Novel biomarkers for the diagnosis of frontotemporal dementia

Poster Presentations P3

S520

(TMT) A and B, denomination (DO80), digit span tests, battery of frontal function were performed in AD patients. All patients gave their informed consent. CSF biomarkers (Ab 1-42, tau and p181 tau) were assessed after lumbar puncture in the month following cognitive tests. Spearman correlation coefficients between CSF biomarkers and the different cognitive tests were calculated. Generalized linear models adjusted for age, sex and level of education were used for multivariable analysis. Results: MMSE scores were inversely associated with the levels of CSF Ab 1-42 (Spearman’s rho ¼ 0.28, p ¼ 0.03) and TMT B scores were associated with tau (Spearman’s rho ¼ 0.37, p ¼ 0.02) and p181-tau (Spearman’s rho ¼ 0.45, p ¼ 0.004) CSF levels. These associations remained after adjustment for age, sex, and level of education in multivariable analysis. No correlation was observed with the other cognitive tests. Conclusions: Our results show that in a cohort of recently diagnosed AD patients, CSF biomarkers levels can correlate with the results of cognitive functions. A b 1-42 levels are linked to MMSE scores reflecting the global cognitive functions and tau and p181 tau CSF levels are correlated with executive functions assessed by the TMT B test. P3-233

IDENTIFICATION OF BLOOD TRANSCRIPTOMIC SIGNATURES IN ALZHEIMER PATIENTS RELATED TO EHT0202 TREATMENT RESPONSE

Richard Einstein, ExonHit Therapeutics, Inc, Gaithersburg, MD, USA. Contact e-mail: [email protected] Background: To date, treatment response in Alzheimer’s Disease (AD) is mainly assessed using psychometric scales. Due to recent development of new biotechnology tools, drug response can be measured also by interrogating genomic expression profiles. The current study is based on the identification of blood-based transcriptomic signatures using Exonhit’s proprietary SpliceArrayÔ technology in AD patients who were enrolled in a clinical trial to assess EHT0202, a new compound sharing both potential disease-modifying and symptomatic properties. Methods: EHT0202/002 study was a randomized, double-blind, placebo-controlled, parallel group, phase IIA study aimed at determining the clinical safety, tolerability and exploratory efficacy of EHT0202 (40 and 80mg bid) as adjunctive therapy to one cholinesterase inhibitor (AchEI) over a 3-month period in mild to moderate AD patients. One of research endpoint was to determine prospective blood expression signatures using the SpliceArrayÔ technology in relation with treatment response to EHT0202 using specific scales measuring clinical cognitive impairment. During the study, blood samples were collected from each randomized patient at different time-points notably before study treatment initiation and at study completion. Results: Sixty AD patients (20 in each of the 3 study groups) having either responded or declined during the study period with regards to ADAS-cog total score, have been selected. Another 20 cognitive impairment free subjects served as controls. Both cross-sectional and longitudinal analyses will be performed in order to determine prospective blood transcriptomic signatures using the SpliceArrayÔ technology in relation to treatment response of EHT0202. Determination of potential signatures for the identification / differentiation of patients treated with either AChEI (donepezil or galantamine), or a combination of AChEI and EHT0202 will be also considered. Conclusions: Pharmacogenomics depends on specific analysis from clinical trials to determine the proper selection of biomarkers for the identification of patients who can beneifit from treatment. In addition, the identification of change in disease state of patients is integral in this assessment. These results, from the pilot study, will be presented. P3-234

DIFFERENTIAL MASS SPECTROMETRY IDENTIFIES CANDIDATE MARKERS FOR ALZHEIMER’S DISEASE IN HUMANS

Ronald C. Hendrickson1, Cloud Paweletz1, Andy Liaw1, Qinghua Song1, Anita Lee1, Jenny Li1, Fanyu Meng1, Ekaterina G. Deyanova1, Matthew Mazur1, Robert E. Settlage1, Matt Wiener1, Xuemei Zhao1, Jeffrey L. Seeburger1, Jeffrey Sachs1, Vijay Modur1, Elizabeth King2, Catherine Joachim2, Mark Shearman1, Keith Soper1, David A. Smith2, William Potter1, Ken Koblan1, Nathan A. Yates1, 1Merck, Rahway, NJ, USA; 2 University of Oxford, Oxford, United Kingdom. Contact e-mail: [email protected]

Background: High Resolution Differential Mass Spectrometry (dMS) is an open discovery platform that has the ability to identify and detect quantitative peptide changes in complex mixtures such as CSF without the need for antibody reagents. Three important elements of the high resolution dMS approach for biomarker research include speed, relevance to biology, and a clear translation path for clinical evaluation. Methods: Here, we describe the use of micro-capillary LC coupled with high resolution mass spectrometry (MS) to identify markers that can distinguish pathologically confirmed AD CSF from age-matched non-demented controls. CSF biochemical biomarkers have the potential to provide objective information on disease status and may be used to design efficient clinical trials for novel therapies that alter the rate of disease progression. Results: Neurosecratory protein VGF and neuronal pentraxin receptor protein are two examples of AD markers that were discovered in a rapid pilot study that examined the CSF from 20 subjects . Neuronal pentraxin receptor is an integral membrane protein involved in the pathway for transport of taipoxin into synapses and may be involved in the clearance of synaptic debris. Conclusions: Changes in these and other AD markers have been confirmed in an independent cross-sectional cohort and also show statistically significant rates of change in a separate longitudinal study. Because these markers are able to be detected by mass spectrometry without the need for antibody reagents, MS based assays provide a clear path for testing AD markers in the clinic. P3-235

BETA-AMYLOID LINKED TO GOLD NANOPARTICLES SCAVENGES BETA-AMYLOID OLIGOMERS FROM SERA

Brian M. Austen1, Richard Aggrey1, Eleonore Cerasoli2, Paulina Rakowska2, Max Ryadnov2, Robert Lawrence1, 1St. George’s University of London, London, United Kingdom; 2National Physical Laboratory, Teddington, United Kingdom. Contact e-mail: sghk200@sgul. ac.uk Background: beta-Amyloid oligomers are known to block hippocampal synaptic plasticity, involved in new memory formation, and have been identified in Alzheimer’s patients’ brain. We have shown previously by a specific ELISA that patients’ sera contain higher levels of amyloid oligomers than age-matched controls. Our aim was to identify the serum oligomers by mass spectrometry after binding to nanoparticle affinity reagents. Methods: beta-Amyloid (Cys26) 40 was synthesised by Fmoc solid phase chemistry and purified by HPLC. It was linked to gold nanoparticles (nps) of 50 10 or 5nm diameter. Results: The reagents were washed free of excess Amyloid-40 (Cys26), and incubated with sera from an Alzheimr’s Disease (AD) patient. The nanoparticles analysed in a Bruker Ultraflex Malditof, mass spectrometer, and gave rise to a peak at M/H+ 4348.5.consistent with an oxidised form of beta-amyloid 40 in the patients serum. Mass analysis of the nanoparticle complex incubated with oligomeric synthetic betaamyloid-40 gave rise to the wild-type amyloid40 at M/H+ at 4329.4, In contrast nps incubated with control serum (Sigma) did not show an amyloid peak Conclusions: Isolation on nanoparticles derivatized with Cys-amyloid-40 enabled affinity isolation and identification of the beta-amyloid species in an AD patient’s sera, confirming analyses obtained by ELISA; the use of nanoparticles enabled a precise mass identification of the oxidised amyloid species in sera, and could give rise to more accurate diagnoses of the disease at early stages in future. P3-236

NOVEL BIOMARKERS FOR THE DIAGNOSIS OF FRONTOTEMPORAL DEMENTIA

Anja H. Simonsen1, George Paraskevas2, Elisabeth Kapaki2, Gunhild Waldemar1, 1Memory Disorders Research Group, Rigshospitalet, Copenhagen, Denmark; 2Department of Neurology, Eginition University Hospital, Athens, Greece. Contact e-mail: [email protected]. dk Background: Frontotemporal dementia (FTD) is a neurodegenerative disease, which is thought to occur in 2-5% of all dementia cases. Personality changes and behavioural disturbances are dominating features early in the disease course with development of cognitive disturbances in the later stages

Poster Presentations P3 of the disease. There is an unmet need for diagnostic biomarkers for FTD. The objective of this hypothesis generating study was to discover new candidate protein markers for early, differential diagnosis of FTD that may improve the performance of current diagnostic assays. Methods: Surface Enhanced Laser Desorption/Ionization (SELDI) TOF-MS and three different chromatographic surfaces were used to differentially profile proteins and peptides in CSF samples from 20 patients with FTD (age range 43-76 yrs) and 34 healthy controls (age range 42-86 yrs). Diagnosis was made using DSM-IV and ICD-10 criteria. Results: A total of 33 candidate biomarkers separating FTD from healthy aging were found with ROC values above 0.7 and p values below 0.01. Among them were fragments of the neurosecretory protein VGF as well as the neuroendochrine peptide Chromogranin B and a member of the complement system C3a. Conclusions: This novel panel of biomarkers could potentially be used to improve early diagnosis of FTD as well as to provide complementary information to help decision making in the development of disease modifying compounds. Disclosure for all authors: All of the authors have seen and approved this abstract. P3-237

POTENTIAL EARLY DIAGNOSIS OF ALZHEIMERS DISEASE USING CHANGES OF THE ENTERIC NERVOUS SYSTEM

Karl H. Scha¨fer1, Maryse Letiembre2, Sandra Semar3, Markus Klotz3, Alex Liu2, Klaus Fassbender2, Tony Wyss-Coray4, Walter SchulzSchaeffer5, Chris Van Ginneken6, 1University of Applied Sciences Kaiserslautern, Zweibru¨cken, Germany; 2University of Saarland, Homburg, Germany; 3University of Applied Sciences Kaiseslautern, Zweibru¨cken, Germany; 4VA Stanford, Palo Alto, CA, USA; 5University of Go¨ttingen, Go¨ttingen, Germany; 6University of Antwerp, Antwerp, Netherlands. Contact e-mail: [email protected] Background: The Enteric nervous system (ENS) is a major part of the peripheral nervous system and much easier accesible as the central nervous system (CNS). In case that changes of the CNS in neurodegenerative diseases occur also in the ENS, there would be a great potential to use this changes for early diagnosis. In Parkinsons disease, there is a reduction of dopaminergic neurons in the gut to be found, but so far there are no reports concerning changes of the ENS in Alzheimers disease. Methods: In this study we investigated changes in the expression of APP in the ENS of several trangenic mice strains, additionally TLR-4, Nestin and GFAP -levels were measured in a double mutant model at different time points (1,5, 2, 3 and 5 month). Motility measurments of gut activity in the adult mice were performed to investigate whether AD alters gut functionality. Immunohistochemical stainings for human APP, as well as for neuronal and glial markers were performed. Smooth muscle tissue with included myenteric plexus from the mouse models were analyzed with RT-PCR. Proteomic analysis (2-D-DIGE) were  were perperformed on brain and gut tissue.Tau and phosphor tau ELISAs formed on gut wall extracts from mice and human. Results: Increased APP levels could be found in all models. In a double APP mutant mouse model, APP was much higher in the gut as to be found in the brain. Nestin, TLR-4 and GFAP-levels rose from 6 weeks to three month and dropped again at 5 month. In these animals, gut motility was significantly altered. The transgenic mice showed weaker but faster contractions. The proteomic analysis revealed several differentially expressed proteins in the brain and also in the gut. The tau/phospho-tau ELISA showed preliminary positive results in individual mice and human gut samples. Conclusions: The early diagnosis of neurodegenerative disease associated pathological proteins in the enteric nervous system seems to be a feasible tool. To evaluate appropriate markers for a reliable diagnosis the onset of the expression of APP and correlated proteins have to be investigated in more detail. P3-238

LATREPIRDINE INCREASES CEREBRAL GLUCOSE UTILIZATION IN AGED MICE

Feng Luo, Prasant Chandran, Nathan Rustay, Vincent Hradil, Gerard B. Fox, Mark Day, Translational Imaging and Biomarkers, Abbott Laboratories, Abbott Park, IL, USA. Contact e-mail: [email protected]

S521

Background: Although the ability of Latrepirdine to improve cognition in Alzheimer’s disease (AD) has been shown in a phase II clinical trial (Doody etal., 2008), its mechanism is unclear. A unique mechanism of action involving stabilisation of mitochondria has been hypothesized, rather than cholinesterase inhibition or NMDA antagonism, which are the mechanisms of the current standards of care (see Rafii & Aisen, 2009). While dysfunction of mitochondria and abnormal energy metabolism are linked to AD pathology (Mancusso et al., 2009), no studies have been reported that investigate Latrepirdine’s effect on cerebral glucose utilization (CGU). The aim of the current study was to assess CGU changes in young and aged mice in vivo using FDG-PET after acute treatment with Latrepirdine. Methods: 8-10 month old C57BL/6 mice and 20 month old B6SJLF2 mice were tested using FDG-PET. Baseline FDG uptake was assessed. A between-group assessment of Latrepirdine’s effects (3mmol/kg) on CGU was used in young mice, while a within-subjects crossover design was employed for aged mice. Experiments were conducted on an Inveon PET/CT (microPET, Siemens Medical Solutions USA, Inc.), a high resolution PET system dedicated to small animal imaging. The animals were fasted for at least 6 hours prior to the study. PET image acquisition started 50 minutes after a bolus injection of 10-15 MBq of 18F-FDG i.v.. Images were reconstructed iteratively using 2DOSEM with the following parameters: a Ramp filter with 0.5 of the Nyquist frequency as the cutoff, 5 iterations, and 128 x 128 array size. Standard uptake value (SUV) was used to quantify CGU in mice. Results: We demonstrate that Latrepirdine significantly enhances CGU in aged B6SJLF2 type mice but not in younger C57BL/6 mice. Increased variability of baseline CGU was observed in aged mice, and the mean baseline CGU was decreased in aged compared to young mice. Conclusions: Latrepirdine enhances CGU in aged mice, lending support for the hypothesis that Latrepirdine may improve energy metabolism in the brain. Monitoring changes in CGU using FDG-PET may represent an imaging biomarker for treatment of AD using Latrepirdine. We highlight the importance of measuring baseline CGU for its potential implications for stratifying subjects. P3-239

META-ANALYSIS OF CSF BIOMARKERS AS PREDICTOR FOR CONVERSION FROM MILD COGNITIVE IMPAIRMENT TO ALZHEIMER’S DISEASE

Stephanie Vos1, Frans Verhey1, Lyzel S. Elias-Sonnenschein1, Pieter Jelle Visser1,2, 1Maastricht University, Maastricht, Netherlands; 2VU University Medical Centre, Amsterdam, Netherlands. Contact e-mail: [email protected] Background: There is an urgent need to diagnose Alzheimer’s disease (AD) in the predementia phase. Cerebrospinal (CSF) biomarkers are increasingly being used to identify early AD. Aim of the present meta-analysis is to evaluate the predictive value of CSF biomarkers to develop AD in patients with mild cognitive impairment (MCI). Methods: Longitudinal studies on CSF biomarkers in MCI patients were investigated. Studies were included if subjects at baseline met criteria for MCI, if data were provided on CSF biomarkers levels at baseline, and if the outcome measure at follow-up was conversion to AD-type dementia. For continuous variables as outcome measure Cohen’s delta was calculated. For dichotomized scores we calculated the sensitivity, specificity, positive predictive value and odds ratio (OR). These dichotomized scores were based on cut-offs provided in the study. If different types of cut-off levels were used within one study, we chose predefined cutoffs over data-driven cut-offs. Results: We identified 20 studies that provided unique data of CSF levels of t-tau, p-tau and Ab1-42 as predictor for AD-type dementia in subjects with MCI. 742 out of 1233 converted to AD during a mean follow up period of 2.4 years. The combination of CSF Ab1-42 and tau had the best predictive accuracy for AD (odds ratio (OR) 14.5, 95% CI 7.8-27.2). The sensitivity of this combination to predict ADtype dementia was 0.87 (95% CI 0.81-0.94), the specificity 0.67 (95% CI 0.53-0.80) and the positive predictive value 0.63 (95% CI 0.53-0.74). Ab1-42, total tau and phosphorylated tau in CSF also predicted conversion, but with lower accuracy (OR 6.7 to 7.1; Cohen’s delta 0.79 to 1.10). Conclusions: The combination of CSF Ab1-42 and tau appears to be a good predictor for AD conversion in MCI patients. Future research should focus on the