Serum neurofilament light chain correlations in patients with a first clinical demyelinating event in the REFLEX study: a post hoc analysis.

Background: In REFLEX, subcutaneous interferon beta-1a (sc IFN ß-1a) delayed the onset of multiple sclerosis (MS) in patients with a first clinical demyelinating event (FCDE). Objectives: This post hoc analysis aimed to determine whether baseline serum neurofilament light (sNfL) chain can predict conversion to MS and whether correlations exist between baseline sNfL and magnetic resonance imaging (MRI) metrics. Methods: sNfL was measured for 494 patients who received sc IFN ß-1a 44 µg once weekly (qw; n = 168), three times weekly (tiw; n = 161), or placebo (n = 165) over 24 months. Median baseline sNfL (26.1 pg/mL) was used to define high/low sNfL subgroups. Hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated using Cox's proportional hazard model to determine factors influencing the risk of conversion to MS. Kaplan-Meier estimates calculated median time-to-conversion to MS (McDonald 2005 criteria) or clinically definite MS (CDMS; Poser criteria). Correlations between sNfL and MRI findings were assessed using Spearman's rank correlation coefficient (r). Results: Multivariable models indicated that high baseline sNfL was associated with the likelihood of converting to MS and inversely to time-to-conversion (HR = 1.3, 95% CI: 1.03-1.64; p = 0.024). Significant additional factors affecting conversion to McDonald MS were on-study treatment (sc IFN ß-1a/placebo; qw: HR = 0.59, 95% CI: 0.46-0.76; tiw: HR = 0.45, 95% CI: 0.34-0.59), classification of FCDE (monofocal/multifocal; HR = 0.69, 95% CI: 0.55-0.85), and most baseline imaging findings (T2 and T1 gadolinium-enhancing [Gd+] lesions; HR = 1.02, 95% CI: 1.01-1.03 and HR = 1.07, 95% CI: 1.03-1.11); all p ⩽ 0.001. Conversion to CDMS showed similar results. At month 24, sNfL was strongly correlated with a mean number of combined unique active (r = 0.71), new T2 (r = 0.72), and new T1 Gd+ (r = 0.60) lesions; weak correlations were observed between sNfL and clinical outcomes for all treatment groups. Conclusion: Higher baseline sNfL was associated with an increased risk of MS conversion, a risk that was mitigated by treatment with sc IFN ß-1a tiw. Trial registration: ClinicalTrials.gov identifier: NCT00404352. Date registered: 28 November 2006.

Neurofilaments as biomarkers in neurological disorders - towards clinical application.

Neurofilament proteins have been validated as specific body fluid biomarkers of neuro-axonal injury. The advent of highly sensitive analytical platforms that enable reliable quantification of neurofilaments in blood samples and simplify longitudinal follow-up has paved the way for the development of neurofilaments as a biomarker in clinical practice. Potential applications include assessment of disease activity, monitoring of treatment responses, and determining prognosis in many acute and chronic neurological disorders as well as their use as an outcome measure in trials of novel therapies. Progress has now moved the measurement of neurofilaments to the doorstep of routine clinical practice for the evaluation of individuals. In this Review, we first outline current knowledge on the structure and function of neurofilaments. We then discuss analytical and statistical approaches and challenges in determining neurofilament levels in different clinical contexts and assess the implications of neurofilament light chain (NfL) levels in normal ageing and the confounding factors that need to be considered when interpreting NfL measures. In addition, we summarize the current value and potential clinical applications of neurofilaments as a biomarker of neuro-axonal damage in a range of neurological disorders, including multiple sclerosis, Alzheimer disease, frontotemporal dementia, amyotrophic lateral sclerosis, stroke and cerebrovascular disease, traumatic brain injury, and Parkinson disease. We also consider the steps needed to complete the translation of neurofilaments from the laboratory to the management of neurological diseases in clinical practice.

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.

Association of Spinal Cord Atrophy and Brain Paramagnetic Rim Lesions With Progression Independent of Relapse Activity in People With MS.

BACKGROUND AND OBJECTIVES: Progression independent of relapse activity (PIRA) is a crucial determinant of overall disability accumulation in multiple sclerosis (MS). Accelerated brain atrophy has been shown in patients experiencing PIRA. In this study, we assessed the relation between PIRA and neurodegenerative processes reflected by (1) longitudinal spinal cord atrophy and (2) brain paramagnetic rim lesions (PRLs). Besides, the same relationship was investigated in progressive MS (PMS). Last, we explored the value of cross-sectional brain and spinal cord volumetric measurements in predicting PIRA. METHODS: From an ongoing multicentric cohort study, we selected patients with MS with (1) availability of a susceptibility-based MRI scan and (2) regular clinical and conventional MRI follow-up in the 4 years before the susceptibility-based MRI. Comparisons in spinal cord atrophy rates (explored with linear mixed-effect models) and PRL count (explored with negative binomial regression models) were performed between: (1) relapsing-remitting (RRMS) and PMS phenotypes and (2) patients experiencing PIRA and patients without confirmed disability accumulation (CDA) during follow-up (both considering the entire cohort and the subgroup of patients with RRMS). Associations between baseline MRI volumetric measurements and time to PIRA were explored with multivariable Cox regression analyses. RESULTS: In total, 445 patients with MS (64.9% female; mean [SD] age at baseline 45.0 [11.4] years; 11.2% with PMS) were enrolled. Compared with patients with RRMS, those with PMS had accelerated cervical cord atrophy (mean difference in annual percentage volume change [MD-APC] -1.41; p = 0.004) and higher PRL load (incidence rate ratio [IRR] 1.93; p = 0.005). Increased spinal cord atrophy (MD-APC -1.39; p = 0.0008) and PRL burden (IRR 1.95; p = 0.0008) were measured in patients with PIRA compared with patients without CDA; such differences were also confirmed when restricting the analysis to patients with RRMS. Baseline volumetric measurements of the cervical cord, whole brain, and cerebral cortex significantly predicted time to PIRA (all p ≤ 0.002). DISCUSSION: Our results show that PIRA is associated with both increased spinal cord atrophy and PRL burden, and this association is evident also in patients with RRMS. These findings further point to the need to develop targeted treatment strategies for PIRA to prevent irreversible neuroaxonal loss and optimize long-term outcomes of patients with MS.

Serum neurofilament light and white matter characteristics in the general population: a longitudinal analysis.

Neurofilament light polypeptide (NfL) is a component of the neuronal cytoskeleton and particularly abundant in large-caliber axons. When axonal injury occurs, NfL is released and reaches the cerebrospinal fluid and the blood. Associations between NfL and white matter alterations have previously been observed in studies based on patients with neurological diseases. The current study aimed to explore the relationship between serum NfL (sNfL) and white matter characteristics in a population-based sample. The cross-sectional associations between sNfL as dependent variable, fractional anisotropy (FA), and white matter lesion (WML) volume were analyzed with linear regression models in 307 community-dwelling adults aged between 35 and 65 years. These analyses were repeated with additional adjustment for the potential confounders age, sex, and body mass index (BMI). Longitudinal associations over a mean follow-up of 5.39 years were analyzed with linear mixed models. The unadjusted cross-sectional models yielded significant associations between sNfL, WML volume, and FA, respectively. However, after the adjustment for confounders, these associations did not reach significance. In the longitudinal analyses, the findings corroborated the baseline findings showing no significant associations between sNfL and white matter macrostructure and microstructure beyond the effects of age. In synopsis with previous studies in patients with acute neurological diseases showing a significant association of sNfL with white matter changes beyond the effects of age, the present results based on a sample from the general population suggest the perspective that changes in sNfL reflect age-related effects that also manifest in altered white matter macrostructure and microstructure.

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.

Ocrelizumab exposure in relapsing-remitting multiple sclerosis: 10-year analysis of the phase 2 randomized clinical trial and its extension.

Open-label extension (OLE) studies help inform long-term safety and efficacy of disease-modifying therapies in multiple sclerosis (MS). We report exploratory analyses from a phase 2 trial on the longest follow-up to date of ocrelizumab-treated patients with relapsing-remitting MS (RRMS). The primary treatment period (PTP) comprised four 24-week treatment cycles; participants were randomized to double-blind ocrelizumab (2000 mg or 600 mg), placebo, or interferon ß-1a (open label) for one cycle, then dose-blinded ocrelizumab 1000 mg or 600 mg for the remaining cycles. The PTP was followed by consecutive assessed and unassessed treatment-free periods (TFPs) and then the OLE (ocrelizumab 600 mg every 24 weeks). Safety and efficacy were prospectively assessed. Of 220 participants randomized, 183 (84%) completed the PTP. After the TFP, 103 entered OLE (median OLE ocrelizumab exposure 6.5 years). Most common adverse events across all periods were infusion-related reactions. MRI activity, annualized relapse rate, and confirmed disability progression (CDP) rates remained low throughout. During the assessed TFP, there was a trend toward less and later B-cell repletion, and later CDP, for patients randomized to ocrelizumab; MRI activity was observed in 16.3% of patients, the earliest 24 weeks after the last ocrelizumab dose. This is the longest follow-up of ocrelizumab-treated patients with RRMS, with no new safety signals emerging during an observation period from 2008 to 2020. Results reinforce the sustained efficacy of long-term ocrelizumab. Reduced disease activity was maintained following interruption of 6-month dosing cycles, with no evidence of rebound.

Neurofilament Light Chain Elevation and Disability Progression in Multiple Sclerosis.

Importance: Mechanisms contributing to disability accumulation in multiple sclerosis (MS) are poorly understood. Blood neurofilament light chain (NfL) level, a marker of neuroaxonal injury, correlates robustly with disease activity in people with MS (MS); however, data on the association between NfL level and disability accumulation have been conflicting. Objective: To determine whether and when NfL levels are elevated in the context of confirmed disability worsening (CDW). Design, Setting, and Participants: This study included 2 observational cohorts: results from the Expression, Proteomics, Imaging, Clinical (EPIC) study at the University of California San Francisco (since 2004) were confirmed in the Swiss Multiple Sclerosis Cohort (SMSC), a multicenter study in 8 centers since 2012. Data were extracted from EPIC in April 2022 (sampling July 1, 2004, to December 20, 2016) and SMSC in December 2022 (sampling June 6, 2012, to September 2, 2021). The study included 2 observational cohorts in tertiary MS centers. All participants of both cohorts with available NfL results were included in the study, and no eligible participants were excluded or declined to participate. Exposure: Association between NfL z scores and CDW. Main Outcome Measures: CDW was defined as Expanded Disability Status Scale (EDSS) worsening that was confirmed after 6 or more months and classified into CDW associated with clinical relapses (CDW-R) or independent of clinical relapses (CDW-NR). Visits were classified in relation to the disability worsening events into CDW(-2) for 2 visits preceding event, CDW(-1) for directly preceding event, CDW(event) for first diagnosis of EDSS increase, and the confirmation visit. Mixed linear and Cox regression models were used to evaluate NfL dynamics and to assess the association of NfL with future CDW, respectively. Results: A total of 3906 EPIC visits (609 participants; median [IQR] age, 42.0 [35.0-50.0] years; 424 female [69.6%]) and 8901 SMSC visits (1290 participants; median [IQR] age, 41.2 [32.5-49.9] years; 850 female [65.9%]) were included. In CDW-R (EPIC, 36 events; SMSC, 93 events), NfL z scores were 0.71 (95% CI, 0.35-1.07; P < .001) units higher at CDW-R(-1) in EPIC and 0.32 (95% CI, 0.14-0.49; P < .001) in SMSC compared with stable MS samples. NfL elevation could be detected preceding CDW-NR (EPIC, 191 events; SMSC, 342 events) at CDW-NR(-2) (EPIC: 0.23; 95% CI, 0.01-0.45; P = .04; SMSC: 0.28; 95% CI, 0.18-0.37; P < .001) and at CDW-NR(-1) (EPIC: 0.27; 95% CI, 0.11-0.44; P < .001; SMSC: 0.09; 95% CI, 0-0.18; P = .06). Those findings were replicated in the subgroup with relapsing-remitting MS. Time-to-event analysis confirmed the association between NfL levels and future CDW-R within approximately 1 year and CDW-NR (in approximately 1-2 years). Conclusions and Relevance: This cohort study documents the occurrence of NfL elevation in advance of clinical worsening and may hint to a potential window of ongoing dynamic central nervous system pathology that precedes the diagnosis of CDW.

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.

Blood neurofilament light levels predict non-relapsing progression following anti-CD20 therapy in relapsing and primary progressive multiple sclerosis: findings from the ocrelizumab randomised, double-blind phase 3 clinical trials.

BACKGROUND: Neurofilament light chain (NfL), a neuronal cytoskeletal protein that is released upon neuroaxonal injury, is associated with multiple sclerosis (MS) relapsing activity and has demonstrated some prognostic ability for future relapse-related disease progression, yet its value in assessing non-relapsing disease progression remains unclear. METHODS: We examined baseline and longitudinal blood NfL levels in 1421 persons with relapsing MS (RMS) and 596 persons with primary progressive MS (PPMS) from the pivotal ocrelizumab MS trials. NfL treatment-response and risk for disease worsening (including disability progression into the open-label extension period and slowly expanding lesions [SELs] on brain MRI) at baseline and following treatment with ocrelizumab were evaluated using time-to-event analysis and linear regression models. FINDINGS: In persons from the RMS control arms without acute disease activity and in the entire PPMS control arm, higher baseline NfL was prognostic for greater whole brain and thalamic atrophy, greater volume expansion of SELs, and clinical progression. Ocrelizumab reduced NfL levels vs. controls in persons with RMS and those with PPMS, and abrogated the prognostic value of baseline NfL on disability progression. Following effective suppression of relapse activity by ocrelizumab, NfL levels at weeks 24 and 48 were significantly associated with long-term risk for disability progression, including up to 9 years of observation in RMS and PPMS. INTERPRETATION: Highly elevated NfL from acute MS disease activity may mask a more subtle NfL abnormality that reflects underlying non-relapsing progressive biology. Ocrelizumab significantly reduced NfL levels, consistent with its effects on acute disease activity and disability progression. Persistently elevated NfL levels, observed in a subgroup of persons under ocrelizumab treatment, demonstrate potential clinical utility as a predictive biomarker of increased risk for clinical progression. Suppression of relapsing biology with high-efficacy immunotherapy provides a window into the relationship between NfL levels and future non-relapsing progression. FUNDING: F. Hoffmann-La Roche Ltd.

Interleukin-3 coordinates glial-peripheral immune crosstalk to incite multiple sclerosis.

Glial cells and central nervous system (CNS)-infiltrating leukocytes contribute to multiple sclerosis (MS). However, the networks that govern crosstalk among these ontologically distinct populations remain unclear. Here, we show that, in mice and humans, CNS-resident astrocytes and infiltrating CD44hiCD4+ T cells generated interleukin-3 (IL-3), while microglia and recruited myeloid cells expressed interleukin-3 receptor-ɑ (IL-3Rɑ). Astrocytic and T cell IL-3 elicited an immune migratory and chemotactic program by IL-3Rɑ+ myeloid cells that enhanced CNS immune cell infiltration, exacerbating MS and its preclinical model. Multiregional snRNA-seq of human CNS tissue revealed the appearance of IL3RA-expressing myeloid cells with chemotactic programming in MS plaques. IL3RA expression by plaque myeloid cells and IL-3 amount in the cerebrospinal fluid predicted myeloid and T cell abundance in the CNS and correlated with MS severity. Our findings establish IL-3:IL-3RA as a glial-peripheral immune network that prompts immune cell recruitment to the CNS and worsens MS.

Association of serum neurofilament light with microglial activation in multiple sclerosis.

BACKGROUND: Translocator protein (TSPO)-PET and neurofilament light (NfL) both report on brain pathology, but their potential association has not yet been studied in multiple sclerosis (MS) in vivo. We aimed to evaluate the association between serum NfL (sNfL) and TSPO-PET-measurable microglial activation in the brain of patients with MS. METHODS: Microglial activation was detected using PET and the TSPO-binding radioligand [11C]PK11195. Distribution volume ratio (DVR) was used to evaluate specific [11C]PK11195-binding. sNfL levels were measured using single molecule array (Simoa). The associations between [11C]PK11195 DVR and sNfL were evaluated using correlation analyses and false discovery rate (FDR) corrected linear regression modelling. RESULTS: 44 patients with MS (40 relapsing-remitting and 4 secondary progressive) and 24 age-matched and sex-matched healthy controls were included. In the patient group with elevated brain [11C]PK11195 DVR (n=19), increased sNfL associated with higher DVR in the lesion rim (estimate (95% CI) 0.49 (0.15 to 0.83), p(FDR)=0.04) and perilesional normal appearing white matter (0.48 (0.14 to 0.83), p(FDR)=0.04), and with a higher number and larger volume of TSPO-PET-detectable rim-active lesions defined by microglial activation at the plaque edge (0.46 (0.10 to 0.81), p(FDR)=0.04 and 0.50 (0.17 to 0.84), p(FDR)=0.04, respectively). Based on the multivariate stepwise linear regression model, the volume of rim-active lesions was the most relevant factor affecting sNfL. CONCLUSIONS: Our demonstration of an association between microglial activation as measured by increased TSPO-PET signal, and elevated sNfL emphasises the significance of smouldering inflammation for progression-promoting pathology in MS and highlights the role of rim-active lesions in promoting neuroaxonal damage.

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.

High serum neurofilament levels are observed close to disease activity events in pediatric-onset MS and MOG antibody-associated diseases.

BACKGROUND: Serum neurofilament light chain (sNfL) is an emerging multiple sclerosis (MS) biomarker which measures neuro-axonal damage. However, understanding its temporal association with disease activity in pediatric-onset MS (POMS) and Myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) remains limited. OBJECTIVE: To investigate the association of sNfL levels and time from disease activity in children with MS and MOGAD. METHODS: POMS and MOGAD cases with onset before 18 years of age were enrolled at the University of California San Francisco (UCSF) Regional Pediatric MS Center. Frequency-matched healthy subjects were recruited from general pediatric clinics. Serum samples were tested for MOG-IgG at Mayo Clinic using a live cell-based fluorescent activated cell sorting assay. sNfL levels were measured using single-molecule array (Simoa) technology measured in pg/mL. Data on demographics, clinical features, MRI, CSF, and treatment data were collected by chart review. RESULTS: We included 201 healthy controls healthy controls, 142 POMS, and 20 confirmed MOGAD cases with available sNfL levels. The median (IQR) age at the time of sampling was 15.6 (3.9), 15.5 (3.1), and 8.8 (4.1) years for controls, POMS, and MOGAD, respectively. Median sNfL levels (pg/ml) were higher in POMS (19.6) and MOGAD (32.7) cases compared to healthy controls (3.9) (p<0.001). sNfL levels ≥100 pg/ml were only detected within four months of a clinical event or MRI activity in both POMS and MOGAD cases. In addition, sNfL levels were higher in POMS patients with new/enlarged T2 and gadolinium-enhanced lesions than those without MRI activity within four months of sampling in POMS cases. CONCLUSION: High sNfL levels were observed close to clinical or MRI events in POMS and MOGAD. Our findings support sNfL as a biomarker of disease activity in pediatric demyelinating disorders.

Evidence of polygenic regulation of the physiological presence of neurofilament light chain in human serum.

Introduction: The measurement of neurofilament light chain (NfL) in blood is a promising biomarker of neurological injury and disease. We investigated the genetic factors that underlie serum NfL levels (sNfL) of individuals without neurological conditions. Methods: We performed a discovery genome-wide association study (GWAS) of sNfL in participants of the German BiDirect Study (N = 1,899). A secondary GWAS for meta-analysis was performed in a small Austrian cohort (N = 287). Results from the meta-analysis were investigated in relation with several clinical variables in BiDirect. Results: Our discovery GWAS identified 12 genomic loci at the suggestive threshold ((p < 1 × 10-5). After meta-analysis, 7 loci were suggestive of an association with sNfL. Genotype-specific differences in sNfL were observed for the lead variants of meta-analysis loci (rs34523114, rs114956339, rs529938, rs73198093, rs34372929, rs10982883, and rs1842909) in BiDirect participants. We identified potential associations in meta-analysis loci with markers of inflammation and renal function. At least 6 protein-coding genes (ACTG2, TPRKB, DMXL1, COL23A1, NAT1, and RIMS2) were suggested as genetic factors contributing to baseline sNfL levels. Discussion: Our findings suggest that polygenic regulation of neuronal processes, inflammation, metabolism and clearance modulate the variability of NfL in the circulation. These could aid in the interpretation of sNfL measurements in a personalized manner.

High serum neurofilament light chain levels correlate with brain atrophy and physical disability in multiple sclerosis.

BACKGROUND AND PURPOSE: Serum neurofilament light chain (sNfL) is a promising biomarker of neuroaxonal damage in persons with multiple sclerosis (pwMS). In cross-sectional studies, sNfL has been associated with disease activity and brain magnetic resonance imaging (MRI) changes; however, it is still unclear to what extent in particular high sNfL levels impact on subsequent disease evolution. METHODS: sNfL was quantified by an ultrasensitive single molecule array (Simoa) in 199 pwMS (median age = 34.2 years, 64.3% female) and 49 controls. All pwMS underwent 3-T MRI to assess global and compartmental normalized brain volumes, T2-lesion load, and cortical mean thickness. Follow-up data and serum samples were available in 144 pwMS (median follow-up time = 3.8 years). Linear and binary logistic models were used to estimate the independent contribution of sNfL for changes in MRI and Expanded Disability Status Scale (EDSS). Age-corrected sNfL z-scores from a normative database of healthy controls were used for sensitivity analyses. RESULTS: High sNfL levels at baseline were associated with atrophy measures of the whole brain (standardized beta coefficient ßj = -0.352, p < 0.001), white matter (ßj = -0.229, p = 0.007), thalamus (ßj = -0.372, p = 0.004), and putamen (ßj = -1.687, p = 0.012). pwMS with high levels of sNfL at baseline and follow-up had a greater risk of EDSS worsening (p = 0.007). CONCLUSIONS: Already single time point elevation of sNfL has a distinct effect on brain volume changes over a short-term period, and repeated high levels of sNfL indicate accumulating physical disability. Serial assessment of sNfL may provide added value in the clinical management of pwMS.

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.

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.

A Multicenter Longitudinal MRI Study Assessing LeMan-PV Software Accuracy in the Detection of White Matter Lesions in Multiple Sclerosis Patients.

BACKGROUND: Detecting new and enlarged lesions in multiple sclerosis (MS) patients is needed to determine their disease activity. LeMan-PV is a software embedded in the scanner reconstruction system of one vendor, which automatically assesses new and enlarged white matter lesions (NELs) in the follow-up of MS patients; however, multicenter validation studies are lacking. PURPOSE: To assess the accuracy of LeMan-PV for the longitudinal detection NEL white-matter MS lesions in a multicenter clinical setting. STUDY TYPE: Retrospective, longitudinal. SUBJECTS: A total of 206 patients with a definitive MS diagnosis and at least two follow-up MRI studies from five centers participating in the Swiss Multiple Sclerosis Cohort study. Mean age at first follow-up = 45.2 years (range: 36.9-52.8 years); 70 males. FIELD STRENGTH/SEQUENCE: Fluid attenuated inversion recovery (FLAIR) and T1-weighted magnetization prepared rapid gradient echo (T1-MPRAGE) sequences at 1.5 T and 3 T. ASSESSMENT: The study included 313 MRI pairs of datasets. Data were analyzed with LeMan-PV and compared with a manual "reference standard" provided by a neuroradiologist. A second rater (neurologist) performed the same analysis in a subset of MRI pairs to evaluate the rating-accuracy. The Sensitivity (Se), Specificity (Sp), Accuracy (Acc), F1-score, lesion-wise False-Positive-Rate (aFPR), and other measures were used to assess LeMan-PV performance for the detection of NEL at 1.5 T and 3 T. The performance was also evaluated in the subgroup of 123 MRI pairs at 3 T. STATISTICAL TESTS: Intraclass correlation coefficient (ICC) and Cohen's kappa (CK) were used to evaluate the agreement between readers. RESULTS: The interreader agreement was high for detecting new lesions (ICC = 0.97, Pvalue < 10-20 , CK = 0.82, P value = 0) and good (ICC = 0.75, P value < 10-12 , CK = 0.68, P value = 0) for detecting enlarged lesions. Across all centers, scanner field strengths (1.5 T, 3 T), and for NEL, LeMan-PV achieved: Acc = 61%, Se = 65%, Sp = 60%, F1-score = 0.44, aFPR = 1.31. When both follow-ups were acquired at 3 T, LeMan-PV accuracy was higher (Acc = 66%, Se = 66%, Sp = 66%, F1-score = 0.28, aFPR = 3.03). DATA CONCLUSION: In this multicenter study using clinical data settings acquired at 1.5 T and 3 T, and variations in MRI protocols, LeMan-PV showed similar sensitivity in detecting NEL with respect to other recent 3 T multicentric studies based on neural networks. While LeMan-PV performance is not optimal, its main advantage is that it provides automated clinical decision support integrated into the radiological-routine flow. EVIDENCE LEVEL: 4 TECHNICAL EFFICACY: Stage 2.

Demographic and disease-related factors impact on cerebrospinal fluid neurofilament light chain levels in multiple sclerosis.

BACKGROUND: Neurofilament light (NfL) levels reflect inflammatory disease activity in multiple sclerosis (MS), but it is less clear if NfL also can serve as a biomarker for MS progression in treated patients without relapses and focal lesion accrual. In addition, it has not been well established if clinically effective treatment re-establishes an age and sex pattern for cerebrospinal fluid NfL (cNfL) as seen in controls, and to what degree levels are affected by disability level and magnetic resonance imaging (MRI) atrophy metrics. METHODS: We included subjects for whom cNfL levels had been determined as per clinical routine or in clinical research, classified as healthy controls (HCs, n = 89), MS-free disease controls (DCs, n = 251), untreated MS patients (uMS; n = 296), relapse-free treated MS patients (tMS; n = 78), and ProTEct-MS clinical trial participants (pMS; n = 41). RESULTS: Using linear regression, we found a positive association between cNfL and age, as well as lower concentrations among women, in all groups, except for uMS patients. In contrast, disability level in the entire MS cohort, or T1 and T2 lesion volumes, brain parenchymal fraction, thalamic fraction, and cortical thickness in the pMS trial cohort, did not correlate with cNfL concentrations. Furthermore, the cNfL levels in tMS and pMS groups did not differ. CONCLUSIONS: In participants with MS lacking signs of inflammatory disease activity, disease modulatory therapy reinstates an age and sex cNfL pattern similar to that of control subjects. No significant association was found between cNfL levels and clinical worsening, disability level, or MRI metrics.