Population-Based Evidence for the Use of Serum Neurofilaments as Individual Diagnostic and Prognostic Biomarkers in Amyotrophic Lateral Sclerosis.

OBJECTIVE: Neurofilament light chains (NfL) and phosphorylated neurofilament heavy chains (pNfH), established as diagnostic and prognostic biomarkers in hospital-based amyotrophic lateral sclerosis (ALS) cohorts, are now surrogate markers in clinical trials. This study extends their evaluation to a population level, with the aim of advancing their full establishment and assessing the transferability of biomarker findings from controlled cohorts to real-world ALS populations. METHODS: We measured serum NfL and pNfH levels in all ALS patients (n = 790) and general population controls (n = 570) with available baseline samples participating in the epidemiological ALS Registry Swabia, providing platform-specific (ELLA™) reference data and Z-scores for controls, as well as reference data, disease-specific Z-scores and longitudinal data in ALS. We evaluated the diagnostic and prognostic utility of neurofilaments and quantified the impact of ALS-related factors and non-ALS confounders. RESULTS: Neurofilaments showed high diagnostic and prognostic utility at the population level, with NfL superior to pNfH. The novel concept of a population-based ALS Z-score significantly improved the prognostic utility compared to absolute raw values. Both biomarkers increased more strongly with age in controls than in ALS, and age adjustment improved diagnostic accuracy. Our data show that disease progression rates, ALS phenotype, body mass index (BMI), and renal function need to be considered when interpreting neurofilament levels; longitudinal neurofilament levels were generally stable in individual patients, especially when adjusted for age and baseline levels. INTERPRETATION: Population-based assessment enhances the utility of particularly serum NfL as a diagnostic and prognostic biomarker in ALS and improves the translation of findings from controlled cohorts to real-world populations. ANN NEUROL 2024.

Blood-CSF barrier integrity in amyotrophic lateral sclerosis.

The integrity of the blood-CSF barrier plays a major role in inflammation, but also in shielding the central nervous system from external and systemic - potentially toxic - factors. Here we report results of measurements of the albumin quotient - which is thought to mirror the integrity of the blood/CSF barrier - in 1059 amyotrophic lateral sclerosis patients. The results were compared with groups of patients suffering from Alzheimer´s disease, facial palsy and tension headache. The albumin quotient, an accepted measure of the blood/CSF barrier integrity, was not significantly different from control populations. In addition, we found that the albumin quotient correlated with survival of the patients; this effect was mainly driven by male patients and influenced by age, BMI and diabetes mellitus. We conclude that the blood/CSF barrier is intact in this large cohort of ALS patients and that the albumin quotient correlates with survival. Whether this is important for the pathogenesis of the disease, requires mechanistic studies.

Single cell transcriptomics of cerebrospinal fluid cells from patients with recent-onset narcolepsy.

Narcolepsy is a rare cause of hypersomnolence and may be associated or not with cataplexy, i.e. sudden muscle weakness. These forms are designated narcolepsy-type 1 (NT1) and -type 2 (NT2), respectively. Notable characteristics of narcolepsy are that most patients carry the HLA-DQB1*06:02 allele and NT1-patients have strongly decreased levels of hypocretin-1 (synonym orexin-A) in the cerebrospinal fluid (CSF). The pathogenesis of narcolepsy is still not completely understood but the strong HLA-bias and increased frequencies of CD4+ T cells reactive to hypocretin in the peripheral blood suggest autoimmune processes in the hypothalamus. Here we analyzed the transcriptomes of CSF-cells from twelve NT1 and two NT2 patients by single cell RNAseq (scRNAseq). As controls, we used CSF cells from patients with multiple sclerosis, radiologically isolated syndrome, and idiopathic intracranial hypertension. From 27,255 CSF cells, we identified 20 clusters of different cell types and found significant differences in three CD4+ T cell and one monocyte clusters between narcolepsy and multiple sclerosis patients. Over 1000 genes were differentially regulated between patients with NT1 and other diseases. Surprisingly, the most strongly upregulated genes in narcolepsy patients as compared to controls were coding for the genome-encoded MTRNR2L12 and MTRNR2L8 peptides, which are homologous to the mitochondria-encoded HUMANIN peptide that is known playing a role in other neurological diseases including Alzheimer's disease.

Primary lateral sclerosis: application and validation of the 2020 consensus diagnostic criteria in an expert opinion-based PLS cohort.

BACKGROUND: Validation of the 2020 consensus criteria for primary lateral sclerosis (PLS) is essential for their use in clinical practice and future trials. METHODS: In a large cohort of patients diagnosed with PLS by expert opinion prior to the new criteria with detailed clinical baseline evaluation (n=107) and longitudinal follow-up (n=63), we applied the new diagnostic criteria and analysed the clinical phenotype, electromyography (EMG), diagnostic accuracy and prognosis, adding neurofilaments and MRI as potential biomarkers. RESULTS: The criteria for definite PLS were met by 28% and those for probable PLS by 19%, whereas 53% did not meet the full criteria at baseline, mainly due to the time, EMG and region criteria. Patients not meeting the criteria had less generalised upper motor neuron involvement but were otherwise similar in demographic and clinical characteristics. All patients with definite and probable PLS maintained PLS diagnosis during follow-up, while four patients not meeting the criteria developed clinical lower motor neuron involvement. Definite PLS cases showed improved survival compared with probable PLS and patients who did not meet the criteria. Despite a clinical PLS phenotype, fibrillation potentials/positive sharp waves and fasciculations in one or more muscles were a frequent EMG finding, with the extent and prognostic significance depending on disease duration. Serum neurofilament light and a multiparametric MRI fibre integrity Z-score correlated with clinical parameters and were identified as potential biomarkers. CONCLUSION: Validation of the 2020 PLS consensus criteria revealed high diagnostic certainty and prognostic significance, supporting their value for research and clinical practice.

Dynamics of synaptic damage in severe traumatic brain injury revealed by cerebrospinal fluid SNAP-25 and VILIP-1.

BACKGROUND: Biomarkers of neuronal, glial cells and inflammation in traumatic brain injury (TBI) are available but they do not specifically reflect the damage to synapses, which represent the bulk volume of the brain. Experimental models have demonstrated extensive involvement of synapses in acute TBI, but biomarkers of synaptic damage in human patients have not been explored. METHODS: Single-molecule array assays were used to measure synaptosomal-associated protein-25 (SNAP-25) and visinin-like protein 1 (VILIP-1) (along with neurofilament light chain (NFL), ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), glial fibrillar acidic protein (GFAP), interleukin-6 (IL-6) and interleukin-8 (IL-8)) in ventricular cerebrospinal fluid (CSF) samples longitudinally acquired during the intensive care unit (ICU) stay of 42 patients with severe TBI or 22 uninjured controls. RESULTS: CSF levels of SNAP-25 and VILIP-1 are strongly elevated early after severe TBI and decline in the first few days. SNAP-25 and VILIP-1 correlate with inflammatory markers at two distinct timepoints (around D1 and then again at D5) in follow-up. SNAP-25 and VILIP-1 on the day-of-injury have better sensitivity and specificity for unfavourable outcome at 6 months than NFL, UCH-L1 or GFAP. Later elevation of SNAP-25 was associated with poorer outcome. CONCLUSION: Synaptic damage markers are acutely elevated in severe TBI and predict long-term outcomes, as well as, or better than, markers of neuroaxonal injury. Synaptic damage correlates with initial injury and with a later phase of secondary inflammatory injury.

Serum neurofilament light chain and glial fibrillary acidic protein for predicting response to apheresis in steroid-refractory multiple sclerosis relapses.

BACKGROUND AND PURPOSE: The predictive value of serum neurofilament light chain (sNfL) and serum glial fibrillary acidic protein (sGFAP) for apheresis outcome in steroid-refractory multiple sclerosis (MS) relapse has not yet been evaluated. METHODS: We used pre- and postapheresis serum samples from 38 participants of the IAPEMS trial (clinicaltrials.gov: NCT02671682), which investigated the use of immunoadsorption versus plasma exchange for the treatment of steroid-refractory MS attacks. Response to apheresis was classified based on improvement on (i) the Expanded Disability Status Scale (EDSS), (ii) the affected functional system scores (FSS) of the EDSS, or (iii) the visual acuity for patients with optic neuritis, 4 weeks postapheresis. sNFL and sGFAP were measured by single molecule arrays. RESULTS: Preprocedural sGFAP levels could discriminate between responders and nonresponders, determined by FSS improvement (p = 0.017). In multivariate logistic regression analysis, younger age (odds ratio [OR] = 0.781, 95% confidence interval [CI] = 0.635-0.962, p = 0.020) and lower sGFAP levels (OR = 0.948, 95% CI = 0.903-0.995, p = 0.031) could predict response to apheresis in the overall cohort. We could observe a trend towards a favourable apheresis outcome with higher sNfL levels (OR = 1.413, 95% CI = 0.965-2.069, p = 0.076). Analysis of the sNfL-to-sGFAP ratio showed an OR of 1.924 (95% CI = 1.073-3.451, p = 0.028) for predicting apheresis response. The ratio showed a better predictive value than the individual parameters. Neither biomarker was affected by the number of steroid cycles preapheresis. CONCLUSIONS: Lower sGFAP levels, a higher sNfL-to-sGFAP ratio, and younger age are associated with a favourable apheresis outcome.

Quantification of blood glial fibrillary acidic protein using a second-generation microfluidic assay. Validation and comparative analysis with two established assays.

OBJECTIVES: Increased levels of glial fibrillary acidic protein (GFAP) in blood have been identified as a valuable biomarker for some neurological disorders, such as Alzheimer's disease and multiple sclerosis. However, most blood GFAP quantifications so far were performed using the same bead-based assay, and to date a routine clinical application is lacking. METHODS: In this study, we validated a novel second-generation (2nd gen) Ella assay to quantify serum GFAP. Furthermore, we compared its performance with a bead-based single molecule array (Simoa) and a homemade GFAP assay in a clinical cohort of neurological diseases, including 210 patients. RESULTS: Validation experiments resulted in an intra-assay variation of 10 %, an inter-assay of 12 %, a limit of detection of 0.9 pg/mL, a lower limit of quantification of 2.8 pg/mL, and less than 20 % variation in serum samples exposed to up to five freeze-thaw cycles, 120 h at 4 °C and room temperature. Measurement of the clinical cohort using all assays revealed the same pattern of GFAP distribution in the different diagnostic groups. Moreover, we observed a strong correlation between the 2nd gen Ella and Simoa (r=0.91 (95 % CI: 0.88-0.93), p<0.0001) and the homemade immunoassay (r=0.77 (95 % CI: 0.70-0.82), p<0.0001). CONCLUSIONS: Our results demonstrate a high reliability, precision and reproducibility of the 2nd gen Ella assay. Although a higher assay sensitivity for Simoa was observed, the new microfluidic assay might have the potential to be used for GFAP analysis in daily clinical workups due to its robustness and ease of use.

CXCL13 in Cerebrospinal Fluid: Clinical Value in a Large Cross-Sectional Study.

C-X-C-motif chemokine ligand 13 (CXCL13) in cerebrospinal fluid (CSF) is increasingly used in clinical routines, although its diagnostic specificity and divergent cut-off values have been defined so far mainly for neuroborreliosis. Our aim was to evaluate the value of CSF-CXCL13 as a diagnostic and treatment response marker and its role as an activity marker in a larger disease spectrum, including neuroborreliosis and other neuroinflammatory and malignant CNS-disorders. Patients who received a diagnostic lumbar puncture (LP) (n = 1234) between July 2009 and January 2023 were included in our retrospective cross-sectional study. The diagnostic performance of CSF-CXCL13 for acute neuroborreliosis was highest at a cut-off of 428.92 pg/mL (sensitivity: 92.1%; specificity: 96.5%). In addition, CXCL13 levels in CSF were significantly elevated in multiple sclerosis with clinical (p = 0.001) and radiographic disease activity (p < 0.001). The clinical utility of CSF-CXCL13 appears to be multifaceted. CSF-CXCL13 is significantly elevated in patients with neuroborreliosis and shows a rapid and sharp decline with antibiotic therapy, but it is not specific for this disease and is also highly elevated in less common subacute neuroinfectious diseases, such as neurosyphilis and cryptococcal meningitis or in primary/secondary B-cell lymphoma.

Multiple Sclerosis and Clostridium perfringens Epsilon Toxin: Is There a Relationship?

Recent research has suggested a link between multiple sclerosis and the gut microbiota. This prospective pilot study aimed to investigate the composition of the gut microbiota in MS patients, the presence of Clostridium perfringens epsilon toxin in the serum of MS patients, and the influence of disease-modifying drugs (DMDs) on epsilon toxin levels and on the microbiota. Epsilon toxin levels in blood were investigated by two methods, a qualitative ELISA and a highly sensitive quantitative ELISA. Neither epsilon toxin nor antibodies against it were detected in the analyzed serum samples. 16S ribosomal RNA sequencing was applied to obtain insights into the composition of the gut microbiota of MS patients. No significant differences in the quantity, diversity, and the relative abundance of fecal microbiota were observed in the gut microbiota of MS patients receiving various DMDs, including teriflunomide, natalizumab, ocrelizumab, and fingolimod, or no therapy. The present study did not provide evidence supporting the hypothesis of a causal relationship between Clostridium perfringens epsilon toxin and multiple sclerosis.

HSV-1 and Cellular miRNAs in CSF-Derived Exosomes as Diagnostically Relevant Biomarkers for Neuroinflammation.

Virus-associated chronic inflammation may contribute to autoimmunity in a number of diseases. In the brain, autoimmune encephalitis appears related to fluctuating reactivation states of neurotropic viruses. In addition, viral miRNAs and proteins can be transmitted via exosomes, which constitute novel but highly relevant mediators of cellular communication. The current study questioned the role of HSV-1-encoded and host-derived miRNAs in cerebrospinal fluid (CSF)-derived exosomes, enriched from stress-induced neuroinflammatory diseases, mainly subarachnoid hemorrhage (SAH), psychiatric disorders (AF and SZ), and various other neuroinflammatory diseases. The results were compared with CSF exosomes from control donors devoid of any neuroinflammatory pathology. Serology proved positive, but variable immunity against herpesviruses in the majority of patients, except controls. Selective ultrastructural examinations identified distinct, herpesvirus-like particles in CSF-derived lymphocytes and monocytes. The likely release of extracellular vesicles and exosomes was most frequently observed from CSF monocytes. The exosomes released were structurally similar to highly purified stem-cell-derived exosomes. Exosomal RNA was quantified for HSV-1-derived miR-H2-3p, miR-H3-3p, miR-H4-3p, miR-H4-5p, miR-H6-3p, miR-H27 and host-derived miR-21-5p, miR-146a-5p, miR-155-5p, and miR-138-5p and correlated with the oxidative stress chemokine IL-8 and the axonal damage marker neurofilament light chain (NfL). Replication-associated miR-H27 correlated with neuronal damage marker NfL, and cell-derived miR-155-5p correlated with oxidative stress marker IL-8. Elevated miR-138-5p targeting HSV-1 latency-associated ICP0 inversely correlated with lower HSV-1 antibodies in CSF. In summary, miR-H27 and miR-155-5p may constitute neuroinflammatory markers for delineating frequent and fluctuating HSV-1 replication and NfL-related axonal damage in addition to the oxidative stress cytokine IL-8 in the brain. Tentatively, HSV-1 remains a relevant pathogen conditioning autoimmune processes and a psychiatric clinical phenotype.

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.

Serum glial fibrillary acidic protein and disability progression in progressive multiple sclerosis.

OBJECTIVE: Progression prediction is a significant unmet need in people with progressive multiple sclerosis (pwPMS). Studies on glial fibrillary acidic protein (GFAP) have either been limited to single center with relapsing MS or were based solely on Expanded Disability Status Scale (EDSS), which limits its generalizability to state-of-the-art clinical settings and trials applying combined outcome parameters. METHODS: Serum GFAP and NfL (neurofilament light chain) were investigated in EmBioProMS participants with primary (PP) or secondary progressive MS. Six months confirmed disability progression (CDP) was defined using combined outcome parameters (EDSS, timed-25-foot walk test (T25FW), and nine-hole-peg-test (9HPT)). RESULTS: 243 subjects (135 PPMS, 108 SPMS, age 55.5, IQR [49.7-61.2], 135 female, median follow-up: 29.3 months [17.9-40.9]) were included. NfL (age-) and GFAP (age- and sex-) adjusted Z scores were higher in pwPMS compared to HC (p < 0.001 for both). 111 (32.8%) CDP events were diagnosed in participants with ≥3 visits (n = 169). GFAP Z score >3 was associated with higher risk for CDP in participants with low NfL Z score (i.e., ≤1.0) (HR: 2.38 [1.12-5.08], p = 0.025). In PPMS, GFAP Z score >3 was associated with higher risk for CDP (HR: 2.88 [1.21-6.84], p = 0.016). Risk was further increased in PPMS subjects with high GFAP when NfL is low (HR: 4.31 [1.53-12.13], p = 0.006). INTERPRETATION: Blood GFAP may help identify pwPPMS at risk of progression. Combination of high GFAP and low NfL levels could distinguish non-active pwPMS with particularly high progression risk.

Clinical characterization of common pathogenic variants of SOD1-ALS in Germany.

Pathogenic variants in the Cu/Zn superoxide dismutase (SOD1) gene can be detected in approximately 2% of sporadic and 11% of familial amyotrophic lateral sclerosis (ALS) patients in Europe. We analyzed the clinical phenotypes of 83 SOD1-ALS patients focusing on patients carrying the most frequent (likely) pathogenic variants (R116G, D91A, L145F) in Germany. Moreover, we describe the effect of tofersen treatment on ten patients carrying these variants. R116G patients showed the most aggressive course of disease with a median survival of 22.0 months compared to 198.0 months in D91A and 87.0 months in L145F patients (HR 7.71, 95% CI 2.89-20.58 vs. D91A; p < 0.001 and HR 4.25, 95% CI 1.55-11.67 vs. L145F; p = 0.02). Moreover, R116G patients had the fastest median ALSFRS-R progression rate with 0.12 (IQR 0.07-0.20) points lost per month. Median diagnostic delay was 10.0 months (IQR 5.5-11.5) and therefore shorter compared to 57.5 months (IQR 14.0-83.0) in D91A (p < 0.001) and 21.5 months (IQR 5.8-38.8) in L145F (p = 0.21) carriers. As opposed to D91A carriers (50.0%), 96.2% of R116G (p < 0.001) and 100.0% of L145F (p = 0.04) patients reported a positive family history. During tofersen treatment, all patients showed a reduction of neurofilament light chain (NfL) serum levels, independent of the SOD1 variant. Patients with SOD1-ALS carrying R116G, D91A, or L145F variants show commonalities, but also differences in their clinical phenotype, including a faster progression rate with shorter survival in R116G, and a comparatively benign disease course in D91A carriers.

Association of Complement Factors With Disability Progression in Primary Progressive Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: The complement system is known to play a role in multiple sclerosis (MS) pathogenesis. However, its contribution to disease progression remains elusive. The study investigated the role of the complement system in disability progression of patients with primary progressive MS (PPMS). METHODS: Sixty-eight patients with PPMS from 12 European MS centers were included in the study. Serum and CSF levels of a panel of complement components (CCs) were measured by multiplex enzyme-linked immunosorbent assay at a baseline time point (i.e., sampling). Mean (SD) follow-up time from baseline was 9.6 (4.8) years. Only one patient (1.5%) was treated during follow-up. Univariable and multivariable logistic regressions adjusted for age, sex, and albumin quotient were performed to assess the association between baseline CC levels and disability progression in short term (2 years), medium term (6 years), and long term (at the time of the last follow-up). RESULTS: In short term, CC played little or no role in disability progression. In medium term, an elevated serum C3a/C3 ratio was associated with a higher risk of disability progression (adjusted OR 2.30; 95% CI 1.17-6.03; p = 0.040). By contrast, increased CSF C1q levels were associated with a trend toward reduced risk of disability progression (adjusted OR 0.43; 95% CI 0.17-0.98; p = 0.054). Similarly, in long term, an elevated serum C3a/C3 ratio was associated with higher risk of disability progression (adjusted OR 1.81; 95% CI 1.09-3.40; p = 0.037), and increased CSF C1q levels predicted lower disability progression (adjusted OR 0.41; 95% CI 0.17-0.86; p = 0.025). DISCUSSION: Proteins involved in the activation of early complement cascades play a role in disability progression as risk (elevated serum C3a/C3 ratio) or protective (elevated CSF C1q) factors after 6 or more years of follow-up in patients with PPMS. The protective effects associated with C1q levels in CSF may be related to its neuroprotective and anti-inflammatory properties.

Effects of tofersen treatment in patients with SOD1-ALS in a "real-world" setting - a 12-month multicenter cohort study from the German early access program.

Background: In April 2023, the antisense oligonucleotide tofersen was approved by the U.S. Food and Drug Administration (FDA) for treatment of SOD1-amyotrophic lateral sclerosis (ALS), after a decrease of neurofilament light chain (NfL) levels had been demonstrated. Methods: Between 03/2022 and 04/2023, 24 patients with SOD1-ALS from ten German ALS reference centers were followed-up until the cut-off date for ALS functional rating scale revised (ALSFRS-R), progression rate (loss of ALSFRS-R/month), NfL, phosphorylated neurofilament heavy chain (pNfH) in cerebrospinal fluid (CSF), and adverse events. Findings: During the observation period, median ALSFRS-R decreased from 38.0 (IQR 32.0-42.0) to 35.0 (IQR 29.0-42.0), corresponding to a median progression rate of 0.11 (IQR -0.09 to 0.32) points of ALSFRS-R lost per month. Median serum NfL declined from 78.0 pg/ml (IQR 37.0-147.0 pg/ml; n = 23) to 36.0 pg/ml (IQR 22.0-65.0 pg/ml; n = 23; p = 0.02), median pNfH in CSF from 2226 pg/ml (IQR 1061-6138 pg/ml; n = 18) to 1151 pg/ml (IQR 521-2360 pg/ml; n = 18; p = 0.02). In the CSF, we detected a pleocytosis in 73% of patients (11 of 15) and an intrathecal immunoglobulin synthesis (IgG, IgM, or IgA) in 9 out of 10 patients. Two drug-related serious adverse events were reported. Interpretation: Consistent with the VALOR study and its Open Label Extension (OLE), our results confirm a reduction of NfL serum levels, and moreover show a reduction of pNfH in CSF. The therapy was safe, as no persistent symptoms were observed. Pleocytosis and Ig synthesis in CSF with clinical symptoms related to myeloradiculitis in two patients, indicate the potential of an autoimmune reaction. Funding: No funding was received towards this study.

Prospective study validating a multidimensional treatment decision score predicting the 24-month outcome in untreated patients with clinically isolated syndrome and early relapsing-remitting multiple sclerosis, the ProVal-MS study.

INTRODUCTION: In Multiple Sclerosis (MS), patients´ characteristics and (bio)markers that reliably predict the individual disease prognosis at disease onset are lacking. Cohort studies allow a close follow-up of MS histories and a thorough phenotyping of patients. Therefore, a multicenter cohort study was initiated to implement a wide spectrum of data and (bio)markers in newly diagnosed patients. METHODS: ProVal-MS (Prospective study to validate a multidimensional decision score that predicts treatment outcome at 24 months in untreated patients with clinically isolated syndrome or early Relapsing-Remitting-MS) is a prospective cohort study in patients with clinically isolated syndrome (CIS) or Relapsing-Remitting (RR)-MS (McDonald 2017 criteria), diagnosed within the last two years, conducted at five academic centers in Southern Germany. The collection of clinical, laboratory, imaging, and paraclinical data as well as biosamples is harmonized across centers. The primary goal is to validate (discrimination and calibration) the previously published DIFUTURE MS-Treatment Decision score (MS-TDS). The score supports clinical decision-making regarding the options of early (within 6 months after study baseline) platform medication (Interferon beta, glatiramer acetate, dimethyl/diroximel fumarate, teriflunomide), or no immediate treatment (> 6 months after baseline) of patients with early RR-MS and CIS by predicting the probability of new or enlarging lesions in cerebral magnetic resonance images (MRIs) between 6 and 24 months. Further objectives are refining the MS-TDS score and providing data to identify new markers reflecting disease course and severity. The project also provides a technical evaluation of the ProVal-MS cohort within the IT-infrastructure of the DIFUTURE consortium (Data Integration for Future Medicine) and assesses the efficacy of the data sharing techniques developed. PERSPECTIVE: Clinical cohorts provide the infrastructure to discover and to validate relevant disease-specific findings. A successful validation of the MS-TDS will add a new clinical decision tool to the armamentarium of practicing MS neurologists from which newly diagnosed MS patients may take advantage. Trial registration ProVal-MS has been registered in the German Clinical Trials Register, `Deutsches Register Klinischer Studien` (DRKS)-ID: DRKS00014034, date of registration: 21 December 2018; https://drks.de/search/en/trial/DRKS00014034.