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 three Main Workshops and seven 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 2) covers the recommendations on ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry. 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 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 9 and 11 (2022), respectively.

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.

Background: Quizartinib, a once-daily, oral, highly potent and selective FLT3 inhibitor, demonstrated a significant improvement in survival vs SC in FLT3-ITD-positive R/R AML in the global, randomized, phase 3 QuANTUM-R study (Cortes et al. Lancet Oncol, 2019; NCT02039726). Patients with R/R FLT3-ITD-positive AML were randomized 2:1 to receive single agent quizartinib or investigator's choice of pre-selected SC. We investigated the effects of baseline co-mutations and FLT3-ITD VAF on overall survival (OS) and response (composite complete remission [CRc]) to quizartinib and SC in QuANTUM-R. Methods: We analyzed 37 recurrently mutated genes in AML in baseline bone marrow samples from 304 patients (82.8% of ITT population [N = 367; quizartinib, n = 245; SC, n = 122]) 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 separately 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, 5 key co-mutations were detected: DNMT3Amut (n = 182/304 [59.9%]), NPM1mut (n = 168/304 [55.3%]), TET2mut (n = 98/304 [32.2%]), IDH1/2mut (n = 49/304 [16.1%]) and CEBPAmut (n = 46/304 [15.1%]). Median OS was numerically longer with quizartinib vs SC in patients with DNMT3Amut, TET2mut, IDH1/2mut and NPM1mut, but not CEBPAmut (Table). CRc rates were numerically higher with quizartinib vs SC for each of the 5 key baseline co-mutations. For single gene mutations, the longest median OS was seen in patients with CEBPAmut treated with quizartinib or SC (37 and 37.6 weeks, respectively). As the majority of NPM1mut patients were also DNMT3Amut (138/168, 82%), we examined various permutations of these two mutations. Patients with NPM1wt/DNMT3Amut had significantly longer median OS with quizartinib vs SC (39.3 vs 19.6 weeks, respectively; HR, 0.239; P = 0.003 [Table]) while NPM1mut/DNMT3Amut patients had lower and similar median OS between the 2 arms (23.6 vs 23.4 weeks, respectively). Quizartinib treatment showed significantly longer median OS vs SC in patients with high FLT3-ITD VAF (23.9 vs 17 weeks respectively; HR, 0.689, P = 0.0148), while the median OS in patients with low FLT3-ITD VAF was similar (34.1 vs 26.6 weeks, respectively; HR, 0.857, P = 0.535). Conclusions: This is the first evaluation of the effect of baseline co-mutations on clinical outcomes in a large trial of R/R AML patients with FLT3-ITD mutations treated with quizartinib. Key co-mutations identified in this analysis were found to potentially impact treatment response and OS with quizartinib, relative to SC. Despite relatively low CRc rates in patients with IDH1/2mut, this group-as well as those with NPM1wt-derived the greatest OS benefit from quizartinib compared with SC on QuANTUM-R. CEBPA mutations were associated with high CRc rates and relatively long median OS, regardless of treatment arm. Patients with NPM1mut had a higher CRc rate with quizartinib vs SC, but this did not translate into longer survival on either arm compared with NPM1wt. A high allelic burden of FLT3-ITD at the time of salvage therapy was associated with relatively poorer median OS; quizartinib significantly improved survival of patients with high FLT3-ITD VAF relative to SC. Although these results require confirmation in an independent dataset, the modulatory effects of baseline co-mutations on treatment response and OS with quizartinib appear to differ from other FLT3 inhibitors. Our results indicate that a subset of R/R AML patients may particularly derive clinical benefit from quizartinib. Table Disclosures Perl: Daiichi Sankyo: Consultancy, Honoraria, Other, Research Funding; Arog: Consultancy, Other: Non-financial support included travel costs for advisory board meetings.; AbbVie: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings.; Actinium Pharmaceuticals: Consultancy, Honoraria, Other: Clinical Advisory Board member, Research Funding; Agios: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Non-financial support included travel costs for advisory board meetings.; Jazz: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings.; NewLink Genetics: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings.; Takeda: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings.; Bayer: Research Funding; BioMed Valley Discoveries: Research Funding; FujiFilm: Research Funding; Novartis: Honoraria, Other: Advisory board, Non-financial support included travel costs for advisory board meetings as well as a medical writing company that assisted with manuscript preparation/submission and slide deck assembly for academic meeting presentations of the data., Research Funding; Astellas: Consultancy, Honoraria, Other: Non-financial support included travel costs for advisory board meetings as well as a medical writing company that assisted with manuscript preparation/submission and slide deck assembly for academic meeting presentations of trial data., Research Funding. Cortes:Astellas Pharma: Consultancy, Honoraria, Research Funding; Jazz Pharmaceuticals: Consultancy, Research Funding; Merus: Consultancy, Honoraria, Research Funding; Forma Therapeutics: Consultancy, Honoraria, Research Funding; Biopath Holdings: Consultancy, Honoraria; BiolineRx: Consultancy; Pfizer: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Sun Pharma: Research Funding; Immunogen: Consultancy, Honoraria, Research Funding. Ganguly:Kite Pharma: Honoraria, Other: Advisory Board; Janssen: Honoraria, Other: Advisory Board; Seattle Genetics: Speakers Bureau; Daiichi Sankyo: Research Funding. Khaled:Omeros: Consultancy; Alexion: Consultancy, Speakers Bureau; Daiichi Sankyo: Other: Travel support. Krämer:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Research Funding; BMS: Research Funding. Martinelli:Novartis: Consultancy, Other: trial grant; Roche: Consultancy, Other: trial grant; Janssen: Consultancy, Other: trial grant; Incyte: Consultancy, Other: trial grant; Pfizer: Consultancy, Other: trial grant; Amgen: Consultancy, Other: trial grant; Celgene: Consultancy, Honoraria, Other: trial grant; Ariad: Consultancy, Other: trial grant; Abbvie: Consultancy, Honoraria, Other: trial grant; Daiichi Sankyo: Consultancy, Honoraria. Russell:Pfizer Inc: Consultancy, Honoraria, Speakers Bureau; Astellas: Consultancy, Honoraria, Speakers Bureau; Jazz: Consultancy, Honoraria, Speakers Bureau; DSI: Consultancy, Honoraria, Speakers Bureau. Chang:Daiichi Sankyo: Employment. Mires:Daiichi Sankyo: Employment. Kato:Daiichi Sankyo, Inc.: Employment; Celgene: Employment, Equity Ownership. Zhang:Daiichi Sankyo: Employment. Korkhov:Precision for Medicine, Inc.: Employment; Daiichi Sankyo: Consultancy. Wang:Precision for Medicine, Inc.: Employment; Daiichi Sankyo: Consultancy. Günnel:Precision for Medicine, Inc.: Employment; Daiichi Sankyo: Consultancy. Sumi:Daiichi Sankyo, Inc.: Employment. Isoyama:Daiichi Sankyo Co, Ltd: Employment. Lesegretain:Daiichi-Sankyo Inc.: Employment, Equity Ownership. Berisha:Daiichi Sankyo: Employment. Dos Santos:Daiichi Sankyo: Employment. Levis:Agios: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Novartis: Consultancy, Research Funding; FUJIFILM: Consultancy, Research Funding; Menarini: Consultancy, Honoraria; Astellas: Consultancy, Research Funding; Daiichi Sankyo Inc: Consultancy, Honoraria.

The presence of MRD in patients with acute myeloid leukemia (AML) who are in morphologic remission has been shown to be a powerful predictor of eventual relapse. FMS-like tyrosine kinase 3 internal tandem duplications (FLT3-ITD) confer a negative prognostic impact by increasing the risk of relapse. However, the ability to detect these mutations in remission bone marrow specimens is hampered by the limited sensitivity of conventional polymerase chain reaction (PCR)-based assays, which detect approximately only 1 of every 100 (1%) mutant cells. To address this problem, we have developed a novel NGS-based MRD assay for the detection of FLT3-ITD mutations.

The 2017 11th Workshop on Recent Issues in Bioanalysis took place in Los Angeles/Universal City, California, on 3-7 April 2017 with participation of close to 750 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule analysis involving LC-MS, hybrid ligand-binding assay (LBA)/LC-MS and LBA approaches. This 2017 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 2017 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations for large-molecule bioanalysis, biomarkers and immunogenicity using LBA. Part 1 (LC-MS for small molecules, peptides and small molecule biomarkers) and Part 2 (hybrid LBA/LC-MS for biotherapeutics and regulatory agencies' inputs) are published in volume 9 of Bioanalysis, issues 22 and 23 (2017), respectively.

Introduction Acute myeloid leukemia (AML) is a heterogeneous disease with multiple factors influencing long-term outcome. FLT3 (FMS-like tyrosine kinase 3) mutations (predominantly internal tandem duplication [ITD]) are reported in ~25% of patients with AML and have been associated with poorer outcomes compared with FLT3 non-mutated AML (Kottaridis et al. Blood. 2001). Targeting these mutations with tyrosine kinase inhibitors has become an active area of research. However, patients must undergo molecular testing to identify the presence of this mutation. The specimen of choice for such testing is bone marrow (BM; aspirate or biopsy), as the site of origin of the disease. However, BM testing is invasive and sometimes cannot be performed repeatedly in a timely manner. A simple technique using easily obtainable biological specimens from a source such as peripheral blood (PB) may be desirable if the results accurately reflect status in the active disease site (i.e., BM). We investigated whether a PB specimen could be utilized for FLT3 -ITD determination and if the characteristics of the ITD mutations identified in PB were consistent with those in BM samples from the same patient. Methods QuANTUM-R is a global, randomized, phase 3 study to assess the efficacy of quizartinib, a selective FLT3 inhibitor, in patients with FLT3 -ITD-mutated relapsed or refractory (R/R) AML. Paired samples of BM and PB were collected from individual patients as part of the screening process for QuANTUM-R. One to 3 mL each of BM and PB were collected on the same day (window Day -14 to Day 0) from each patient prior to receipt of any study treatment. A statistically powered number of patients (minimum 95) with paired BM and PB samples were selected during a defined period of screening in the phase 3 study. Samples were subjected to DNA isolation followed by PCR and fragment size analysis. Each sample was assessed for the presence of ITD mutation; moreover, the length and allelic frequency of the ITD mutation were measured for each sample. The assay was validated down to a limit of detection (LoD) of 1%. Correlation between the data obtained from BM and PB was assessed using Deming regression analysis and Bland-Altman plot constructed to assess concordance between the 2 sample types. Results Paired samples from 107 patients (median age 58y; 32% were ≥65y; 57% were female) were included. Sixteen samples had no ITD mutations in either BM or PB; 3 had ITD only in the BM sample with no corresponding ITD in the paired PB sample (despite having circulating blasts), and 88 had ITD in both BM and PB. In all 88 cases, the length of the ITD was identical in both specimen types. The sizes of ITD observed in this patient population were between 9 and 198 bases; 21/88 samples with ITD demonstrated the presence of multiple ITDs, with samples from 2 patients exhibiting 3 distinct ITDs, each of whose sizes were the same in BM and PB. Across all samples with ITD, the measured ITD allelic ratio ranged from 1% to 96%, with a similar or slightly lower ratio measured in the PB compared to the BM. In 23 (26%) cases, the allelic ratio measured in the PB was lower than that of the BM by ≥10%. Blast counts for these samples were also measured and there was no correlation between blast count and allelic ratio in either specimen type. High correlation between PB and BM samples for the presence of FLT3 -ITD was observed when regressing PB Ratio on BM Ratio via Deming regression analysis, and the following values were obtained: R 2 = 0.866; 95% CI for the slope, 0.98 to 1.12; and Bland-Altman difference plot for allelic ratio against difference between PB and BM showed good random scatter around average bias of -5.2%, with 95% CI including 0.0% (95% CI, -27.6 to 17.2). Conclusions FLT3 -ITD mutation testing is performed in all AML patients at diagnosis for prognostic purposes and to guide therapeutic decisions. As FLT3 inhibitors are developed for clinical use, regular monitoring of patients9 residual disease burden/response to therapy through assessment of FLT3 status may become an important element of monitoring benefit from effective therapy. Mutation assessment is currently performed on BM aspirate or biopsy based on the common belief that PB is not adequate for this assessment. Our results of FLT3 -ITD mutation analysis in paired BM and PB samples collected from >100 patients with R/R AML show that PB specimens have a high degree of concordance with BM specimens for assessment of FLT3 mutation while being less invasive. Disclosures Khaled: Daiichi Sankyo, Inc: Other: Travel Support; City of Hope: Research Funding. Ganguly: Amgen: Other: Advisory Board; Seattle Genetics: Speakers Bureau. Perl: Seattle Genetics: Other: Advisory board; Daiichi Sankyo: Consultancy; Astellas: Consultancy; Novartis: Other: Advisory Board; Pfizer: Other: Advisory Board; Actinium Pharmaceuticals: Other: Scientific Advisory Board; Arog Pharmaceuticals: Consultancy; Asana Biosciences: Other: Scientific advisory board. Kobayashi: Daiichi Sankyo, Inc.: Employment. Berisha: Daiichi Sankyo, Inc.: Employment. Lameh: Daiichi Sankyo, Inc.: Research Funding; Navigate BioPharma Service Inc.,: Employment. Martinelli: Celgene: Consultancy; Pfizer: Consultancy; Amgen: Consultancy; Ariad: Consultancy; Incyte-Teva: Speakers Bureau.

Multiplex ligand binding assays (LBAs) are increasingly being used to support many stages of drug development. The complexity of multiplex assays creates many unique challenges in comparison to single-plexed assays leading to various adjustments for validation and potentially during sample analysis to accommodate all of the analytes being measured. This often requires a compromise in decision making with respect to choosing final assay conditions and acceptance criteria of some key assay parameters, depending on the intended use of the assay. The critical parameters that are impacted due to the added challenges associated with multiplexing include the minimum required dilution (MRD), quality control samples that span the range of all analytes being measured, quantitative ranges which can be compromised for certain targets, achieving parallelism for all analytes of interest, cross-talk across assays, freeze-thaw stability across analytes, among many others. Thus, these challenges also increase the complexity of validating the performance of the assay for its intended use. This paper describes the challenges encountered with multiplex LBAs, discusses the underlying causes, and provides solutions to help overcome these challenges. Finally, we provide recommendations on how to perform a fit-for-purpose-based validation, emphasizing issues that are unique to multiplex kit assays.

The amyloid-β peptide (Aβ)-in particular, the 42-amino acid form, Aβ1-42-is thought to play a key role in the pathogenesis of Alzheimer's disease (AD). Thus, several therapeutic modalities aiming to inhibit Aβ synthesis or increase the clearance of Aβ have entered clinical trials, including γ-secretase inhibitors, anti-Aβ antibodies, and amyloid-β precursor protein cleaving enzyme inhibitors. A unique class of small molecules, γ-secretase modulators (GSMs), selectively reduce Aβ1-42 production, and may also decrease Aβ1-40 while simultaneously increasing one or more shorter Aβ peptides, such as Aβ1-38 and Aβ1-37. GSMs are particularly attractive because they do not alter the total amount of Aβ peptides produced by γ-secretase activity; they spare the processing of other γ-secretase substrates, such as Notch; and they do not cause accumulation of the potentially toxic processing intermediate, β-C-terminal fragment. This report describes the translation of pharmacological activity across species for two novel GSMs, (S)-7-(4-fluorophenyl)-N2-(3-methoxy-4-(3-methyl-1H-1,2,4-triazol-1-yl)phenyl)-N4-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidine-2,4-diamine (BMS-932481) and (S,Z)-17-(4-chloro-2-fluorophenyl)-34-(3-methyl-1H-1,2,4-triazol-1-yl)-16,17-dihydro-15H-4-oxa-2,9-diaza-1(2,4)-cyclopenta[d]pyrimidina-3(1,3)-benzenacyclononaphan-6-ene (BMS-986133). These GSMs are highly potent in vitro, exhibit dose- and time-dependent activity in vivo, and have consistent levels of pharmacological effect across rats, dogs, monkeys, and human subjects. In rats, the two GSMs exhibit similar pharmacokinetics/pharmacodynamics between the brain and cerebrospinal fluid. In all species, GSM treatment decreased Aβ1-42 and Aβ1-40 levels while increasing Aβ1-38 and Aβ1-37 by a corresponding amount. Thus, the GSM mechanism and central activity translate across preclinical species and humans, thereby validating this therapeutic modality for potential utility in AD.

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.

The pharmacokinetics, pharmacodynamics, safety, and tolerability of BMS-932481, a γ-secretase modulator (GSM), were tested in healthy young and elderly volunteers after single and multiple doses. BMS-932481 was orally absorbed, showed dose proportionality after a single dose administration, and had approximately 3-fold accumulation after multiple dosing. High-fat/caloric meals doubled the Cmax and area under the curve and prolonged Tmax by 1.5 hours. Consistent with the preclinical pharmacology of GSMs, BMS-932481 decreased cerebrospinal fluid (CSF) Aβ39, Aβ40, and Aβ42 while increasing Aβ37 and Aβ38, thereby providing evidence of γ-secretase enzyme modulation rather than inhibition. In plasma, reductions in Aβ40 and Aβ42 were observed with no change in total Aβ; in CSF, modest decreases in total Aβ were observed at higher dose levels. Increases in liver enzymes were observed at exposures associated with greater than 70% CSF Aβ42 lowering after multiple dosing. Although further development was halted due to an insufficient safety margin to test the hypothesis for efficacy of Aβ lowering in Alzheimer's disease, this study demonstrates that γ-secretase modulation is achievable in healthy human volunteers and supports further efforts to discover well tolerated GSMs for testing in Alzheimer's disease and other indications.

OBJECTIVE:Cerebrospinal fluid (CSF) neurofilament light chain (NfL) concentration is elevated in neurological disorders, including frontotemporal degeneration (FTD). We investigated the clinical correlates of elevated CSF NfL levels in FTD.METHODS:CSF NfL, amyloid-β1-42 (Aβ42), tau, and phosphorylated tau concentrations were compared in 47 normal controls (NC), 8 asymptomatic gene carriers (NC2) of FTD-causing mutations, and 79 FTD (45 behavioral variant frontotemporal dementia [bvFTD], 18 progressive nonfluent aphasia [PNFA], 16 semantic dementia [SD]), 22 progressive supranuclear palsy, 50 Alzheimer disease, 6 Parkinson disease, and 17 corticobasal syndrome patients. Correlations between CSF analyte levels were performed with neuropsychological measures and the Clinical Dementia Rating scale sum of boxes (CDRsb). Voxel-based morphometry of structural magnetic resonance images determined the relationship between brain volume and CSF NfL.RESULTS:Mean CSF NfL concentrations were higher in bvFTD, SD, and PNFA than other groups. NfL in NC2 was similar to NC. CSF NfL, but not other CSF measures, correlated with CDRsb and neuropsychological measures in FTD, but not in other diagnostic groups. Analyses in 2 independent FTD cohorts and a group of autopsy-verified or biomarker-enriched cases confirmed the larger group analysis. In FTD, gray and white matter volume negatively correlated with CSF NfL concentration, such that individuals with the highest NfL levels exhibited the most atrophy.INTERPRETATION:CSF NfL is elevated in symptomatic FTD and correlates with disease severity. This measurement may be a useful surrogate endpoint of disease severity in FTD clinical trials. Longitudinal studies of CSF NfL in FTD are warranted.

Background Elevated CSF τ is considered a biomarker of neuronal injury in newly developed Alzheimer's disease (AD) and mild cognitive impairment (MCI) criteria. However, previous studies have failed to detect alterations of τ species in other primary tauopathies. We assessed CSF τ protein abnormalities in AD, a tauopathy with prominent Aβ pathology, and progressive supranuclear palsy (PSP), a primary tauopathy characterised by deposition of four microtubule-binding repeat (4R) τ with minimal Aβ pathology. Methods 26 normal control (NC), 37 AD, and 24 patients with PSP participated in the study. AD and PSP were matched for severity using the clinical dementia rating sum of boxes (CDR-sb) scores. The INNO BIA AlzBio3 multiplex immunoassay was used to measure CSF Aβ, total τ, and ptau181. Additional, novel ELISAs targeting different N-terminal and central τ epitopes were developed to examine CSF τ components and to investigate interactions between diagnostic group, demographics and genetic variables. Results PSP had lower CSF N-terminal and C-terminal τ concentrations than NC and AD measured with the novel τ ELISAs and the standard AlzBio3 τ and ptau assays. AD had higher total τ and ptau levels than NC and PSP. There was a gender by diagnosis interaction in AD and PSP for most τ species, with lower concentrations for male compared to female patients. Conclusions CSF τ fragment concentrations are different in PSP compared with AD despite the presence of severe τ pathology and neuronal injury in both disorders. CSF τ concentration likely reflects multiple factors in addition to the degree of neuronal injury.

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.

Tau concentration in CSF has been shown to correlate with Alzheimer's disease (AD) progression and is a key measurement in the assessment of AD risk and status. 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 for the measurement of tau in CSF on the VITROS Immunodiagnostic Systems. The objective of this study was to demonstrate the stability of the assay reagents over a 29 week period. One lot of VITROS Tau assay reagent was stored at 2–8°C and tested at 4 timepoints over a 29 week period to demonstrate the stability of the reagents. At each timepoint, the reagents were calibrated and used to test two CSF pools containing approximately 400 and 2700pg/mL of endogenous tau. The percent change in the measured tau level for each CSF pool at each timepoint was calculated. The %CV was also calculated across all timepoints for both CSF pools. For the 29 week study, the mean percent differences in measured CSF tau levels vs. time zero were -3.7% and -2.0% for the low and high CSF pools, respectively. The individual percent differences from time zero for both CSF pools across all timepoints ranged from -8.1% to -0.6%. The pg/mL %CV's across all timepoints were 3.8% and 1.6% for the low and high CSF pools, respectively. The VITROS Immunodiagnostic Products tau assay has demonstrated consistent results with reagents stored at 2–8°C for over 6 months.

OBJECTIVE: To compare CSF tau protein abnormalities in Alzheimer's disease (AD), a tauopathy with prominent Aβ pathology, and progressive supranuclear palsy (PSP), a primary 4 repeat tauopathy with minimal Aβ pathology.

Beta amyloid 1–42 (Aβ42) concentration in cerebrospinal fluid (CSF) has been shown to correlate with Alzheimer's disease (AD) progression and is a key measurement in identification of people at risk for progressing to 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 Amyloid Beta 42 Assay (AB-42)1 under development for measurement of Aβ42 in CSF on VITROS Immunodiagnostic Systems. The objective of this study was to demonstrate consistent performance of the assay across lots and instrument platforms. Two lots of VITROS AB-42 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 1.001, 14.0, and 0.998 respectively. The lot-to-lot comparison (Lot 2 - 3600 vs. Lot 1 - 3600) slope, intercept, and correlation (r) results were 0.992, -5.69, and 0.999 respectively. Across lots and systems (Lot 2 - ECiQ vs. Lot 1 - 3600) slope, intercept, and correlation (r) results were 1.014, -0.84, and 0.998 respectively. The mean doses of the controls (n = 8 per control) ranged from 55.0 - 1899 pg/mL with an overall %CV from 1.6% - 5.0%. The mean doses of the individual CSF samples (n = 4 per CSF) ranged from 41.8 - 1236 pg/mL with an overall %CV from 1.0% - 25.3%. Only one CSF sample with a concentration >100 pg/mL had a CV >10%. The VITROS Immunodiagnostic Products Amyloid Beta 42 Assay 1 has demonstrated consistent results across reagent lots and across systems.

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.

As the aging population grows, it has become increasingly important to carefully characterize amnestic mild cognitive impairment (aMCI), a preclinical stage of Alzheimer’s disease (AD). Functional magnetic resonance imaging (fMRI) is a valuable tool for monitoring disease progression in selectively vulnerable brain regions associated with AD neuropathology. However, the reliability of fMRI data in longitudinal studies of older adults with aMCI is largely unexplored. To address this, amnestic aMCI participants completed two visual working tasks, a delayed-recognition task and a oneback task, on three separate scanning sessions over a three-month period. Test-retest reliability of the fMRI blood oxygen level dependent (BOLD) activity was assessed using an intraclass correlation (ICC) analysis approach. Results indicated that brain regions engaged during the task displayed greater reliability across sessions compared to regions that were not utilized by the task. During When brain regions Duringtask-engagement, differential reliability scores were observed across the brain such that the frontal lobe, medial temporal lobe, and subcortical structures exhibited fair to moderate reliability (ICC = 0.3 – 0.6), while temporal, parietal, and occipital regions exhibited moderate to good reliability (ICC = 0.4 – 0.7). Additionally, reliability across brain regions was more stable when three fMRI sessions were used in the ICC calculation relative to two fMRI sessions. In conclusion, the fMRI BOLD signal is reliable across scanning sessions in this population and thus a useful tool for tracking longitudinal change in observational and interventional studies in aMCI.