Glial and neuroaxonal biomarkers in a multiple sclerosis (MS) cohort

The desire to treat patients with MS early and to target the correct therapy to patients is hampered by a lack of useful prognostic biomarkers that can predict disease progression, severity, and responses to treatment. In addition, clinical trials of new drugs require biomarkers that can predict response to therapy over the course of a few years trial. These biomarkers need to be rapid, relatively inexpensive, and have the potential to be uniformly administered across multiple centres and clinicians. To date, there have been many studies into potential biomarkers for MS. Only serial Magnetic Resonance Imaging (MRI) have emerged as having clinical utility in longitudinal or prospective studies. Small cohort, cross-sectional studies and moderate cohort longitudinal studies have provided a panel of possible biomarkers for MS. Neurofilament light (NfL-l) protein is the light subunit of a structural component in the neuronal axons and increased levels in the cerebrospinal fluid (CSF) reflect axonal degeneration. Tau protein is a microtubule binding protein that contributes to the stability of microtubules. The binding of tau to microtubules is reduced by increases in the phosphorylation state of tau. Hyperphosphorylation of tau disrupts microtubules and leads to degeneration of neurons. Detection of NfL-l protein, tau and phospho tau protein in the CSF in considered to reflect the degree of axonal damage in the central nervous system. Glial fibrillary acidic protein (GFAP) is secreted from astrocytic and is a well-established marker of reactive astrogliosis. Extensive astrocytosis leads to the formation of the astroglial scar, that plays a role in the progression of disease.

The aim of the study is to test neuronal (as NfL-l, tau and phospho tau) and astrocytic activation biomarkers (GFAP) and correlate with clinical characteristics of multiple sclerosis patients.

This observational case-control study included patients with MS and controls with other neurological conditions. The MS patients were separated in two groups: Relapsing patients included patients with Relapsing Remitting MS and CIS (Clinically Isolated Syndrome) while the progressive group included primary (PPMS) and secondary progressive patients (SPMS). CSF was collected according to international standards of collection and storage for biomarkers. CSF levels of b-amyloid, tau, phospho tau, NfL and GFAP were determined using enzyme-linked immunosorbent assay. EDSS (Expanded Disability Status Scale) was performed by a neurostatus trained and certified neurologist. The clinical data (sex, age, duration of disease, type of disease, EDSS, previous disease modifying therapy, previous use of steroids and previous use of statins) of the patients were correlated with the measured proteins. There are available studies that showed reduced levels in CSF NfL-l after natalizumab use and serum NfL-l after fingolimod use, hence patients of high efficacy medication as monoclonal antibody natalizumab were excluded from the study and fingolimod patients were kept to a minimum. Follow up EDSS was measured 6-12 months after sampling. We present normally distributed variables using means and standard deviations and non-normally distributed variables using medians along with their ranges. Spearman's correlation coefficients were used to analyse correlations between the CSF's biomarker concentrations, and both unadjusted and Bonferroni adjusted p-values are reported. Orthogonal projection to latent structure discriminant analysis (OPLS-DA) was also used to find differences in terms of CSF metabolites between the relapsing and remitting patients [CIS, RR, PR versus PP, SP patients]. The OPLS-DA algorithm finds the projection direction, score vector, that gives the largest covariance between the variables and the pre-defined classes (i.e. relapsing and remitting phases) and that maximizes the separation between the classes. The variables that are found to have an influence on the projection (VIP: Variable importance on the projection plots) and that contribute to discriminate between the classes are summarized in a VIP bar-plot. The higher the VIP bar, the more influential is the variable on the model. We used linear regression to examine the association of the biomarkers with EDSS-change, :adjusting for age. A global validation test was used in each model to examine if the assumptions are met. All statistical analyses were performed in R.

87 MS patients (aged 41.1±11.96) enrolled in the study and 21 controls (aged 44.17±12.8). The female/male ratio was 8 females/12 males in the controls and 64 females/20 males in the patients' group. From the patients' cohort 86% (75 patients) were relapsing forms and 14% (12 patients) were progressive forms of the disease. From the total sample 60 patients had disease duration more than a year and 48 less or equal to 1 year. 31 (28.9%) patients had received prior corticosteroid course and 15 patients (13.9%) had prior statin use. In the MS cohort 31 patients (28.7%) had received previous first line disease modifying treatment. The EDSS that showed mild disability without effect of mobility on grades below 4, affected the majority of the sample with EDSS 1-3 in 70% of the patients. 27% of the patients had EDSS scale score 3.5-6.