Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) characterized by demyelination and axonal loss [1
]. The current concept of MS pathology is based on infiltrating immune B- and T-cells via the blood–brain barrier, local antibody production and activation of glial cells [2
]. These processes are thought to lead to primary demyelination followed by neurodegeneration [2
]. In recent years, several neurochemical markers have been established for the characterization of pathological molecular processes. One of the most extensively investigated markers for neuroaxonal loss is neurofilament light chain (NfL) [4
]. NfL is one of four neurofilament subunits and the most abundant one, making it a popular target for neurological diseases [7
]. Here, NfL showed superior sensitivity for MS than the phosphorylated subunit of neurofilament [8
Initially investigated using standard immunoassays, NfL in the cerebrospinal fluid (CSF) from MS patients was found to correlate with disease course and activity [9
]. In the early phase of the disease, it has a prognostic value [5
] and can be used as a treatment response marker [13
]. However, NfL is not specific for MS, is rather a general marker for neurodegenerative processes [14
] and changes with the normal aging brain [16
], which needs to be considered when looking at NfL changes over time.
As detection methods were developed over the years, highly sensitive immunoassays became available and allowed the analysis of brain-derived proteins, not only in the CSF, but in serum as well [17
]. Beyond showing a good correlation with CSF values, serum NfL has already thoroughly been investigated in MS [18
], i.e., it has been shown to correlate with clinical and radiological disease activity (relapses, new/enlarged T2 lesions and gadolinium-enhancing lesions in magnetic resonance imaging (MRI)) [19
]. The most important advantage of serum analyses is the possibility of serial sampling and consecutive analysis of biomarkers. Thus, NfL is regularly used in clinical trials to monitor therapy efficacy, and it is on the footsteps of being used as a secondary outcome parameter in clinical trials [23
In most studies, group effects of treatments on neurofilaments are investigated, which already indicate the applicability of serum NfL as a therapy response marker [22
] and as a prognostic marker for long-term clinical outcomes in MS [25
]. However, longitudinal data of intraindividual NfL levels over disease course under immunomodulatory therapies in well-characterized MS patients are widely missing and only described rarely [26
In this study, we analyzed consecutive samples of MS patients in the early phase of the disease, before and after the initiation of disease-modifying treatment with either glatiramer acetate or interferon-beta over a follow-up period of 24 months. Serum NfL levels at each visit were correlated to clinical outcome parameters (relapse and Expanded Disability Status Scale (EDSS)), serum cytokine profile, cognitive functions and MRI parameters of disease activity and progression.
The aim of this study was (a) to show the effect of immunomodulatory therapies on serum NfL levels in MS patients over disease course, (b) to evaluate the relationship between NfL and MRI parameters reflecting disease progression, such as T2 lesion load, (c) to evaluate possible correlation with cognitive functions and (d) to compare serum NfL levels with the serum cytokine profile.
Neurodegeneration and axonal loss are major hallmarks of MS [2
]. NfL has been extensively investigated as a biomarker for those molecular processes [6
]. Initially NfL was exclusively analyzed in CSF, but with improved analytical sensitivity, serum analyses became possible as well [17
]. Serum NfL shows a good correlation with CSF level and thereby offers a window to monitor axonal loss in MS patients consecutively [18
]. Therefore, numerous studies including serum NfL in MS are available and it is used frequently in clinical trials [9
]. However, longitudinal serum NfL assessments are scarce [26
], especially in individual MS patients before and after initiation of first-line therapies. For this purpose, we aimed at characterizing the influence of those therapies on serum NfL levels.
Our data suggested that serum NfL may be suitable as a marker for therapy responsiveness based on the following findings: (a) sNfL levels stayed at a consistent low level or even dropped significantly in relapse-free patients over time and (b) sNfL levels after 9 and 12 months were significantly lower in patients without relapse within 12 months compared with patients suffering from a relapse during this time period.
However, we want to point out that most MS patients in the early phase of the disease, which is the case for most of our patients, show serum NfL levels that are within a normal age-adjusted range [16
Furthermore, our data showed that serum NfL levels were associated with relapses as they were higher in time points with a present relapse compared with non-relapse time points.
The individual serum NfL courses showed that effects that were seen on a group basis did not always hold for every individual. Although serum NfL levels increased during the event of a relapse and decreased after high-dose corticosteroid therapy in most patients, there were exceptions (e.g., patient 16). More consistently, in our cohort, we observed that serum NfL levels stayed at a constant low level in therapy-responsive patients, which might be helpful in therapy monitoring of patients treated with first-line therapies. Our data suggested that this effect can be seen after 9 months. Whereas a sampling interval of 3 or even 6 months seems appropriate in patients without disease activity, other studies of highly active and more severely affected patients suggest a sampling interval and serum NfL testing every month [26
]. We did not observe a positive correlation between serum NfL and EDSS for all time points, which is not surprising as this was also not seen in other studies [31
] or only described in larger cohorts and with patients more severely affected by the disease and accordingly with higher EDSS [22
]. The same was true for the correlation of serum NfL and PASAT as cognitive functions are only mildly affected in the early phase of the disease [33
]. Even though, in a previous study, we observed an association of a more active disease course with higher levels of pro-inflammatory cytokines and lower levels of anti-inflammatory cytokines in a subpopulation of our study cohort [29
], there was no correlation of serum NfL with any of the observed cytokines in the present study.
We also want to discuss the shortcomings of this study. As this was a retrospective analysis of serum NfL in a prospectively collected cohort, pre-analytical effects on serum NfL outcomes must be considered as samples were stored for more than 10 years. However, the observed values were in the same range as those of comparable patients [30
] and of particular interest as no other therapies were available at this time and thereby we were able to monitor long-term outcomes of serum NfL in this specific study population. We can also not completely rule out spontaneous processes or regression that influences serum NfL (sNfL) levels, as we did not include untreated, stable MS patients. As this was an explorative study, these findings need to be confirmed in independent studies and it is desirable to have more detailed MRI data (e.g., number of gadolinium-enhanced lesions, atrophy, etc.) and complete data sets for every patient in those future studies because, for example, T1-hypointense lesions explain the severity of clinical disability better than T2-hyperintense white matter lesions and gadolinium-enhancing lesions correlate better with active disease status. Missing correlation with EDSS was similar to previous findings [31
]. However, we were also unable to detect any correlations with the analyzed cytokines. This might be due to the small sample size or that inflammatory processes were either not present in patients or not displayed in the serum of those patients.
Monitoring of subclinical disease activity using MRI is an established procedure in the care of MS patients. Due to the method’s invasiveness, this is not possible for CSF examination, although CSF parameters are appropriate to reflect intrathecal inflammatory processes. Serum NfL appears to be a promising marker for monitoring subclinical disease activity, as demonstrated in this cohort with longitudinal data collection under the same therapy over 24 months. However, this effect may not be seen in every patient as shown in our single-patient illustrations. In a heterogeneous disease like MS, a single biomarker is not sufficient to completely monitor and evaluate therapy efficacy. For this reason, all available information, clinical and paraclinical, should be gathered and taken into account for clinical decision making.
In summary, our study presents the first results on the effect of first-line therapies on serum NfL levels in mildly affected MS patients over 24 months. Here, serum NfL seems especially helpful in detecting therapy-responsive patients, but we also want to address the need for identifying factors that might influence serum NfL values. Among others, this includes processes involved in the transport of NfL from the CSF into serum as well as NfL clearance. The more we know about non-disease-related mechanisms that affect serum NfL, the better we can model serum NfL courses and identify real changes that are caused by pathological processes.