACE inhibition in a transgenic mouse model of Alzheimer’s disease.

In this study, we investigated the effects of chronic administration of an inhibitor of the β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) on Alzheimer-related pathology by multitracer PET imaging in transgenic APPPS1-21 (TG) mice. Methods: Wild-type (WT) and TG mice received vehicle or BACE inhibitor (60 mg/kg) starting at 7 wk of age. Outcome measures of brain metabolism, neuroinflammation, and amyloid-β pathology were obtained through small-animal PET imaging with 18F-FDG, 18F-peripheral benzodiazepine receptor (18F-PBR), and 18F-florbetapir (18F-AV45), respectively. Baseline scans were acquired at 6–7 wk of age and follow-up scans at 4, 7, and 12 mo. 18F-AV45 uptake was measured at 8 and 13 mo of age. After the final scans, histologic measures of amyloid-β (4G8), microglia (ionized calcium binding adaptor molecule 1), astrocytes (glial fibrillary acidic protein), and neuronal nuclei were performed. Results: TG mice demonstrated significant age-associated increases in 18F-AV45 uptake. An effect of treatment was observed in the cortex (P = 0.0014), hippocampus (P = 0.0005), and thalamus (P < 0.0001). Histology confirmed reduction of amyloid-β pathology in TG-BACE mice. Regardless of treatment, TG mice demonstrated significantly lower 18F-FDG uptake than WT mice in the thalamus (P = 0.0004) and hippocampus (P = 0.0332). Neuronal nucleus staining was lower in both TG groups in the thalamus and cortex. 18F-PBR111 detected a significant age-related increase in TG mice (P < 0.0001) but did not detect the treatment-induced reduction in activated microglia as demonstrated by histology. Conclusion: Although 18F-FDG, 18F-PBR111, and 18F-AV45 all detected pathologic alterations between TG and WT mice, only 18F-AV45 could detect an effect of BACE inhibitor treatment. However, changes in WT binding of 18F-AV45 undermine the specificity of this effect.

In Vivo Amyloid-β Imaging in the APPPS1–21 Transgenic Mouse Model with a 89Zr-Labeled Monoclonal Antibody

Introduction: The accumulation of amyloid-β is a pathological hallmark of Alzheimer’s disease and is a target for molecular imaging probes to aid in diagnosis and disease monitoring. This study evaluated the feasibility of using a radiolabeled monoclonal anti-amyloid-β antibody (JRF/AβN/25) to non-invasively assess amyloid-β burden in aged transgenic mice (APPPS1–21) with μPET imaging. Methods: We investigated the antibody JRF/AβN/25 that binds to full-length Aβ. JRF/AβN/25 was radiolabeled with a [89Zr]-desferal chelate and intravenously injected into 12–13 month aged APPPS1–21 mice and their wild-type (WT) controls. Mice underwent in vivo μPET imaging at 2, 4, and 7 days post injection and were sacrificed at the end of each time point to assess brain penetrance, plaque labeling, biodistribution, and tracer stability. To confirm imaging specificity we also evaluated brain uptake of a non-amyloid targeting [89Zr]-labeled antibody (trastuzumab) as a negative control, additionally we performed a competitive blocking study with non-radiolabeled Df-Bz-JRF/AβN/25 and finally we assessed the possible confounding effects of blood retention. Results: Voxel-wise analysis of μPET data demonstrated significant [89Zr]-Df-Bz-JRF/AβN/25 retention in APPPS1–21 mice at all time points investigated. With ex vivo measures of radioactivity, significantly higher retention of [89Zr]-Df-Bz-JRF/AβN/25 was found at 4 and 7 days pi in APPPS1–21 mice. Despite the observed genotypic differences, comparisons with immunohistochemistry revealed that in vivo plaque labeling was low. Furthermore, pre-treatment with Df-Bz-JRF/AβN/25 only partially blocked [89Zr]-Df-Bz-JRF/AβN/25 uptake indicative of a high contribution of non-specific binding. Conclusion: Amyloid plaques were detected in vivo with a radiolabeled monoclonal anti-amyloid antibody. The low brain penetrance of the antibody in addition to non-specific binding prevented an accurate estimation of plaque burden. However, it should be noted that [89Zr]-Df-Bz-JRF/AβN/25 nevertheless demonstrated in vivo binding and strategies to increase brain penetrance would likely achieve better results.

PHARMACODYNAMICS OF THE ORAL BACE INHIBITOR JNJ-54861911 IN EARLY ALZHEIMER'S DISEASE

cebo were studied. Comprehensive neuropathological assessments were performed ranging from 4 months to 14 years after the trial. Large coronal paraffin sections of cerebral hemisphere were immunostained for Ab, scanned, then (i) the entire neocortical ribbon was scored for plaques in a CERAD-like manner (frequent1⁄43, moderate1⁄42, sparse1⁄41, none1⁄40) and (ii) false coloured to permit visualisation of staining patterns at low power. Results: 16/21 had a distribution of tangles and at least some residual Ab pathology indicating the cause of dementia had been AD, whereas 5/21 had alternative causes for dementia (PSP1⁄41, DLB1⁄41, VaD1⁄41and FTDTDP431⁄42). 18/21 had received the active agent and 3/21 the placebo. Scanning and false colouration of large Ab immunostained sections generated images suitable for comparison with amyloid PET scans as used in current immunotherapy trials. 13/15 AD subjects receiving the active agent had evidence of plaque removal (almost complete removal1⁄45, extensive patches of removal1⁄44, small patches of removal1⁄44, no plaque removal1⁄42). In the immunised AD group the mean plaque score was 1.46 (range1⁄40.05-2.9) vs. 2.4 for the single placebo AD case. There was a significant inverse correlation between peripheral blood anti-Ab titres and plaque scores. Conclusions: AD patients actively immunised against Ab can remain virtually plaque–free for up to 14 years. Nearly a quarter of the patients examined in this study did not have neuropathologically verified AD. Neuropathology follow up of patients in therapeutic trials for AD provides valuable information on the cause of dementia and effects of treatment.

Mass spectrometric characterization of intact desferal-conjugated monoclonal antibodies for immuno-positron emission tomography imaging

RATIONALEnImmuno-PET imaging may prove to be a diagnostic and progression/intervention biomarker for Alzheimers disease (AD) with improved sensitivity and specificity. Immuno-PET imaging is based on the coupling of an antibody with a chelator that captures a radioisotope thus serving as an in-vivo PET ligand. A robust and quality controlled process for linking the chelator to the-antibody is fundamental for the success of this approach.nnnMETHODSnThe structural integrities of two monoclonal antibodies (trastuzumab and JRF/AβN/25) and the quantity of desferal-based chelator attached following modification of the antibodies were assessed by online desalting and intact mass analysis. Enzymatic steps for the deglycosylation and removal of C-terminal lysine was performed sequentially and in a single tube to improve intact mass data.nnnRESULTSnIntact mass analysis demonstrated that inclusion of enzymatic processing was critical to correctly derive the quantity of chelator linked to the monoclonal antibodies. For trastuzumab, enzymatic cleaving of the glycans was sufficient, whilst additional removal of the C-terminal lysine was necessary for JRF/AβN/25 to ensure reproducible assessment of the relatively low amount of attached chelator.nnnCONCLUSIONSnAn efficient intact mass analysis-based process was developed to reproducibly determine the integrity of monoclonal antibodies and the quantity of attached chelator. This technique could serve as an essential quality control approach for the development and production of immuno-PET tracers.

Young to middle-aged dogs with high basal Aβ42 CSF level demonstrate learning impairment compared to dogs with low basal CSF Aβ42

reported [Barthel2011]. Results: Cross-sectional effect size was larger for SUVR methods including subcortical white matter as a reference region, and highest using a composite reference region including subcortical white matter, brainstem, and whole-cerebellum (Cohen’s D1⁄43.88). Lowest effect size was obtained using the cerebellar grey reference (2.66). Accuracy was higher using the composite reference and a cutpoint of 0.845 (sensitivity1⁄40.979, specificity1⁄40.978), compared to the cerebellar grey referencewith a cutpoint of 1.346 (sensitivity1⁄40.917, specificity1⁄40.956). The Freesurfer method performed slightly better than the AALmethod using the cerebellar grey reference. Conclusions: Comparing SUVR cutpoints to visual assessment, an SUVR method using a subcortical white matter reference region appears superior to methods using cerebellar references. These results can be influenced by the visual assessment method used which focuses on comparing the target intensity in the cortical gray matter versus the subcortical white matter. [Landau2013] J Nucl Med 54:1–8. [Barthel2011] Lancet Neurol 10:424-35.