Early diagnosis of Alzheimer’s disease by combination of

1. Early diagnosis of Alzheimer’s disease by combination of cerebrospinal fluid core biomarkers and Visinin-like protein-1 (VILIP-1)G. Šimić1*, M. Babić1, D. Bažadona1, F. Borovečki2, D. Čerimagić1, N. Dejanović1, S. Hajnšek2, N. Henigsberg1, M. Jazvinšćak Jembrek3, N. Jovanov Milošević1, S. Kalanj-Bognar1, S. Kiđemet-Piskač1, N. Mimica1, M. Mustapić3, D. Mück Šeler3, G. Nedić Erjavec3, M. Nikolac Perković3, R. Petrović2, N. Pivac3, P. Presečki1, M. Radoš1, G. Stanić1, D. Švob Štrac3, Ž. Vogrinc2, Ž. Vukelić1, Patrick R. Hof4 1 University of Zagreb School of Medicine, Croatian Institute for Brain Research, Croatia 2 Clinical Hospital Center Zagreb, Croatia 3Ruđer Bošković Institute, Zagreb, Croatia 4Fishberg Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, USA Croatian Science Foundation Abstract Mild cognitive impairment (MCI) is a syndrome characterized by cognitive impairment without dementia, which primarily affects episodic memory. Patients with MCI often have an initial stage of Alzheimer's disease (AD). In this study we compared the effectiveness of 6 CSF biomarkers(Aβ1-42, total tau, p-tau 181, p-tau 199, p-tau 231 and VILIP-1) in differentiation of AD patients from healthy controls (HC). Biomarker levels among AD, MCI, and HC, were compared using the Kruskal-Wallis test (Aβ1-42: (χ2=10.763; df=2; p=0.005); total tau: (χ2=34.182; df=2; p<0.001); p-tau 181: (χ2=28.329; df=2; p<0.001); p-tau 231: (χ2=28.215; df=2; p<0.001); p-tau 199: (χ2=24.101; df=2; p<0.001);VILIP-1: (χ2=15.588; df=2; p<0.001)), followed by the Mann-Whitney U test for pairwise comparisons (Aβ1-42: AD vs MCI (U=1032; Z=-3.366; p=0.001); AD vs HC(U=823.5; Z=-0.860; p=0.390); MCI vs HC (U=347.5; Z=-1.224; p=0.221); total tau: AD vs MCI(U=1110; Z=-4.786; p<0.001); AD vs HC (U=428.5; Z=-4.336; p<0.001); MCI vs HC (U=391; Z=-1.508; p=0.132); p-tau 231: AD vs MCI (U=183; Z=-3.712; p<0.001); AD vs HC(U=48; Z=-4.816; p<0.001); MCI vs HC (U=141.5; Z=-1.820; p=0.069); p-tau 181: AD vs MCI (U=878; Z=-2.892; p=0.004); AD vs HC (U=200; Z=-4.793; p<0.001); MCI vs HC (U=152; Z=-3.363; p=0.001); p-tau 199: AD vs MCI (U=197.5; Z=-3.861; p<0.001); AD vs HC (U=69; Z=-4.160; p<0.001); MCI vs HC (U=158.5; Z=-0.990; p=0.322); VILIP-1: AD vs MCI (U=831; Z=-3.295; p=0.001); AD vs HC (U=300.5; Z=-2.931; p=0.003); MCI vs HC (U=279.5; Z=-0.467; p=0.641). Phospho-tau biomarkers, especially p-tau 199, reached the highest specificity, sensitivity and area under curve (AUC), as revealed by ROC analysis. MCI patients with a high risk of AD development were detected based on cut-off levels for p-tau biomarkers. Five MCI patients (21,8%) had increased both p-tau 199 and p-tau 181, while 8 MCI patients (34,8 %) had increased p-tau 199 only. Additionally, 5 MCI patients also had increased both p-tau 199 and p-tau 231.These 5 MCI will be further monitored as they are at risk of AD development. VILIP-1 is not as reliable as phospho-tau biomarkers, but adds to the overall specificity, sensitivity and AUC in differentiating AD from MCI and HC. Background Materials and methods Results and conclusions Boxes represent the median, the 25th and the 75thpercentiles, bars indicate the range of data distribution. Circles representoutliers (values more than 1.5 box-length from the 75th/25thpercentile). Alzheimer’s disease (AD) is the major primarycause of dementia. Ferri and collaboratorsestimated that 24 million people suffered fromdementia in 2005, with this number reaching81 million by 2040. Clinical diagnosis ofAD, which is still based on symptomatology, isaccurate in only 63 to 90% of dementia cases. A growing number of potential treatmentsfor AD are in different phases of preclinicaland clinical research and thus much effort isdedicated to identify reliable biomarkers toenable an accurate diagnosis of AD. Three main cerebrospinal fluid (CSF)biomarkers of AD, amyloid β1-42 (Aβ1-42), totaltau (tTau), and phosphorylated forms of tau(PTau) reflect two major neuropathologicalhallmarks of AD - neurofibrillary tangles andsenile plaques. These CSF biomarkers arealtered in early stages of AD, even before theoccurrence of the first dementia symptoms,and permit to differentiate patients withprodromal AD (i.e. those with mild cognitiveimpairment, MCI) who often progress to AD,from healthy controls. CSF biomarkers arealso used for differentiation of AD from otherprimary causes of dementia, such as vasculardementia, frontotemporal dementia (FTD), anddementia with Lewy bodies. Reductionof Aβ1-42 in CSF of AD patients is explained byAβ1-42 aggregation into senile plaques, increaseof tTau reflects neuronal degeneration,while elevation of pTau is a consequence ofneurofibrillary degeneration and consequenttangles formation in the brain. Althoughnumerous studies in which diagnostic accuracyof CSF biomarkers was analyzed have beenpublished, an ideal biomarker (with specificityand sensitivity over 85%) could not yet bedefined. All patients wererecruited from the University Hospital Centre,Zagreb, underwent complete blood testsincluding electrolytes, albumin, thyroidfunction, levels of vitamin B12, VDRL testfor syphilis, MMSE, and neurological examination.After exclusion of patients with secondarycauses of dementia, selected patients,upon signing the informed consent,underwent lumbar puncture. CSF was taken in the L3/L4 or L4/L5intervertebral spaces, always between 9 a.m.and 11 a.m., and collected in polypropylenetubes. Leukocyte and erythrocyte cell counts, lactate, glucose, total protein concentration,TPHA, and IgG index were alsodetermined in native CSF.CSFsamples were centrifuged for 10 minutes at 2,000 g, dispensed into 150 μl aliquots andstored at -80 degrees Celsius. CSF Aβ1-42, total tau and p-tau 181 levels were determined using Innogenetics ELISA kits (INNOTEST hTAU Ag, INNOTEST PHOSPHO-TAU(181P) and INNOTEST β-AMYLOID1-42). P-tau 199 CSF levels and level of tau phosphorylation on epitope 231 were measured using Invitrogen ELISA kits, while VILIP-1 (Visinin like protein-1) levels were determined using Biovendor ELISA kit. Protein concentrations were calculated in GraphPad Prism 5.0 software (San Diego, CA, USA) using 4-parameter algorithm. Plates were washed in an automatic washer. Statistical analysis: Aβ1-42, total tau, p-tau 181, p-tau 199, p-tau 231 and VILIP-1 levels among AD, MCI, and HC, were compared using a Kruskal-Wallis test, followed by the Mann-Whitney U test for pairwise comparisons. We compared the effectiveness of 6 CSF biomarkers (Aβ1-42, total tau, p-tau 181, p-tau 199, p-tau 231 and VILIP-1) in differentiation of AD patients from HC. Phospho-tau biomarkers, especially p-tau 199, reached the highest specificity, sensitivity and AUC. MCI patients with a high risk of AD development were detected based on cut-off levels for p-tau biomarkers. Five MCI patients (21,73%) had increased both p-tau 199 and p-tau 181, while 8 MCI patients (34,78 %) had increased p-tau 199 only. Additionally 5 MCI patients also had increased both p-tau 199 and p-tau 231. These 5 MCI will be further monitored as they are at risk of AD development. VILIP-1 is not as reliable as phospho-tau biomarkers, but adds to the overall specificity, sensitivity and AUC in differentiating AD from MCI and HC. cut-off (p-tau 199) = 2,58 pg/ml cut-off (p-tau 181) = 37,03 pg/ml 5 MCI patients (21,7%) had increased both p-tau 199 and p-tau 181, while 8 MCI patients (34,8 %) had increased p-tau 199 only. Five MCI patients also had increased both p-tau 199 and p-tau 231 (see below). Alzheimer's disease (AD) is the major primary cause of dementia. Mild cognitive impairment (MCI) is a syndrome characterized by cognitive impairment without dementia, which primarily affects episodic memory. About 12% of MCI patients however have an initial stage of AD. There is an urgent need for novel CSF biomarkers that would improve the early diagnosis of AD. Visinin-like protein-1 (VILIP-1), a neuron-specific intracellular calcium sensor, is a novel promising biomarker of AD. VILIP-1 was previously identified as a marker of neuronal injury. Thus, the objective of this study was to assess if combination of cerebrospinal fluid VILIP-1 and core CSF biomarkers could improve early diagnosis of AD. cut-off (p-tau 199) = 2,58 pg/ml cut-off (p-tau 231) = 1,281U/ml Acknowledgements All patients signedstandard Patient Consent Form approved by theEthical Committee of the University HospitalCenter Zagreb (approval no. 01-20/53-1/2006signed on 26th June 2006). This research wasalso approved by the central Ethical Committeeof the University of Zagreb Medical School(case no. 380-59/11-500-77/90, class 641-01/11-02 signed on 19th May 2011).The workis funded by the Croatian Science Foundationgrant no. 09/16 “Detection and trackingof biological markers for early therapeuticintervention in sporadic Alzheimer’s disease”to G.Š., and in part by NIH grant P50 AG005138 to P.R.H.The authors declare no conflict of interest.