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.

Serum neurofilament light levels in natalizumab-treated patients with multiple sclerosis who switch to extended interval dosing from every-4-week dosing in real-world clinical practice.

BACKGROUND: Serum levels of neurofilament light chain (sNfL) are a potentially useful biomarker for assessing the efficacy of multiple sclerosis (MS) treatments. OBJECTIVE: To compare levels of sNfL in patients with MS who switched from natalizumab every 4 weeks (Q4W) to extended interval dosing (EID) and patients who remained on Q4W dosing in real-world clinical practice. METHODS: This was a retrospective analysis of samples from patients treated with natalizumab from 2010 to 2015 at a single center in the United States. Levels of sNfL were compared in patients who stayed on Q4W dosing or who switched to EID (parallel-arm analyses) and during Q4W and EID periods in patients who switched to EID (pre- and post-switch analyses). RESULTS: The analysis included 139 patients (Q4W: n = 79; EID: n = 60). After adjustment, levels of sNfL did not significantly differ between patients who remained on Q4W dosing and those who switched to EID in parallel-arm analyses (adjusted Q4W-EID difference = 0.51 pg/mL; p = 0.60) or pre- and post-switch analyses (adjusted difference = 0.96 pg/mL; p = 0.10). CONCLUSION: These sNfL biomarker results suggest that the effectiveness of natalizumab is maintained in patients who switch from Q4W dosing to EID.

Development of a Highly Sensitive Neurofilament Light Chain Assay on an Automated Immunoassay Platform.

Background: Neurofilament light chain (NfL) is an axonal cytoskeletal protein that is released into the extracellular space following neuronal or axonal injury associated with neurological conditions such as multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and other diseases. NfL is detectable in the cerebrospinal fluid (CSF) and blood. Numerous studies on MS have demonstrated that NfL is correlated with disease activity, predicts disease progression, and is reduced by treatment with MS disease-modifying drugs, making NfL an attractive candidate to supplement existing clinical and imaging measures in MS. However, for NfL to achieve its potential as a clinically useful biomarker for clinical decision-making or drug development, a standardized, practical, and widely accessible assay is needed. Our objective was to develop a novel NfL assay on an automated, globally available immunoassay platform and validate its performance. Methods: A prototype NfL assay was first developed and evaluated on the ADVIA Centaur® XP immunoassay system from Siemens Healthineers. The lower limit of quantitation (LLoQ), within-lab precision, assay range, cross-reactivity with neurofilament medium and heavy chains, and effect of interfering substances were determined. NfL assay values in serum and CSF were compared with radiological and clinical disease activity measures in patients with MS and ALS, respectively. This assay was further optimized to utilize serum, plasma, and CSF sample types on the Atellica® IM system and transferred to Siemens' CLIA laboratory where it was analytically validated as a laboratory-developed test (LDT). Results: In this study, an LLoQ of 1.85 pg/mL, within-lab precision <6%, and an assay range of up to 646 pg/mL were demonstrated with the serum prototype assay. Cross-reactivity of <0.7% with the neurofilament medium and heavy chains was observed. Serum and CSF NfL assay values were associated with radiological and clinical disease activity measures in patients with MS and ALS, respectively. The optimized version of the NfL assay demonstrated specimen equivalence with additional plasma tube types and was analytically validated as an LDT. Conclusion: The analytical performance of the NfL assay fulfilled all acceptance criteria; therefore, we suggest that the assay is acceptable for use in both research and clinical practice settings to determine elevated NfL levels in patients.

Measuring treatment response to advance precision medicine for multiple sclerosis.

OBJECTIVE: To assess the independent contributions of clinical measures (relapses, Expanded Disability Status Scale [EDSS] scores, and neuroperformance measures) and nonclinical measures (new brain magnetic resonance imaging [MRI] activity and serum neurofilament light chain [sNfL] levels) for distinguishing natalizumab-treated from placebo-treated patients. METHODS: We conducted post hoc analyses using data from the AFFIRM trial of natalizumab for multiple sclerosis. We used multivariable regression analyses with predictors (EDSS progression, no relapse, new or enlarging MRI activity, brain atrophy, sNfL levels, and neuroperformance worsening) to identify measures that independently discriminated between treatment groups. RESULTS: The multivariable model that best distinguished natalizumab from placebo was no new or enlarging T2 or gadolinium-enhancing activity on MRI (odds ratio; 95% confidence interval: 7.2; 4.7-10.9), year 2 sNfL levels <97.5th percentile (4.1; 2.6-6.2), and no relapses in years 0-2 (2.1; 1.5-3.0). The next best-fitting model was a two-component model that included no MRI activity and sNfL levels <97.5th percentile at year 2. There was little difference between the three- and two-component models. INTERPRETATION: Nonclinical measures (new MRI activity and sNfL levels) discriminate between treatment and placebo groups similarly to or better than clinical outcomes composites and have implications for patient monitoring.

A pharmacokinetic and biomarker study of delayed-release dimethyl fumarate in subjects with secondary progressive multiple sclerosis: evaluation of cerebrospinal fluid penetration and the effects on exploratory biomarkers.

BACKGROUND: Biomarkers are a useful and reliable measure of disease activity in many fields of medicine. Axonal and glial biomarkers in multiple sclerosis (MS) are being applied more often as technology is improving and becoming increasingly reliable. Nonclinical studies have shown dimethyl fumarate (DMF) to have cytoprotective and anti-inflammatory effects. The purpose of this study is to explore the pharmacokinetics (PK) of DMF (by measuring MMF, the active compound) in serum and cerebrospinal fluid (CSF) as well as relevant biomarker data for patients with secondary progressive MS (pwSPMS) and whether there is objective evidence for neuroprotection in pwSPMS treated with DMF. METHODS: Sixteen pwSPMS had serum and cerebrospinal fluid (CSF) evaluation for PK studies levels of MMF at various time points after ingestion of DMF. The CSF biomarkers neurofilament light chain (NfL), glial fibrillary acidic protein (GFAP), ubiquitin carboxyo-terminal hydrolase isozyme L1 (UCH-L1) and total tau (T-tau) were measured at baseline, week 6 and week 28 after initiating DMF with a starting dose of 120 mg twice daily for 4 weeks, followed by a maintenance dose of 240 mg twice daily. Clinical correlation of these patients with EDSS and MRI at these same time periods were made with the biomarkers. Four normal human volunteers had CSF studies for biomarkers at baseline. RESULTS: PK data showed CSF MMF concentration 11% of plasma with Tmax of plasma at 5 hr and Tmax of CSF at 7 hr. Biomarker data showed that CS NfL and to a lesser extent, GFAP, but not UCH-L1 nor T-tau showed relevant changes with clinical data. Some pwSPMS receiving DMF showed clinical improvements in Expanded Disability Status Scale (EDSS). Biomarker changes, but not MRI, correlated with clinical measures in this group of pwSPMS over the observation period. CONCLUSIONS: PK data showed that the Tmax of CSF MMF peaked only 2 hours later than that of plasma with 11% measured in the CSF so that MMF readily crossed the blood brain barrier allowing potential direct penetration into the brain. NfL CSF levels, and to a lesser extent, GFAP CSF levels, showed correlation to disease activity in pwSPMS . These data suggest that DMF may have some benefit in reducing disease activity in pwSPMS if studied for a longer duration and larger well-controlled studies are warranted. DMF was reasonably well tolerated but 3 of the 16 patients did discontinue DMF at 6 weeks due to persistent side effects. NfL appeared to be more clinically relevant biomarker than brain MRI in this this group during the 28-week study period.

N-protein presents early in blood, dried blood and saliva during asymptomatic and symptomatic SARS-CoV-2 infection.

The COVID-19 pandemic continues to have an unprecedented impact on societies and economies worldwide. There remains an ongoing need for high-performance SARS-CoV-2 tests which may be broadly deployed for infection monitoring. Here we report a highly sensitive single molecule array (Simoa) immunoassay in development for detection of SARS-CoV-2 nucleocapsid protein (N-protein) in venous and capillary blood and saliva. In all matrices in the studies conducted to date we observe >98% negative percent agreement and >90% positive percent agreement with molecular testing for days 1-7 in symptomatic, asymptomatic, and pre-symptomatic PCR+ individuals. N-protein load decreases as anti-SARS-CoV-2 spike-IgG increases, and N-protein levels correlate with RT-PCR Ct-values in saliva, and between matched saliva and capillary blood samples. This Simoa SARS-CoV-2 N-protein assay effectively detects SARS-CoV-2 infection via measurement of antigen levels in blood or saliva, using non-invasive, swab-independent collection methods, offering potential for at home and point of care sample collection.

Temporal profile of serum neurofilament light in multiple sclerosis: Implications for patient monitoring.

OBJECTIVE: To understand how longitudinal serum neurofilament light chain (sNfL) patterns can inform its use as a prognostic biomarker in multiple sclerosis (MS) and evaluate whether sNfL reflects MS disease activity and disease-modifying therapy usage. METHODS: This was a post hoc analysis of longitudinal data and samples from the ADVANCE trial (NCT00906399) of patients with relapsing-remitting MS (RRMS). sNfL was measured every 3 months for 2 years, then every 6 months for 4 years. Regression models explored how sNfL data predicted 4-year values of brain volume, expanded disability status scale score, and T2 lesions. sNfL levels were assessed in those receiving placebo, peginterferon beta-1a, and those with disease activity. RESULTS: Baseline sNfL was a predictor of 4-year brain atrophy and development of new T2 lesions. Clinical (p = 0.02) and magnetic resonance imaging (MRI) (p < 0.01) outcomes improved in those receiving peginterferon beta-1a whose sNfL decreased to <16 pg/mL after 12 months versus those whose sNfL remained ⩾16 pg/mL. Mean sNfL levels decreased in peginterferon beta-1a-treated patients and increased in placebo-treated patients (-9.5% vs. 6.8%; p < 0.01). sNfL was higher and more variable in patients with evidence of active MS. CONCLUSION: These data support sNfL as a prognostic and disease-monitoring biomarker for RRMS.

Association of Serum Neurofilament Light Levels With Long-term Brain Atrophy in Patients With a First Multiple Sclerosis Episode.

Importance: Data are needed on the potential long-term prognostic association of serum neurofilament light in multiple sclerosis (MS). Objective: To evaluate serum neurofilament light as a biomarker associated with long-term disease outcomes in clinically isolated syndrome. Design, Setting, and Participants: This post hoc cohort study used data from the Controlled High-Risk Avonex Multiple Sclerosis Prevention Study, a 36-month, multicenter, placebo-controlled interferon ß-1a randomized clinical trial conducted from April 1996 to March 2000, and its long-term (5- and 10-year) extension study from February 2001 to March 2009. Participants included individuals with a symptomatic initial demyelinating event and brain magnetic resonance imaging (MRI) lesions suggestive of MS. Data were analyzed from April 2017 through 2019. Exposure: The variable of interest was naturally occurring serum neurofilament light concentration. Main Outcomes and Measures: Gadolinium-enhancing (Gd+) lesion number, T2 lesion volume, and brain parenchymal fraction, a measure of brain atrophy were measured at baseline and 5 and 10 years. Multivariate regression models evaluated whether age, sex, and baseline covariates, including serum neurofilament light, brain parenchymal fraction, Expanded Disability Status Scale, Gd+ lesion count, and T2 lesion volume, were associated with brain parenchymal fraction changes over 5 and 10 years. Results: Among 308 included participants (mean [SD] age, 33.2 [7.6] years; 234 [76.0%] women), baseline serum neurofilament light concentrations were associated with Gd+ lesions (Spearman r = 0.41; P < .001) and T2 lesion volume (Spearman r = 0.42; P < .001). Among covariates for brain parenchymal fraction change, serum neurofilament light concentration had the greatest correlation with change in brain parenchymal fraction at 5 years (Spearman r = -0.38; P < .001) and was the only variable associated with brain parenchymal fraction at 10 years (Spearman r = -0.45; P < .001). Participants in the highest vs lowest baseline serum neurofilament light tertiles showed brain parenchymal fraction reduction at 5 years (-1.83% [95% CI, -1.49% to -2.18%] vs -0.95% [95% CI, -0.78% to -1.12%]; P < .001) and 10 years (-3.54% [95% CI, -2.90% to -4.17%] vs -1.90% [95% CI, -1.43% to -2.37%]; P < .001). At 5 years, 6 of 45 participants (13.3%) in the highest neurofilament tertile and 2 of 52 participants (3.8%) in the lowest neurofilament tertile achieved an Expanded Disability Status Scale score of 3.5 or greater. Conclusions and Relevance: This cohort study found that higher baseline serum neurofilament light levels were associated with increased brain atrophy over 5 and 10 years. These findings suggest that serum neurofilament light could be a biomarker associated with disease severity stratification in early MS and may help to guide intervention.

Serum neurofilament light as a biomarker in progressive multiple sclerosis.

There is an unmet need in multiple sclerosis (MS) therapy for treatments to stop progressive disability. The development of treatments may be accelerated if novel biomarkers are developed to overcome the limitations of traditional imaging outcomes revealed in early phase trials. In January 2019, the International Progressive MS Alliance convened a standing expert panel to consider potential tissue fluid biomarkers in MS in general and in progressive MS specifically. The panel focused their attention on neurofilament light chain (NfL) in serum or plasma, examining data from both relapsing and progressive MS. Here, we report the initial conclusions of the panel and its recommendations for further research. Serum NfL (sNfL) is a plausible marker of neurodegeneration that can be measured accurately, sensitively, and reproducibly, but standard procedures for sample processing and analysis should be established. Findings from relapsing and progressive cohorts concur and indicate that sNfL concentrations correlate with imaging and disability measures, predict the future course of the disease, and can predict response to treatment. Importantly, disease activity from active inflammation (i.e., new T2 and gadolinium-enhancing lesions) is a large contributor to sNfL, so teasing apart disease activity from the disease progression that drives insidious disability progression in progressive MS will be challenging. More data are required on the effects of age and comorbidities, as well as the relative contributions of inflammatory activity and other disease processes. The International Progressive MS Alliance is well positioned to advance these initiatives by connecting and supporting relevant stakeholders in progressive MS.

Blood neurofilament light levels segregate treatment effects in multiple sclerosis.

OBJECTIVE: To determine factors (including the role of specific disease modulatory treatments [DMTs]) associated with (1) baseline, (2) on-treatment, and (3) change (from treatment start to on-treatment assessment) in plasma neurofilament light chain (pNfL) concentrations in relapsing-remitting multiple sclerosis (RRMS). METHODS: Data including blood samples analyses and long-term clinical follow-up information for 1,261 Swedish patients with RRMS starting novel DMTs were analyzed using linear regressions to model pNfL and changes in pNfL concentrations as a function of clinical variables and DMTs (alemtuzumab, dimethyl fumarate, fingolimod, natalizumab, rituximab, and teriflunomide). RESULTS: The baseline pNfL concentration was positively associated with relapse rate, Expanded Disability Status Scale score, Age-Related MS Severity Score, and MS Impact Score (MSIS-29), and negatively associated with Symbol Digit Modalities Test performance and the number of previously used DMTs. All analyses, which used inverse propensity score weighting to correct for differences in baseline factors at DMT start, highlighted that both the reduction in pNfL concentration from baseline to on-treatment measurement and the on-treatment pNfL level differed across DMTs. Patients starting alemtuzumab displayed the highest reduction in pNfL concentration and lowest on-treatment pNfL concentrations, while those starting teriflunomide had the smallest decrease and highest on-treatment levels, but also starting from lower values. Both on-treatment pNfL and decrease in pNfL concentrations were highly dependent on baseline concentrations. CONCLUSION: Choice of DMT in RRMS is significantly associated with degree of reduction in pNfL, which supports a role for pNfL as a drug response marker.

Neurofilament light levels are associated with long-term outcomes in multiple sclerosis.

BACKGROUND: Neurofilament light chain (NfL) is a promising marker of disease activity/treatment response in multiple sclerosis (MS), although its predictive value for long-term clinical outcomes remains unclear. OBJECTIVE: We measured NfL from a phase 3 trial in relapsing-remitting MS and investigated its association with outcomes after 8 and 15 years. METHODS: NfL concentrations were measured by single molecule array assay in cerebrospinal fluid (CSF) from MS patients (n = 235) in a 2-year randomized clinical trial (RCT) of intramuscular interferon ß-1a, and in serum (n = 164) from the extension study. RESULTS: Year 2 CSF and Year 3 serum NfL were associated with brain parenchymal fraction (BPF) change over 8 years (p < 0.0001, r = -0.46; p < 0.05. r = -0.36, respectively) and were predictive of reaching Expanded Disability Status Scale (EDSS) ⩾ 6.0 at Year 8 (odds ratio (OR) (upper vs lower tertile) = 3.4; 95% confidence interval (CI) = 1.2-9.9, p < 0.05; OR = 11.0, 95% CI = 2.0-114.6; p < 0.01, respectively). Serum NfL concentration (Year 4) was predictive of reaching EDSS score ⩾6.0 at 15 years (OR (upper vs lower tertile) = 4.9; 95% CI = 1.4-20.4; p < 0.05). NfL concentrations were complementary to 2-year BPF change in predicting long-term outcomes. CONCLUSION: Serum and CSF NfL concentrations were associated with long-term clinical outcomes in MS patients and are promising biomarkers for disease severity stratification supporting treatment decisions.

Dimethyl fumarate decreases neurofilament light chain in CSF and blood of treatment naïve relapsing MS patients.

OBJECTIVES: In a prospective phase IV trial of the first-line oral treatment dimethyl fumarate (DMF), we examined dynamics of neurofilament light (NFL) chain in serum, plasma and cerebrospinal fluid (CSF) samples collected over 12 months from relapsing-remitting multiple sclerosis (RRMS) patients. NFL changes were related to disease activity. METHODS: We examined NFL levels by single-molecule array in 88 CSF, 348 plasma and 131 sera from treatment-naïve RRMS patients (n=52), healthy controls (n=23) and a placebo group matched by age, sex and NFL (n=52). Plasma/sera were collected at baseline, and 1, 3, 6 and 12 months after DMF. CSF samples were collected at baseline and 12 months after DMF. RESULTS: NFL concentration in CSF, plasma and serum correlated highly (p<0.0001 for all), but plasma levels were only 76.9% of paired serum concentration. After 12 months of DMF treatment, NFL concentration decreased by 73%, 69% and 55% in the CSF, serum and plasma (p<0.0001, respectively). Significant reduction in blood was observed after 6 and 12 months treatment compared with baseline (p<0.01 and p<0.0001, respectively) and to placebo (p<0.0001). Patients with NFL above the 807.5 pg/mL cut-off in CSF had 5.0-times relative risk of disease activity (p<0.001). CONCLUSIONS: This study provides Class II evidence that first-line DMF reduces NFL in both blood and CSF after 6 months and normalises CSF levels in 73% of patients. High NFL concentration in CSF after a year reflected disease activity. NFL levels were higher in serum than in plasma, which should be considered when NFL is used as a biomarker.

Reversibility of the effects of natalizumab on peripheral immune cell dynamics in MS patients.

OBJECTIVE: To characterize the reversibility of natalizumab-mediated changes in pharmacokinetics/pharmacodynamics in patients with multiple sclerosis (MS) following therapy interruption. METHODS: Pharmacokinetic/pharmacodynamic data were collected in the Safety and Efficacy of Natalizumab in the Treatment of Multiple Sclerosis (AFFIRM) (every 12 weeks for 116 weeks) and Randomized Treatment Interruption of Natalizumab (RESTORE) (every 4 weeks for 28 weeks) studies. Serum natalizumab and soluble vascular cell adhesion molecule-1 (sVCAM-1) were measured using immunoassays. Lymphocyte subsets, α4-integrin expression/saturation, and vascular cell adhesion molecule-1 (VCAM-1) binding were assessed using flow cytometry. RESULTS: Blood lymphocyte counts (cells/L) in natalizumab-treated patients increased from 2.1 × 109 to 3.5 × 109. Starting 8 weeks post last natalizumab dose, lymphocyte counts became significantly lower in patients interrupting treatment than in those continuing treatment (3.1 × 109 vs 3.5 × 109; p = 0.031), plateauing at prenatalizumab levels from week 16 onward. All measured cell subpopulation, α4-integrin expression/saturation, and sVCAM changes demonstrated similar reversibility. Lymphocyte counts remained within the normal range. Ex vivo VCAM-1 binding to lymphocytes increased until ≈16 weeks after the last natalizumab dose, then plateaued, suggesting reversibility of immune cell functionality. The temporal appearance of gadolinium-enhancing lesions was consistent with pharmacodynamic marker reversal. CONCLUSIONS: Natalizumab's effects on peripheral immune cells and pharmacodynamic markers were reversible, with changes starting 8 weeks post last natalizumab dose; levels returned to those observed/expected in untreated patients ≈16 weeks post last dose. This reversibility differentiates natalizumab from MS treatments that require longer reconstitution times. Characterization of the time course of natalizumab's biological effects may help clinicians make treatment sequencing decisions. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that the pharmacodynamic markers of natalizumab are reversed ≈16 weeks after stopping natalizumab.

Population Pharmacokinetics and Target Engagement of Natalizumab in Patients With Multiple Sclerosis.

Natalizumab (humanized immunoglobulin G4 antibody targeting alpha-4 integrins) is a highly efficacious treatment for relapsing-remitting multiple sclerosis (RRMS) that has been in clinical use since 2006. However, natalizumab pharmacokinetic (PK) characteristics and concentration alpha-4 integrin saturation relationships have not been well described in the scientific literature. Therefore, clinical data from 11 studies were pooled and analyzed to characterize the PK and pharmacodynamic (PD) properties of natalizumab in RRMS subjects. Natalizumab PK was best described using a 2-compartment model with linear first-order and Michaelis-Menten elimination. Subcutaneous absorption of natalizumab was characterized using first-order absorption with lag time. The relationship between natalizumab concentration and alpha-4 integrin saturation (PD) was best described by a direct response model with a sigmoidal effect on alpha-4 integrin saturation mediated by a maximum effect relationship with natalizumab concentrations. Covariate analysis showed that body weight, product formulations, and the presence of antinatalizumab antibodies were the main covariates affecting natalizumab PK, whereas age and formulations affected PD. The use of simulations based on a pharmacokinetic-pharmacodynamic model showed that covariates, although statistically significant, are not expected to have any clinical impact at the approved clinical dosing regimen of natalizumab (300 mg once every 4 weeks).

Stability and predictive value of anti-JCV antibody index in multiple sclerosis: A 6-year longitudinal study.

BACKGROUND: Risk of natalizumab-related progressive multifocal leukoencephalopathy is associated with the presence of anti-JC-virus (JCV) antibodies. OBJECTIVE: To investigate the longitudinal evolution of anti-JCV antibody index and to determine the predictive value of baseline anti-JCV antibody index for long-term stability of anti-JCV antibody status. METHODS: MS patients from the MS centre of Medical University of Innsbruck, who had serum sampling for a time period of 4-6 years at intervals of 6±3 months, were included in this retrospective, longitudinal study. Anti-JCV antibody serological status and index were determined by 2-step second-generation anti-JCV antibody assay. RESULTS: 154 patients were included in this study. Median follow-up time was 63.7 months, with median 11 samples available per patient. At baseline, 111 (72.1%) patients were anti-JCV antibody positive. Baseline anti-JCV antibody index significantly correlated with age (R = 0.22, p = 0.005); there was no difference with respect to sex, disease duration or previously used disease-modifying treatment. During follow-up anti-JCV antibody status changed from negative to positive or vice versa in 17% of patients. In seronegative patients at baseline, baseline anti-JCV antibody index was significantly lower in those remaining seronegative at follow-up compared to those converting to seropositivity (median 0.16 vs. 0.24, p = 0.002). In seropositive patients at baseline, index was higher in those remaining seropositive compared to those reverting to seronegativity (2.6 vs. 0.45, p<10-7). Baseline anti-JCV antibody index >0.90 predicted stable positive serostatus (sensitivity 88.7%, specificity 96.5%) and <0.20 stable negative serostatus (sensitivity 61.3%, specificity 97.6%). CONCLUSIONS: Anti-JCV antibody index remained relatively stable over 6-year follow-up with annual serostatus change of ~3%. Baseline anti-JCV antibody index predicted stable negative and stable positive JCV serostatus.

A Randomized Trial Evaluating Various Administration Routes of Natalizumab in Multiple Sclerosis.

The study's primary objective was to compare the pharmacokinetics (PK) and pharmacodynamics (PD) of single subcutaneous (SC) or intramuscular (IM) 300-mg doses of natalizumab with IV 300-mg doses of natalizumab in patients with multiple sclerosis (MS). Secondary objectives included investigation of the safety, tolerability, and immunogenicity of repeated SC and IM natalizumab doses. DELIVER was a 32-week, open-label, multicenter study of natalizumab-naive patients with relapsing-remitting MS (RRMS) or secondary progressive MS (SPMS) randomized to receive 300 mg natalizumab by SC injection, IM injection, or IV infusion. PK and PD were evaluated over 8 weeks after the first natalizumab treatment (Part 1) and over 24 weeks with repeated dosing every 4 weeks, beginning at week 8 (Part 2). Seventy-six patients (24 with RRMS and 52 with SPMS) were enrolled in DELIVER. Following SC or IM administration of natalizumab, peak serum concentrations were approximately 40% of those observed with IV administration and showed no major differences in elimination characteristics. Mean bioavailability relative to IV administration was 57.1% to 71.3% with SC administration and 48.7% with IM administration; mean trough serum concentrations were similar with SC or IV administration and lower with IM administration. Following single or multiple doses of natalizumab, PD response was comparable across administration routes and disease stages. No meaningful differences were observed across administration groups in the incidence or nature of overall adverse events, serious adverse events, administration site reactions, hypersensitivity reactions, or antinatalizumab antibodies. These findings support the comparability of PD measures of natalizumab administered IV, SC, or IM.

CD62L is not a reliable biomarker for predicting PML risk in natalizumab-treated R-MS patients.

OBJECTIVE: To assess if the percentage of CD3(+)CD4(+)CD62L(+) cells in cryopreserved peripheral blood mononuclear cells (PBMCs) (here termed %CD62L) can predict risk of developing progressive multifocal leukoencephalopathy (PML) and better inform the physician for benefit-risk assessment of natalizumab treatment decisions in a global setting. METHODS: Cryopreserved PBMCs from 21 natalizumab-treated patients who developed PML and 104 matched natalizumab-treated patients with multiple sclerosis (MS) without PML collected as a part of Biogen clinical trials were retrospectively examined for CD3, CD4, CCR7, CD45RA, and CD62L by flow cytometry. RESULTS: In this cohort, %CD62L in natalizumab-treated patients did not predict PML risk. Natalizumab-treated patients with MS without PML showed highly variable %CD62L upon serial sampling. In the STRATA study, the distribution of %CD62L in samples collected more than 6 months before a PML diagnosis, at diagnosis, and in natalizumab-treated patients without PML overlapped. No statistical threshold for risk could be determined. In addition, we demonstrated that lymphocyte viability strongly affects %CD62L, supporting previous reports that %CD62L is inherently unstable following cryopreservation and is sensitive to sample collection. CONCLUSION: Data from this well-controlled cohort of natalizumab-treated patients indicate that %CD62L is not a biomarker of PML risk.

Lymphotoxin-LIGHT pathway regulates the interferon signature in rheumatoid arthritis.

A subset of patients with autoimmune diseases including rheumatoid arthritis (RA) and lupus appear to be exposed continually to interferon (IFN) as evidenced by elevated expression of IFN induced genes in blood cells. In lupus, detection of endogenous chromatin complexes by the innate sensing machinery is the suspected driver for the IFN, but the actual mechanisms remain unknown in all of these diseases. We investigated in two randomized clinical trials the effects on RA patients of baminercept, a lymphotoxin-beta receptor-immunoglobulin fusion protein that blocks the lymphotoxin-αß/LIGHT axis. Administration of baminercept led to a reduced RNA IFN signature in the blood of patients with elevated baseline signatures. Both RA and SLE patients with a high IFN signature were lymphopenic and lymphocyte counts increased following baminercept treatment of RA patients. These data demonstrate a coupling between the lymphotoxin-LIGHT system and IFN production in rheumatoid arthritis. IFN induced retention of lymphocytes within lymphoid tissues is a likely component of the lymphopenia observed in many autoimmune diseases. ClinicalTrials.gov NCT00664716.

Anti-JC virus antibody levels in serum or plasma further define risk of natalizumab-associated progressive multifocal leukoencephalopathy.

OBJECTIVE: The increased risk of progressive multifocal leukoencephalopathy (PML) with natalizumab treatment is associated with the presence of anti-JC virus (JCV) antibodies. We analyzed whether anti-JCV antibody levels, measured as index, may further define PML risk in seropositive patients. METHODS: The association between serum or plasma anti-JCV antibody levels and PML risk was examined in anti-JCV antibody-positive multiple sclerosis (MS) patients from natalizumab clinical studies and postmarketing sources. For PML and non-PML patients, the probabilities of having an index below and above a range of anti-JCV antibody index thresholds were calculated using all available data and applied to the PML risk stratification algorithm. Longitudinal stability of anti-JCV antibody index was also evaluated. RESULTS: Anti-JCV antibody index data were available for serum/plasma samples collected >6 months prior to PML diagnosis from 71 natalizumab-treated PML patients and 2,522 non-PML anti-JCV antibody-positive patients. In patients with no prior immunosuppressant use, anti-JCV antibody index distribution was significantly higher in PML patients than in non-PML patients (p < 0.0001). Among patients who were anti-JCV antibody negative at baseline in the AFFIRM and STRATIFY-1 trials, 97% remained consistently negative or below an index threshold of 1.5 over 18 months. Retrospective analyses of pre-PML samples collected longitudinally from PML patients displayed sustained higher anti-JCV antibody index over time. INTERPRETATION: Anti-JCV antibody levels in serum/plasma, measured as index, may differentiate PML risk in anti-JCV antibody-positive MS patients with no prior immunosuppressant use. Continued evaluation of anti-JCV antibody index and PML risk is warranted.