Tau concentration in cerebrospinal fluid (CSF) has been shown to correlate with Alzheimer's disease (AD) progression and is a key measurement in the identification of people at risk for progressing to Alzheimer's disease (AD) and the proper management of people with AD. The use of such markers for clinical purposes requires assays that can deliver consistent analytical and clinical performance. We report on the VITROS ® Immunodiagnostic Products Tau Assay 1 assay under development for the measurement of tau in CSF on the VITROS Immunodiagnostic Systems. The objective of this study was to demonstrate consistent performance of the assay across lots and across instrument platforms. Two lots of VITROS Tau assay reagent were tested on the VITROS ® ECiQ and VITROS ® 3600 Immunodiagnostic Systems to evaluate lot-to-lot and system-to-system variability. Fifty individual CSF samples and controls were run in singleton with controls run at the beginning and end of each test. The results from the VITROS 3600 using Lot 1 reagent were used as the control condition and linear regression was performed, as well as the %CV calculated across all runs. The system-to-system comparison (Lot 1 - ECiQ vs. Lot 1 - 3600) slope, intercept, and correlation (r) results were 0.9913, 2.87, and 1.000 respectively. The lot-to-lot comparison (Lot 2 - 3600 vs. Lot 1 - 3600) slope, intercept, and correlation (r) results were 0.979, -8.70, and 0.999 respectively. Across lots and systems (Lot 2 - ECiQ vs. Lot 1 - 3600) slope, intercept, and correlation (r) results were 0.981, 0.65, and 1.000 respectively. The mean doses of the controls (n = 8 per control) ranged from 64.8 - 4145 pg/mL with an overall %CV from 0.9% - 4.1%. The mean doses of the individual CSF samples (n = 4 per CSF) ranged from 56.2 - 1412 pg/mL with a %CV from 0.5% - 5.8%, overall 2.4%CV across lots and systems. The VITROS Immunodiagnostic Products Tau Assay 1 has demonstrated consistent results across reagent lots and across systems.
Measurement of Alzheimer's disease (AD) biomarkers in clinical cerebrospinal fluid (CSF) samples has been challenging with existing assays due to inter-lot variability, matrix interferences, and inconsistent quantitation. Here we describe the development and analytical validation of an assay to measure Total Tau in human CSF using Fit-for-Purpose and CLSI principles. Elements of assay development and performance are presented as well as results of multi-lot validation. Monoclonal antibodies were evaluated and selected based on sensitivity, specificity, affinity, and performance characteristics in human CSF. Critical reagents were subjected to biochemical and functional characterization to verify purity, integrity, and lot-to-lot consistency. The assay was developed with MSD's MULTI-ARRAY® technology and optimized to minimize CSF matrix effects and interferences. Analytical validation was assessed across three independent kit lots to verify performance including sensitivity, accuracy, and precision using matrix-based validation samples. Tests were conducted by multiple analysts over multiple runs and days. Dilution linearity and spike recovery were measured using well-curated normal and AD CSF samples. The assay demonstrated good sensitivity, performance, and inter-lot reproducibility and differentiated between normal and AD CSF samples. The average LLOD determined from 54 runs over three independent kit lots was 10 pg/mL with a quantitative range of 30 to 8000 pg/mL. The precision, accuracy, and total error were determined from matrix-based controls with typical precision of <10% CV (inter-plate) and <5% CV (intra-plate). Dilution linearity and spike recovery testing demonstrated quantitation of Tau protein over the range of the assay and minimal interference from human CSF matrix. Furthermore, the assay detected 6 isoforms of human Tau and was tolerant of significant CSF contamination with hemolyzed blood. Data also showed that the assay measured elevated levels of Tau in AD samples compared to healthy samples. A new assay was developed and analytically validated to measure Tau in human CSF. The result is an assay with good analytical performance characteristics and lot to lot reproducibility with the ability to distinguish Tau levels between healthy and AD CSF samples. This assay will support ongoing efforts to standardize testing of biomarkers for AD and other tauopathies.
Gamma-secretase produces several ß-amyloid (Aß) species that accumulate in the AD brain, making this enzyme a prime target for Aß-lowering strategies. BMS-708163 is an oral gamma-secretase inhibitor (GSI) designed for the selective inhibition of Aß production. Phase 1 studies showed that BMS-708163 caused a dose-dependent reduction in CSF Aß40 and Aß42 levels at ≥ 50 mg/day in healthy individuals. In a separate study, BMS-708163 caused a dose-dependent increase in CSF Aß1-14, 1-15 and 1-16 levels in mild-to-moderate AD patients, indicating increased alpha-secretase cleavage of Aß. The current study evaluated the effects of BMS-708163 on CSF Aß38, Aß40, Aß42, tau, and p-tau levels in AD patients. CN156013 was a randomized, double-blind, placebo-controlled, 24-week phase 2 trial designed to assess the safety and tolerability of BMS-708163 in patients with mild-to-moderate AD. Secondary analyses included an evaluation of the pharmacodynamic (PD) effects of BMS-708163 on CSF biomarkers. CSF at trough was collected in a subset of patients (n = 10-13/group) at baseline, week 12, and week 24. Levels of Aß38 (not reported), Aß40, and Aß42 were measured using assays from Mesoscale Discovery and levels of tau and p-tau were measured using the AlzBio3 assay from Innogenetics. BMS-708163 caused dose-dependent decreases in trough CSF Aß40 levels. Compared with placebo, statistically significant reductions in trough CSF Aß40 levels were observed at 100 mg/day (25%) and 125 mg/day (45%). A comparison to phase 1 data showed that BMS-708163 caused similar PD effects on trough CSF Aß40 levels in healthy individuals and AD patients. Based on these data, peak CSF Aß40 levels were estimated to be lowered 10% at 25 mg/day and 20-25% at 50 mg/day. The overall pattern of trough CSF Aß42 levels was similar to that of CSF Aß40. However, the variability in changes of CSF Aß42 was much greater due to assay variability and disease-related changes. We also observed non-statistically significant reductions in mean CSF tau and p-tau of ∼10-20% at 50 mg/day. The Aß biomarker results provide evidence that BMS-708163 engages its target in a dose-dependent manner and is a centrally active GSI in mild-to-moderate patients with AD at all doses tested.
The use of low Aβ42 and high Tau levels in cerebrospinal fluid (CSF) has emerged as a useful tool to enrich for mild cognitively impaired (MCI) patients at risk of progressing to dementia of the Alzheimer's type and for dementia patients likely to possess amyloid plaque and neurofibrillary tangle pathology. Use of these tests in the clinical trial setting has been challenging as the majority of assays were designed specifically as research use only (RUO) tools. The RUO tests were not designed to address significant matrix effects associated with CSF Aβ42 and lack of batch-batch robustness. The current study describes the clinical performance of improved CSF Aβ42 and Tau assays in differentiating Alzheimer's disease (AD) and MCI patients from normal controls (C). CSF samples were collected from 50 AD patients, 50 MCI patients and 50 controls. For the AD group, inclusion was based upon a probable diagnosis using NINCDS-ADRDA criteria with an MMSE between 14-23. For MCI, inclusion was based upon demonstration of an amnestic memory impairment using education adjusted scoring on the Wechsler memory Scale Revised. CSF was collected in polypropylene tubes and stored at -80C. Improved research use only kits from MSD were utilized for analysis of CSF Aβ42 and Tau per manufacturer's instructions. The control group was significantly younger than the MCI and AD groups with mean ages of 58, 67 and 70 respectively. Gender distribution was evenly matched across groups. CSF Tau levels were significantly elevated in MCI and AD compared to controls. CSF Aβ42 levels were low in AD and MCI compared to controls with significantly lower levels in AD compared to MCI. There were no significant differences in Tau between MCI and AD. Use of the ratio of Tau/Aβ42 showed good sensitivity and specificity in differentiating AD from controls with a sensitivity of 70% and specificity of 92%. Improved versions of the CSF Aβ42 and Tau assays showed good clinical performance in differentiating AD and MCI from controls. Current results are consistent with literature reporting low levels of CSF Aβ42 and high Tau in patients with AD.
Abstract Introduction: We evaluated the impact of baseline (BL) co-mutations and FLT3-ITD VAF on overall survival (OS) and response (composite complete remission [CRc]) to quizartinib and SC in the phase III QuANTUM-R trial. Methods: We analyzed 37 recurrently mutated genes in AML in BL samples from 304 patients (pts) (83% of ITT population) with R/R FLT3-ITD-positive AML using next-generation sequencing and a customized Archer® Core Myeloid panel. Positive mutation status was defined as ≥1 mutation detected in the gene region using a VAF cutoff of 2.7%. FLT3-ITD VAF was measured by the Navigate BioPharma FLT3 Mutation Assay (polymerase chain reaction-based, VAF cutoff of 3%). Low and high FLT3-ITD VAF were defined as ≤25% and >25%, respectively. Results: In addition to FLT3-ITD, the prevalence of key BL co-mutations were 59.9% for DNMT3Amut and 55.3% for NPM1mut. Pts with DNMT3Amut treated with quizartinib had significantly longer OS vs SC (6.3 and 5.4 mos, respectively; hazard ratio [HR], 0.652), p<.05). Pts with NPM1mut treated with quizartinib had a higher CRc rate than with SC, but similar OS (5.1 vs 4.7 mos, respectively; HR, 0.954, p=.82). Pts with NPM1wt/DNMT3Amut treated with quizartinib had the highest CRc rate and longest median OS (9.0 and 4.5 mos, respectively; HR, 0.239, p=.003). OS benefit with quizartinib relative to SC was more pronounced among pts with high FLT3-ITD VAF than low FLT3-ITD VAF. The OS benefit with quizartinib in pts with NPM1wt/DNMT3Amut was maintained in both low and high FLT3-ITD VAF groups. Similarly, for other DNMT3A/NPM1 co-permutations, OS in both low and high FLT3-ITD VAF groups was consistent with OS in the co-mutation group. Conclusions: Key co-mutations identified here potentially affect treatment response and OS with quizartinib vs SC. Our results suggest that molecular subsets of R/R AML pts may particularly derive clinical benefit from quizartinib. TableCRc, %Median OS, monthsQuizartinibSCQuizartinibSCHR95% CIITT Population (N = 367)a48276.24.70.760.58-0.98Single Gene Analyses (n = 304)bDNMT3Amut (n = 182)52376.35.40.6520.44-0.97DNMT3Awt (n = 122)40246.04.60.8490.53-1.37NPM1mut (n = 168)48395.14.70.9540.63-1.44NPM1wt (n = 136)47218.55.10.4850.31-0.76TET2mut (n = 98)34326.22.90.6640.38-1.16TET2wt (n = 206)54306.35.40.7280.51-1.05CEBPAmut (n = 46)44428.58.71.9220.80-4.62CEBPAwt (n = 258)48296.24.50.6130.45-0.84IDH1/2mut (n = 49)32275.53.70.4270.20-0.92IDH1/2wt (n = 255)51316.55.10.750.54-1.04Double Gene Analyses (n = 304)NPM1wt/DNMT3Amut (n = 44)61279.04.50.2390.09-0.61NPM1mut/DNMT3Amut (n = 138)50405.45.40.8370.52-1.34FLT3-ITD VAF AnalysesFLT3-ITD high VAF50195.53.90.6890.51-0.93FLT3-ITD low VAF43467.96.10.8570.53-1.40FLT3-ITD VAF Analyses in Selected MutationsDNMT3Amut high VAF53215.82.70.6260.40-0.98DNMT3Amut low VAF526910.26.40.7370.36-1.51NPM1wt/DNMT3Amut high VAF6409.01.50.01790.002-0.16NPM1wt/DNMT3Amut low VAF555011.36.20.3720.11-1.23aN = 367; quizartinib, n = 245; SC, n = 122bBaseline bone marrow samples were available and viable from 304 of 367 pts in the ITT population Citation Format: Alexander E. Perl, Jorge E. Cortes, Siddhartha Ganguly, Samer K. Khaled, Alwin Krämer, Giovanni Martinelli, Nigel H. Russell, Ken C. Chang, Kazunobu Kato, Yuhu Yan, Li-An Xu, Sergey Korkhov, Tobias Günnel, Hiroyuki Sumi, Arnaud Lesegretain, Flora Berisha, Derek Mires, Aziz Benzohra, Takeshi Isoyama, Cedric Dos Santos, Mark J. Levis. Effect of co-mutations and FLT3-ITD variant allele frequency (VAF) on response to quizartinib or salvage chemotherapy (SC) in relapsed/refractory (R/R) acute myeloid leukemia (AML) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 784.
The 2016 10th Workshop on Recent Issues in Bioanalysis (10th WRIB) took place in Orlando, Florida with participation of close to 700 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. WRIB was once again a weeklong event - A Full Immersion Week of Bioanalysis for PK, Biomarkers and Immunogenicity. As usual, it is specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecules involving LCMS, hybrid LBA/LCMS, and LBA approaches, with the focus on PK, biomarkers and immunogenicity. This 2016 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. This White Paper is published in 3 parts due to length. This part (Part 3) discusses the recommendations for large molecule bioanalysis using LBA, biomarkers and immunogenicity. Parts 1 (small molecule bioanalysis using LCMS) and Part 2 (Hybrid LBA/LCMS and regulatory inputs from major global health authorities) have been published in the Bioanalysis journal, issues 22 and 23, respectively.
CN156018 is an ongoing randomized, double blinded, placebo-controlled Phase II study designed to examine safety and tolerability of avagacestat in patients with predementia AD (PDAD) over a 2 year treatment period. Patients with PDAD were characterized as having objective cognitive impairment as well as low CSF Aβ42 and high tau concentrations. CSF biomarkers were utilized as an enrichment strategy to identify a subset of mild cognitively impaired patients more likely to progress to dementia of the Alzheimer's type and more likely to have increased brain amyloid burden. A multiplex immunoassay (Alzbio3) approved for research-use-only and manufactured by Innogenetics-Fujirebio was utilized to determine CSF Aβ42 and Tau eligibility concentrations for the study. Preliminary analyses with Alzbio3 data showed good correlation with baseline cognitive status and brain amyloid burden. The current study examined the performance of novel improved versions of the Mesoscale Discovery (MSD) assays for CSF Aβ42 and Tau using baseline samples from CN156018 by assessing correlations with Alzbio 3 data, ApoE and baseline cognitive status. Baseline CSF samples from randomized (153) and non-randomized (37) patients were analyzed for CSF Aβ42 and Tau levels using the MSD assays. Correlation analyses were done to compare MSD results to Alzbio3 and to determine whether MSD CSF biomarker concentrations also correlated with ApoE and baseline cognitive status. Levels of CSF Aβ42 correlated more highly between the Alzbio3 and MSD assays than did Tau levels. Preliminary analyses suggest that levels of CSF Aβ42 using both assays were lower in subjects with an ApoE4 allele that is consistent with literature reports. MSD CSF Aβ42 and Tau/Aβ42 ratio data showed statistically significant correlations with baseline ADAS-cog and Free and Cued Selective Reminding Test (FCSRT) scores. CSF Aβ42 and Tau/Aβ42 levels as measured by improved MSD assays showed good correlation with Alzbio3 data. In addition, CSF Aβ42 levels measured by both the Alzbio3 and MSD assays were lower in patients with one or more ApoE4 alleles consistent with literature reports. MSD CSF Aβ42 and Tau/Aβ42 levels correlated with baseline cognitive status in the predementia AD population.
The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "Context of Use - COU"); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness. Part 1A (Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC), Part 1B (Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine) and Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) are published in volume 14 of Bioanalysis, issues 9 and 10 (2022), respectively.
Cerebral spinal fluid (CSF) Aβ42, tau and p181tau are widely accepted biomarkers of Alzheimer’s disease (AD). Numerous studies show that CSF tau and p181tau levels are elevated in mild-to-moderate AD compared to age-matched controls. In addition, these increases might predict preclinical AD in cognitively normal elderly. Despite their importance as biomarkers, the molecular nature of CSF tau and ptau is not known. In the current study, reverse-phase high performance liquid chromatography was used to enrich and concentrate tau prior to western-blot analysis. Multiple N-terminal and mid-domain fragments of tau were detected in pooled CSF with apparent sizes ranging from <20 kDa to ~40 kDa. The pattern of tau fragments in AD and control samples were similar. In contrast, full-length tau and C-terminal-containing fragments were not detected. To quantify levels, five tau ELISAs and three ptau ELISAs were developed to detect different overlapping regions of the protein. The discriminatory potential of each assay was determined using 20 AD and 20 age-matched control CSF samples. Of the tau ELISAs, the two assays specific for tau containing N-terminal sequences, amino acids 9-198 (numbering based on tau 441) and 9-163, exhibited the most significant differences between AD and control samples. In contrast, CSF tau was not detected with an ELISA specific for a more C-terminal region (amino acids 159-335). Significant discrimination was also observed with ptau assays measuring amino acids 159-p181 and 159-p231. Interestingly, the discriminatory potential of p181 was reduced when measured in the context of tau species containing amino acids 9-p181. Taken together, these results demonstrate that tau in CSF occurs as a series of fragments and that discrimination of AD from control is dependent on the subset of tau species measured. These assays provide novel tools to investigate CSF tau and ptau as biomarkers for other neurodegenerative diseases.
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