Complement Activation Is Associated With Disease Severity in Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: Histopathologic studies have identified immunoglobulin (Ig) deposition and complement activation as contributors of CNS tissue damage in multiple sclerosis (MS). Intrathecal IgM synthesis is associated with higher MS disease activity and severity, and IgM is the strongest complement-activating immunoglobulin. In this study, we investigated whether complement components (CCs) and complement activation products (CAPs) are increased in persons with MS, especially in those with an intrathecal IgM synthesis, and whether they are associated with disease severity and progression. METHODS: CC and CAP levels were quantified in plasma and CSF of 112 patients with clinically isolated syndrome (CIS), 127 patients with MS (90 relapsing-remitting, 14 primary progressive, and 23 secondary progressive), 31 inflammatory neurologic disease, and 44 symptomatic controls from the Basel CSF databank study. Patients with CIS/MS were followed in the Swiss MS cohort study (median 6.3 years). Levels of CC/CAP between diagnosis groups were compared; in CIS/MS, associations of CC/CAP levels with intrathecal Ig synthesis, baseline Expanded Disability Status Scale (EDSS) scores, MS Severity Score (MSSS), and neurofilament light chain (NfL) levels were investigated by linear regression, adjusted for age, sex, and albumin quotient. RESULTS: CSF (but not plasma) levels of C3a, C4a, Ba, and Bb were increased in patients with CIS/MS, being most pronounced in those with an additional intrathecal IgM production. In CIS, doubling of C3a and C4a in CSF was associated with 0.31 (CI 0.06-0.56; p = 0.016) and 0.32 (0.02-0.62; p = 0.041) increased EDSS scores at lumbar puncture. Similarly, doubling of C3a and Ba in CIS/MS was associated with 0.61 (0.19-1.03; p < 0.01) and 0.74 (0.18-1.31; p = 0.016) increased future MSSS. In CIS/MS, CSF levels of C3a, C4a, Ba, and Bb were associated with increased CSF NfL levels, e.g., doubling of C3a was associated with an increase of 58% (Est. 1.58; CI 1.37-1.81; p < 0.0001). DISCUSSION: CNS-compartmentalized activation of the classical and alternative pathways of complement is increased in CIS/MS and associated with the presence of an intrathecal IgM production. Increased complement activation within the CSF correlates with EDSS, future MSSS, and NfL levels, supporting the concept that complement activation contributes to MS pathology and disease progression. Complement inhibition should be explored as therapeutic target to attenuate disease severity and progression in MS.

Caregivers' attitudes toward blood-based biomarker testing for Alzheimer's disease.

INTRODUCTION: We aimed to evaluate informal caregivers' attitudes toward undergoing and future implementation of blood-based biomarkers (BBBM) testing for Alzheimer's disease (AD). METHODS: We explored caregivers' perspectives, by combining an online survey (n = 107) with a subsequent focus group (n = 7). We used descriptive statistics and thematic content analysis to identify common themes in answers to open-ended survey questions and focus group data. RESULTS: Most caregivers (72.0%) favored BBBM for AD diagnosis. Provided with hypothetical scenarios, confidence in a normal result decreased significantly if experienced symptoms were more severe (mild: 78.5% vs. severe: 48.6%). Caregivers' attitudes toward BBBM for screening purposes significantly improved with prospect of treatment (53.3% vs. 92.5%). Concerns toward BBBM testing included treatment unavailability, increased/prolonged distress, and AD-related stigma. Potential benefits were actionability, explanation for symptoms, and opportunities for better care and future treatment. DISCUSSION: Emerging AD treatment and reduction of AD-related stigma could profoundly increase public interest in BBBM testing for AD. Highlights: Most informal caregivers would want blood-based biomarker (BBBM) testing for Alzheimer's disease (AD) diagnosis.Perceived (dis)advantages were related to diagnosing AD early.With severe symptoms, there was less confidence in normal BBBM results.Treatment availability would significantly increase interest in BBBM testing for AD.Informal caregivers showed uncertainty regarding the meaning of the term "AD."

Neurofilament light protein as a biomarker for spinal muscular atrophy: a review and reference ranges.

Spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality, characterized by progressive neuromuscular degeneration resulting from mutations in the survival motor neuron (SMN1) gene. The availability of disease-modifying therapies for SMA therapies highlights the pressing need for easily accessible and cost-effective blood biomarkers to monitor treatment response and for better disease management. Additionally, the wide implementation of newborn genetic screening programs in Western countries enables presymptomatic diagnosis of SMA and immediate treatment administration. However, the absence of monitoring and prognostic blood biomarkers for neurodegeneration in SMA hinders effective disease management. Neurofilament light protein (NfL) is a promising biomarker of neuroaxonal damage in SMA and reflects disease progression in children with SMA undergoing treatment. Recently, the European Medicines Agency issued a letter of support endorsing the potential utilization of NfL as a biomarker of pediatric neurological diseases, including SMA. Within this review, we comprehensively assess the potential applications of NfL as a monitoring biomarker for disease severity and treatment response in pediatric-onset SMA. We provide reference ranges for normal levels of serum based NfL in neurologically healthy children aged 0-18 years. These reference ranges enable accurate interpretation of NfL levels in children and can accelerate the implementation of NfL into clinical practice.

Development and multi-center validation of a fully automated digital immunoassay for neurofilament light chain: toward a clinical blood test for neuronal injury.

OBJECTIVES: Neurofilament light chain (NfL) has emerged as a promising biomarker for detecting and monitoring axonal injury. Until recently, NfL could only be reliably measured in cerebrospinal fluid, but digital single molecule array (Simoa) technology has enabled its precise measurement in blood samples where it is typically 50-100 times less abundant. We report development and multi-center validation of a novel fully automated digital immunoassay for NfL in serum for informing axonal injury status. METHODS: A 45-min immunoassay for serum NfL was developed for use on an automated digital analyzer based on Simoa technology. The analytical performance (sensitivity, precision, reproducibility, linearity, sample type) was characterized and then cross validated across 17 laboratories in 10 countries. Analytical performance for clinical NfL measurement was examined in individual patients with relapsing remitting multiple sclerosis (RRMS) after 3 months of disease modifying treatment (DMT) with fingolimod. RESULTS: The assay exhibited a lower limit of detection (LLoD) of 0.05 ng/L, a lower limit of quantification (LLoQ) of 0.8 ng/L, and between-laboratory imprecision <10 % across 17 validation sites. All tested samples had measurable NfL concentrations well above the LLoQ. In matched pre-post treatment samples, decreases in NfL were observed in 26/29 RRMS patients three months after DMT start, with significant decreases detected in a majority of patients. CONCLUSIONS: The sensitivity characteristics and reproducible performance across laboratories combined with full automation make this assay suitable for clinical use for NfL assessment, monitoring in individual patients, and cross-comparisons of results across multiple sites.

Methods to Discover and Validate Biofluid-Based Biomarkers in Neurodegenerative Dementias.

Neurodegenerative dementias are progressive diseases that cause neuronal network breakdown in different brain regions often because of accumulation of misfolded proteins in the brain extracellular matrix, such as amyloids or inside neurons or other cell types of the brain. Several diagnostic protein biomarkers in body fluids are being used and implemented, such as for Alzheimer's disease. However, there is still a lack of biomarkers for co-pathologies and other causes of dementia. Such biofluid-based biomarkers enable precision medicine approaches for diagnosis and treatment, allow to learn more about underlying disease processes, and facilitate the development of patient inclusion and evaluation tools in clinical trials. When designing studies to discover novel biofluid-based biomarkers, choice of technology is an important starting point. But there are so many technologies to choose among. To address this, we here review the technologies that are currently available in research settings and, in some cases, in clinical laboratory practice. This presents a form of lexicon on each technology addressing its use in research and clinics, its strengths and limitations, and a future perspective.

Serum neurofilament light chain reference database for individual application in paediatric care: a retrospective modelling and validation study.

BACKGROUND: Neurological conditions represent an important driver of paediatric disability burden worldwide. Measurement of serum neurofilament light chain (sNfL) concentrations, a specific marker of neuroaxonal injury, has the potential to contribute to the management of children with such conditions. In this context, the European Medicines Agency recently declared age-adjusted reference values for sNfL a top research priority. We aimed to establish an age-adjusted sNfL reference range database in a population of healthy children and adolescents, and to validate this database in paediatric patients with neurological conditions to affirm its clinical applicability. METHODS: To generate a paediatric sNfL reference dataset, sNfL values were measured in a population of healthy children and adolescents (aged 0-22 years) from two large cohorts in Europe (the Coronavirus Antibodies in Kids from Bavaria study, Germany) and North America (a US Network of Paediatric Multiple Sclerosis Centers paediatric case-control cohort). Children with active or previous COVID-19 infection or SARS-CoV-2 antibody positivity at the time of sampling, or a history of primary systemic or neurological conditions were excluded. Linear models were used to restrospectively study the effect of age and weight on sNfL concentrations. We modelled the distribution of sNfL concentrations as a function of age-related physiological changes to derive reference percentile and Z score values via a generalised additive model for location, scale, and shape. The clinical utility of the new reference dataset was assessed in children and adolescents (aged 1-19 years) with neurological diseases (epilepsy, traumatic brain injury, bacterial CNS infections, paediatric-onset multiple sclerosis, and myelin oligodendrocyte glycoprotein antibody-associated disease) from the paediatric neuroimmunology clinic at the University of California San Francisco (San Francisco, CA, USA) and the Children's Hospital of the University of Regensburg (Regensburg, Germany). FINDINGS: Samples from 2667 healthy children and adolescents (1336 [50·1%] girls and 1331 [49·9%] boys; median age 8·0 years [IQR 4·0-12·0]) were used to generate the reference database covering neonatal age to adolescence (target age range 0-20 years). In the healthy population, sNfL concentrations decreased with age by an estimated 6·8% per year until age 10·3 years (estimated multiplicative effect per 1 year increase 0·93 [95% CI 0·93-0·94], p<0·0001) and was mostly stable thereafter up to age 22 years (1·00 [0·52-1·94], p>0·99). Independent of age, the magnitude of the effect of weight on sNfL concentrations was marginal. Samples from 220 children with neurological conditions (134 [60·9%] girls and 86 [39·1%] boys; median age 14·7 years [IQR 10·8-16·5]) were used to validate the clinical utility of the reference Z scores. In this population, age-adjusted sNfL Z scores were higher than in the reference population of healthy children and adolescents (p<0·0001) with higher effect size metrics (Cohen's d=1·56) compared with the application of raw sNfL concentrations (d=1·28). INTERPRETATION: The established normative sNfL values in children and adolescents provide a foundation for the clinical application of sNfL in the paediatric population. Compared with absolute sNfL values, the use of sNfL Z score was associated with higher effect size metrics and allowed for more accurate estimation of the extent of ongoing neuroaxonal damage in individual patients. FUNDING: Swiss National Science Foundation, US National Institutes of Health, and the National Multiple Sclerosis Society.

Clinical Value of Longitudinal Serum Neurofilament Light Chain in Prodromal Genetic Frontotemporal Dementia.

BACKGROUND AND OBJECTIVES: Elevated serum neurofilament light chain (NfL) is used to identify carriers of genetic frontotemporal dementia (FTD) pathogenic variants approaching prodromal conversion. Yet, the magnitude and timeline of NfL increase are still unclear. Here, we investigated the predictive and early diagnostic value of longitudinal serum NfL for the prodromal conversion in genetic FTD. METHODS: In a longitudinal observational cohort study of genetic FTD pathogenic variant carriers, we examined the diagnostic accuracy and conversion risk associated with cross-sectional and longitudinal NfL. Time periods relative to prodromal conversion (>3, 3-1.5, 1.5-0 years before; 0-1.5 years after) were compared with values of participants who did not convert. Next, we modeled longitudinal NfL and MRI volume trajectories to determine their timeline. RESULTS: We included 21 participants who converted (5 chromosome 9 open-reading frame 72 [C9orf72], 10 progranulin [GRN], 5 microtubule-associated protein tau [MAPT], and 1 TAR DNA-binding protein [TARDBP]) and 61 who did not (20 C9orf72, 30 GRN, and 11 MAPT). Participants who converted had higher NfL levels at all examined periods before prodromal conversion (median values 14.0-18.2 pg/mL; betas = 0.4-0.7, standard error [SE] = 0.1, p < 0.046) than those who did not (6.5 pg/mL) and showed further increase 0-1.5 years after conversion (28.4 pg/mL; beta = 1.0, SE = 0.1, p < 0.001). Annualized longitudinal NfL change was only significantly higher in participants who converted (vs. participants who did not) 0-1.5 years after conversion (beta = 1.2, SE = 0.3, p = 0.001). Diagnostic accuracy of cross-sectional NfL for prodromal conversion (vs. nonconversion) was good-to-excellent at time periods before conversion (area under the curve range: 0.72-0.92), improved 0-1.5 years after conversion (0.94-0.97), and outperformed annualized longitudinal change (0.76-0.84). NfL increase in participants who converted occurred earlier than frontotemporal MRI volume change and differed by genetic group and clinical phenotypes. Higher NfL corresponded to increased conversion risk (hazard ratio: cross-sectional = 6.7 [95% CI 3.3-13.7]; longitudinal = 13.0 [95% CI 4.0-42.8]; p < 0.001), but conversion-free follow-up time varied greatly across participants. DISCUSSION: NfL increase discriminates individuals who convert to prodromal FTD from those who do not, preceding significant frontotemporal MRI volume loss. However, NfL alone is limited in predicting the exact timing of prodromal conversion. NfL levels also vary depending on underlying variant-carrying genes and clinical phenotypes. These findings help to guide participant recruitment for clinical trials targeting prodromal genetic FTD.

Neurofilament Light and Its Association With CNS Involvement in Patients With Classic Infantile Pompe Disease.

BACKGROUND AND OBJECTIVES: Enzyme replacement therapy (ERT) has substantially improved the outcome of classic infantile Pompe disease, an inheritable muscle disease previously fatal at infancy. However, under treatment, patients develop white matter abnormalities and neurocognitive problems. Therefore, upcoming therapies also target the brain. Currently, biomarkers reflecting CNS involvement are lacking. We aimed to study the association of neurofilament light (NfL) and CNS involvement. METHODS: To investigate the potential of NfL, we analyzed serum samples of patients with classic infantile Pompe disease who were treated with ERT. The samples were collected at ages of <1, 5, and 10 years, as well as around MRI scans. We compared the outcomes with levels in age- and sex-matched peers. Control samples were originally collected as part of routine blood work in children who underwent small surgeries and stored in the biobank of the Erasmus MC/Sophia Children's Hospital. RESULTS: We analyzed 74 serum samples of 17 patients collected at ages ranging from 22 days to 21.2 years (1-8 samples per patient) and compared these with outcomes of 71 matched peers. In the first year of age, NfL levels in patients and controls were similar (10.3 vs 11.0 pg/mL), but mixed linear model analysis showed a yearly increase of NfL of 6.0% in patients, compared with a decrease of 8.8% in controls (p < 0.001). Higher NfL was associated with lower IQ scores (p = 0.009) and lower processing speed scores (p = 0.001). DISCUSSION: We found significant differences in NfL levels between patients and controls and a good association between NfL and cognition. NfL deserves further exploration as a biomarker for CNS involvement in patients with classic infantile Pompe disease.

Granulocyte activation markers in cerebrospinal fluid differentiate acute neuromyelitis spectrum disorder from multiple sclerosis.

BACKGROUND: Granulocyte invasion into the brain is a pathoanatomical feature differentiating neuromyelitis optica spectrum disorder (NMOSD) from multiple sclerosis (MS). We aimed to determine whether granulocyte activation markers (GAM) in cerebrospinal fluid (CSF) can be used as a biomarker to distinguish NMOSD from MS, and whether levels associate with neurological impairment. METHODS: We quantified CSF levels of five GAM (neutrophil elastase, myeloperoxidase, neutrophil gelatinase-associated lipocalin, matrixmetalloproteinase-8, tissue inhibitor of metalloproteinase-1), as well as a set of inflammatory and tissue-destruction markers, known to be upregulated in NMOSD and MS (neurofilament light chain, glial fibrillary acidic protein, S100B, matrix metalloproteinase-9, intercellular adhesion molecule-1, vascular cellular adhesion molecule-1), in two cohorts of patients with mixed NMOSD and relapsing-remitting multiple sclerosis (RRMS). RESULTS: In acute NMOSD, GAM and adhesion molecules, but not the other markers, were higher than in RRMS and correlated with actual clinical disability scores. Peak GAM levels occurred at the onset of NMOSD attacks, while they were stably low in MS, allowing to differentiate the two diseases for ≤21 days from onset of clinical exacerbation. Composites of GAM provided area under the curve values of 0.90-0.98 (specificity of 0.76-1.0, sensitivity of 0.87-1.0) to differentiate NMOSD from MS, including all anti-aquaporin-4 protein (aAQP4)-antibody-negative patients who were untreated. CONCLUSIONS: GAM composites represent a novel biomarker to reliably differentiate NMOSD from MS, including in aAQP4- NMOSD. The association of GAM with the degree of concurrent neurological impairment provides evidence for their pathogenic role, in turn suggesting them as potential drug targets in acute NMOSD.

A multimodal marker for cognitive functioning in multiple sclerosis: the role of NfL, GFAP and conventional MRI in predicting cognitive functioning in a prospective clinical cohort.

BACKGROUND: Cognitive impairment in people with MS (PwMS) has primarily been investigated using conventional imaging markers or fluid biomarkers of neurodegeneration separately. However, the single use of these markers do only partially explain the large heterogeneity found in PwMS. OBJECTIVE: To investigate the use of multimodal (bio)markers: i.e., serum and cerebrospinal fluid (CSF) levels of neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP) and conventional imaging markers in predicting cognitive functioning in PwMS. METHODS: Eighty-two PwMS (56 females, disease duration = 14 ± 9 years) underwent neuropsychological and neurological examination, structural magnetic resonance imaging, blood sampling and lumbar puncture. PwMS were classified as cognitively impaired (CI) if scoring ≥ 1.5SD below normative scores on ≥ 20% of test scores. Otherwise, PwMS were defined as cognitively preserved (CP). Association between fluid and imaging (bio)markers were investigated, as well as binary logistics regression to predict cognitive status. Finally, a multimodal marker was calculated using statistically important predictors of cognitive status. RESULTS: Only higher NfL levels (in serum and CSF) correlated with worse processing speed (r = - 0.286, p = 0.012 and r = - 0.364, p = 0.007, respectively). sNfL added unique variance in the prediction of cognitive status on top of grey matter volume (NGMV), p = 0.002). A multimodal marker of NGMV and sNfL yielded most promising results in predicting cognitive status (sensitivity = 85%, specificity = 58%). CONCLUSION: Fluid and imaging (bio)markers reflect different aspects of neurodegeneration and cannot be used interchangeably as markers for cognitive functioning in PwMS. The use of a multimodal marker, i.e., the combination of grey matter volume and sNfL, seems most promising for detecting cognitive deficits in MS.

Urine as matrix for analysis of neurofilament light chain is not suitable to distinguish frontotemporal dementia from psychiatric diseases.

The clinical overlap of frontotemporal dementia and primary psychiatric diseases hampers diagnostic distinction, leading to frequent misdiagnosis and diagnostic delay. Neurofilament light chain has shown great potential in CSF and blood for the distinction of frontotemporal dementia from primary psychiatric diseases. Measurement of neurofilament light chain in urine would be even more patient-friendly. We aimed to test the performance of neurofilament light chain urine measurements for diagnostics in frontotemporal dementia and to assess their correlation with serum levels. Fifty-five subjects (n = 19 frontotemporal dementia, n = 19 primary psychiatric diseases and n = 17 controls) were included with available paired urine and serum samples. All subjects underwent standardized extensive diagnostic assessment. Samples were analysed with the ultrasensitive single molecule array neurofilament light chain assay. Neurofilament light chain group comparisons were performed adjusted for age, sex and geriatric depression scale. In the majority of the cohort, neurofilament light chain concentrations were not detectable in urine (n = 6 samples above lower limit of detection (0.038 pg/ml): n = 5 frontotemporal dementia, n = 1 primary psychiatric disease). The frequency of a detectable neurofilament light chain level in urine in the frontotemporal dementia group did not differ from psychiatric disorders (Fisher Exact-test P = 0.180). In the individuals with detectable urine neurofilament light chain values, there was no correlation between the urine and serum neurofilament light chain levels. As expected, serum neurofilament light chain levels were higher in frontotemporal dementia compared to primary psychiatric diseases and controls (P < 0.001), adjusted for age, sex and geriatric depression scale. Receiver operating characteristic curve analysis of serum neurofilament light chain of frontotemporal dementia versus primary psychiatric diseases showed an area under the curve of 0.978 95% confidence interval 0.941-1.000, P < 0.001. Urine is not suitable as a matrix for neurofilament light chain analysis and serum neurofilament light chain is still the most patient-friendly matrix for differentiation between frontotemporal dementia and primary psychiatric diseases.

Serum Glial Fibrillary Acidic Protein Compared With Neurofilament Light Chain as a Biomarker for Disease Progression in Multiple Sclerosis.

Importance: There is a lack of validated biomarkers for disability progression independent of relapse activity (PIRA) in multiple sclerosis (MS). Objective: To determine how serum glial fibrillary acidic protein (sGFAP) and serum neurofilament light chain (sNfL) correlate with features of disease progression vs acute focal inflammation in MS and how they can prognosticate disease progression. Design, Setting, and Participants: Data were acquired in the longitudinal Swiss MS cohort (SMSC; a consortium of tertiary referral hospitals) from January 1, 2012, to October 20, 2022. The SMSC is a prospective, multicenter study performed in 8 centers in Switzerland. For this nested study, participants had to meet the following inclusion criteria: cohort 1, patients with MS and either stable or worsening disability and similar baseline Expanded Disability Status Scale scores with no relapses during the entire follow-up; and cohort 2, all SMSC study patients who had initiated and continued B-cell-depleting treatment (ie, ocrelizumab or rituximab). Exposures: Patients received standard immunotherapies or were untreated. Main Outcomes and Measures: In cohort 1, sGFAP and sNfL levels were measured longitudinally using Simoa assays. Healthy control samples served as the reference. In cohort 2, sGFAP and sNfL levels were determined cross-sectionally. Results: This study included a total of 355 patients (103 [29.0%] in cohort 1: median [IQR] age, 42.1 [33.2-47.6] years; 73 female patients [70.9%]; and 252 [71.0%] in cohort 2: median [IQR] age, 44.3 [33.3-54.7] years; 156 female patients [61.9%]) and 259 healthy controls with a median [IQR] age of 44.3 [36.3-52.3] years and 177 female individuals (68.3%). sGFAP levels in controls increased as a function of age (1.5% per year; P < .001), were inversely correlated with BMI (-1.1% per BMI unit; P = .01), and were 14.9% higher in women than in men (P = .004). In cohort 1, patients with worsening progressive MS showed 50.9% higher sGFAP levels compared with those with stable MS after additional sNfL adjustment, whereas the 25% increase of sNfL disappeared after additional sGFAP adjustment. Higher sGFAP at baseline was associated with accelerated gray matter brain volume loss (per doubling: 0.24% per year; P < .001) but not white matter loss. sGFAP levels remained unchanged during disease exacerbations vs remission phases. In cohort 2, median (IQR) sGFAP z scores were higher in patients developing future confirmed disability worsening compared with those with stable disability (1.94 [0.36-2.23] vs 0.71 [-0.13 to 1.73]; P = .002); this was not significant for sNfL. However, the combined elevation of z scores of both biomarkers resulted in a 4- to 5-fold increased risk of confirmed disability worsening (hazard ratio [HR], 4.09; 95% CI, 2.04-8.18; P < .001) and PIRA (HR, 4.71; 95% CI, 2.05-9.77; P < .001). Conclusions and Relevance: Results of this cohort study suggest that sGFAP is a prognostic biomarker for future PIRA and revealed its complementary potential next to sNfL. sGFAP may serve as a useful biomarker for disease progression in MS in individual patient management and drug development.

Brain-derived neurotrophic factor, neurofilament light and glial fibrillary acidic protein do not change in response to aerobic training in people with MS-related fatigue - a secondary analysis of a randomized controlled trial.

BACKGROUND: Neuroinflammation and neurodegeneration are pathological hallmarks of multiple sclerosis (MS). Brain-derived neurotrophic factor (BDNF), neurofilament light (NfL), and glial fibrillary acidic protein (GFAP) are blood-based biomarkers for neurogenesis, axonal damage and astrocyte reactivity, respectively. We hypothesize that exercise has a neuroprotective effect on MS reflected by normalization of BDNF, NfL and GFAP levels. OBJECTIVES: To investigate the neuroprotective effect of aerobic training (AT) compared to a control intervention on blood-based biomarkers (i.e. BDNF, NfL, GFAP) in people with MS (pwMS). METHODS: In the TREFAMS-AT (Treating Fatigue in Multiple Sclerosis - Aerobic Training) study, 89 pwMS were randomly allocated to either a 16-week AT intervention or a control intervention (3 visits to a MS nurse). In this secondary analysis, blood-based biomarker concentrations were measured in 55 patients using Simoa technology. Changes in pre- and post-intervention concentrations were compared and between-group differences were assessed using analysis of covariance (ANCOVA). Confounding effects of age, sex, MS-related disability assessed using the Expanded Disability Status Scale (EDSS), MS duration, use of disease-modifying medication, and Body Mass Index were considered. RESULTS: Blood samples were available for 30 AT and 25 control group participants (mean age 45.6 years, 71% female, median disease duration 8 years, median EDSS score 2.5). Within-group changes in both study groups were small and non-significant, with the exception of BDNF in the control group (median (interquartile range) -2.1 (-4.7; 0)). No between-group differences were found for any biomarker: BDNF (ß = 0.11, 95%CI (-3.78 to 4.00)), NfL (ß = -0.04, 95%CI (-0.26 to 0.18)), and GFAP (ß = -0.01, 95%CI (-0.16 to 0.15)), adjusted for confounders. CONCLUSION: Aerobic exercise therapy did not result in statistically significant changes in the tested neuro-specific blood-based biomarkers in people with MS. TRIAL REGISTRATION: this study is registered under number ISRCTN69520623 (https://www.isrctn.com/ISRCTN695206).

Serum neurofilament light chain in functionally relevant coronary artery disease and adverse cardiovascular outcomes.

Background: Functionally relevant coronary artery disease (fCAD), causing symptoms of myocardial ischemia, can currently only be reliably detected with advanced cardiac imaging. Serum neurofilament light chain (sNfL) is a biomarker for neuro-axonal injury known to be elevated by cardiovascular (CV) risk factors and cerebrovascular small-vessel diseases. Due to their pathophysiological similarities with fCAD and the link to CV risk factors, we hypothesised that sNfL may have diagnostic and prognostic value for fCAD and adverse cardiovascular outcomes.Methods: Of the large prospective Basel VIII study (NCT01838148), 4'016 consecutive patients undergoing cardiac work-up for suspected fCAD were included (median age 68 years, 32.5% women, 46.9% with history of CAD). The presence of fCAD was adjudicated using myocardial perfusion imaging single-photon emission tomography (MPI-SPECT) and coronary angiography. sNfL was measured using a high-sensitive single-molecule array assay. All-cause and cardiovascular death, myocardial infarction (MI), and stroke/transient ischaemic attack (TIA) during 5-year follow-up were the prognostic endpoints.Results: The diagnostic accuracy of sNfL for fCAD as quantified by the area under the curve (AUC) was low (0.58, 95%CI 0.56-0.60). sNfL was strongly associated with age, renal dysfunction, and body mass index and was a strong and independent predictor of all-cause death, cardiovascular death, and stroke/TIA but not MI. Time-dependent AUC for cardiovascular-death at 1-year was 0.85, 95%CI 0.80-0.89, and 0.81, 95%CI 0.77-0.86 at 2-years.Conclusion: While sNfL concentrations did not show a diagnostic role for fCAD, in contrast, sNfL was a strong and independent predictor of cardiovascular outcomes, including all-cause death, cardiovascular death and stroke/TIA.

Assessing the commutability of candidate reference materials for the harmonization of neurofilament light measurements in blood.

OBJECTIVES: Neurofilament light chain (NfL) concentration in blood is a biomarker of neuro-axonal injury in the nervous system and there now exist several assays with high enough sensitivity to measure NfL in serum and plasma. There is a need for harmonization with the goal of creating a certified reference material (CRM) for NfL and an early step in such an effort is to determine the best matrix for the CRM. This is done in a commutability study and here the results of the first one for NfL in blood is presented. METHODS: Forty paired individual serum and plasma samples were analyzed for NfL on four different analytical platforms. Neat and differently spiked serum and plasma were evaluated for their suitability as a CRM using the difference in bias approach. RESULTS: The correlation between the different platforms with regards to measured NfL concentrations were very high (Spearman's ρ≥0.96). Samples spiked with cerebrospinal fluid (CSF) showed higher commutability compared to samples spiked with recombinant human NfL protein and serum seems to be a better choice than plasma as the matrix for a CRM. CONCLUSIONS: The results from this first commutability study on NfL in serum/plasma showed that it is feasible to create a CRM for NfL in blood and that spiking should be done using CSF rather than with recombinant human NfL protein.

Bioinformatics tools and data resources for assay development of fluid protein biomarkers.

Fluid protein biomarkers are important tools in clinical research and health care to support diagnosis and to monitor patients. Especially within the field of dementia, novel biomarkers could address the current challenges of providing an early diagnosis and of selecting trial participants. While the great potential of fluid biomarkers is recognized, their implementation in routine clinical use has been slow. One major obstacle is the often unsuccessful translation of biomarker candidates from explorative high-throughput techniques to sensitive antibody-based immunoassays. In this review, we propose the incorporation of bioinformatics into the workflow of novel immunoassay development to overcome this bottleneck and thus facilitate the development of novel biomarkers towards clinical laboratory practice. Due to the rapid progress within the field of bioinformatics many freely available and easy-to-use tools and data resources exist which can aid the researcher at various stages. Current prediction methods and databases can support the selection of suitable biomarker candidates, as well as the choice of appropriate commercial affinity reagents. Additionally, we examine methods that can determine or predict the epitope - an antibody's binding region on its antigen - and can help to make an informed choice on the immunogenic peptide used for novel antibody production. Selected use cases for biomarker candidates help illustrate the application and interpretation of the introduced tools.

Age- and disease-specific reference values for neurofilament light presented in an online interactive support interface.

Interpretation of axonal damage biomarker Neurofilament Light chain (NfL) concentrations is difficult due to the lack of age-specific and disease-specific reference values. We here developed an interactive interface to support interpretation of NfL results in human body fluids. We used NfL values of 1698 individuals without a neurological disorder, aged 19-85 years, and patients with MS and dementias. Percentile regression estimates per diagnosis populate interactive graphs, alongside NfL background information (available on: https://mybiomarkers.shinyapps.io/Neurofilament). This accessible interface provides reference for interpretation of the individual patient results for clinicians. It showcases an adaptable method to support interpretation of age-dependent biomarkers in neurology.

A Combination of Neurofilament Light, Glial Fibrillary Acidic Protein, and Neuronal Pentraxin-2 Discriminates Between Frontotemporal Dementia and Other Dementias.

BACKGROUND: The differential diagnosis of frontotemporal dementia (FTD) is still a challenging task due to its symptomatic overlap with other neurological diseases and the lack of biofluid-based biomarkers. OBJECTIVE: To investigate the diagnostic potential of a combination of novel biomarkers in cerebrospinal fluid (CSF) and blood. METHODS: We included 135 patients from the Center for Memory Disturbances, University of Perugia, with the diagnoses FTD (n = 37), mild cognitive impairment due to Alzheimer's disease (MCI-AD, n = 47), Lewy body dementia (PDD/DLB, n = 22), and cognitively unimpaired patients as controls (OND, n = 29). Biomarker levels of neuronal pentraxin-2 (NPTX2), neuronal pentraxin receptor, neurofilament light (NfL) and glial fibrillary acidic protein (GFAP) were measured in CSF, as well as NfL and GFAP in serum. We assessed biomarker differences by analysis of covariance and generalized linear models (GLM). We performed receiver operating characteristics analyses and Spearman correlation to determine biomarker associations. RESULTS: CSF NPTX2 and serum GFAP levels varied most between diagnostic groups. The combination of CSF NPTX2, serum NfL and serum GFAP differentiated FTD from the other groups with good accuracy (FTD versus MCI-AD: area under the curve (AUC) [95% CI] = 0.89 [0.81-0.96]; FTD versus PDD/DLB: AUC = 0.82 [0.71-0.93]; FTD versus OND: AUC = 0.80 [0.70-0.91]). CSF NPTX2 and serum GFAP correlated positively only in PDD/DLB (ρ= 0.56, p < 0.05). NPTX2 and serum NfL did not correlate in any of the diagnostic groups. Serum GFAP and serum NfL correlated positively in all groups (ρ= 0.47-0.74, p < 0.05). CONCLUSION: We show the combined potential of CSF NPTX2, serum NfL, and serum GFAP to differentiate FTD from other neurodegenerative disorders.

Plasma P-Tau181 for the Discrimination of Alzheimer's Disease from Other Primary Dementing and/or Movement Disorders.

Blood phospho-tau181 may offer a useful biomarker for Alzheimer's disease. However, the use of either serum or plasma phospho-tau181 and their diagnostic value are currently under intense investigation. In a pilot study, we measured both serum and plasma phospho-tau181 (pT181-Tau) by single molecule array (Simoa) in a group of patients with Alzheimer's disease and a mixed group of patients with other primary dementing and/or movement disorders. Classical cerebrospinal fluid biomarkers were also measured. Plasma (but not serum) pT181-Tau showed a significant increase in Alzheimer's disease and correlated significantly with cerebrospinal fluid amyloid and pT181-Tau. Receiver operating curve analysis revealed a significant discrimination of Alzheimer's from non-Alzheimer's disease patients, with an area under the curve of 0.83 and an excellent sensitivity but a moderate specificity. Plasma pT181-Tau is not an established diagnostic biomarker for Alzheimer's disease, but it could become one in the future, or it may serve as a screening tool for specific cases of patients or presymptomatic subjects.

A Pragmatic, Data-Driven Method to Determine Cutoffs for CSF Biomarkers of Alzheimer Disease Based on Validation Against PET Imaging.

BACKGROUND AND OBJECTIVES: To elaborate a new algorithm to establish a standardized method to define cutoffs for CSF biomarkers of Alzheimer disease (AD) by validating the algorithm against CSF classification derived from PET imaging. METHODS: Low and high levels of CSF phosphorylated tau were first identified to establish optimal cutoffs for CSF ß-amyloid (Aß) peptide biomarkers. These Aß cutoffs were then used to determine cutoffs for CSF tau and phosphorylated tau markers. We compared this algorithm to a reference method, based on tau and amyloid PET imaging status (ADNI study), and then applied the algorithm to 10 large clinical cohorts of patients. RESULTS: A total of 6,922 patients with CSF biomarker data were included (mean [SD] age: 70.6 [8.5] years, 51.0% women). In the ADNI study population (n = 497), the agreement between classification based on our algorithm and the one based on amyloid/tau PET imaging was high, with Cohen's kappa coefficient between 0.87 and 0.99. Applying the algorithm to 10 large cohorts of patients (n = 6,425), the proportion of persons with AD ranged from 25.9% to 43.5%. DISCUSSION: The proposed novel, pragmatic method to determine CSF biomarker cutoffs for AD does not require assessment of other biomarkers or assumptions concerning the clinical diagnosis of patients. Use of this standardized algorithm is likely to reduce heterogeneity in AD classification.