Evaluating the utility of serum NfL, GFAP, UCHL1 and tTAU as estimates of CSF levels and diagnostic instrument in neuroinflammation and multiple sclerosis.

BACKGROUND: This study aimed to evaluate the utility of neurofilament light chain (NfL), glial fibrillary acidic protein (GFAP), ubiquitin C-terminal hydrolase L1 (UCHL1) and total tau (tTAU) serum concentrations as approximation for cerebrospinal fluid (CSF) concentrations of the respective biomarkers in the context of neuroinflammation and multiple sclerosis (MS). METHODS: NfL, GFAP, UCHL1 and tTAU concentrations in serum and CSF were measured in 183 patients (122 with neuroinflammatory disease and 61 neurological or somatoform disease controls) using the single molecule array HD-1 analyzer (Quanterix, Boston, MA). Spearman's rank correlations were computed between serum and CSF concentrations. In a second step, the effects of age, BMI, gadolinium-enhancing lesions in MRI, integrity of the blood-brain barrier (BBB) and presence of acute relapse were accounted for by computing partial correlations. The analyses were repeated for a subsample consisting of MS phenotype patients only (n = 118). EDSS, MS disease activity and acute relapse were considered as additional covariates. Receiver operating characteristic (ROC) analysis was performed for each serum/CSF biomarker concentration to assess how well the particular biomarker concentration differentiates MS patients from somatoform disease controls. Correlations between serum and CSF levels as well as area under the curve (AUC) values were compared for the different biomarkers using z-test statistics. RESULTS: Serum concentrations correlated positively with CSF levels for NfL (r = 0.705, p < 0.01) as well as for GFAP (r = 0.259, p < 0.01). Correlation coefficients were significantly higher for NfL than for GFAP (z = 5.492, p < 0.01). We found no significant serum-CSF correlations for UCHL1 or tTAU. After adjusting for covariates, the results remained unchanged. In the analysis focusing only on MS patients, the results were replicated. ROC analysis demonstrated similarly acceptable performance of serum and CSF NfL values in differentiating MS phenotype patients from somatoform disease controls. AUC values were significantly higher for serum and CSF NfL compared to other biomarkers. CONCLUSION: NfL and GFAP but not UCHL1 or tTAU serum concentrations are associated with CSF levels of the respective biomarker. NfL exhibits more robust correlations between its serum and CSF concentrations as compared to GFAP independently from BBB integrity, clinical and radiological covariates. Both serum and CSF NfL values differentiate between MS and controls.

Matching proposed clinical and MRI criteria of aggressive multiple sclerosis to serum and cerebrospinal fluid markers of neuroaxonal and glial injury.

BACKGROUND: Definitions of aggressive MS employ clinical and MR imaging criteria to identify highly active, rapidly progressing disease courses. However, the degree of overlap between clinical and radiological parameters and biochemical markers of CNS injury is not fully understood. Aim of this cross-sectional study was to match clinical and MR imaging hallmarks of aggressive MS to serum/CSF markers of neuroaxonal and astroglial injury (neurofilament light chain (sNfL, cNfL), and glial fibrillary acidic protein (sGFAP, cGFAP)). METHODS: We recruited 77 patients with relapsing-remitting MS (RRMS) and 22 patients with clinically isolated syndrome. NfL and GFAP levels in serum and CSF were assessed using a single-molecule-array HD-1-analyzer. A general linear model with each biomarker as a dependent variable was computed. Clinical and imaging criteria of aggressive MS, as recently proposed by the ECTRIMS Consensus Group, were modeled as independent variables. Other demographic, clinical or laboratory parameters, were modeled as covariates. Analyses were repeated in a homogenous subgroup, consisting only of newly diagnosed, treatment-naïve RRMS patients presenting with an acute relapse. RESULTS: After adjusting for covariates and multiplicity of testing, sNfL and cNfL concentrations were strongly associated with the presence of ≥2 gadolinium-enhancing lesions (psNfL = 0.00008; pcNfL = 0.004) as well as the presence of infratentorial lesions on MRI (psNfL = 0.0003; pcNfL < 0.004). No other clinical and imaging criteria of aggressive MS correlated significantly with NfL or GFAP in serum and CSF. In the more homogeneous subgroup, sNfL still was associated with the presence of ≥2 gadolinium-enhancing lesions (psNfL = 0.001), presence of more than 20 T2-lesions (psNfL = 0.049) as well as the presence of infratentorial lesions on MRI (psNfL = 0.034), while cNfL was associated with the presence of ≥2 gadolinium-enhancing lesions (psNfL = 0.011) and presence of more than 20 T2-lesions (psNfL = 0.029). CONCLUSIONS: Among proposed risk factors for an aggressive disease course, MRI findings but not clinical characteristics correlated with sNfL and cNfL as a marker of neuroaxonal injury and should be given appropriate weight considering MS prognosis and therapy. No significant correlation was detected for GFAP alone.

Serum and cerebrospinal fluid BDNF concentrations are associated with neurological and cognitive improvement in multiple sclerosis: A pilot study.

BACKGROUND: Biomarkers of disease activity have been intensively studied in multiple sclerosis (MS) but knowledge on predictors of disability improvement is limited. The aim of this pilot study was to explore whether increased brain-derived neurotrophic factor concentrations in serum and CSF (sBDNF/cBDNF) precede neurological and cognitive improvement in MS. METHODS: In this pilot, monocentric prospective cohort study we collected serum/CSF samples at baseline together with EDSS (n = 36) and cognitive testing (n = 34) in patients with relapsing-remitting/primary progressive MS or clinically isolated syndrome. BDNF was assessed in serum and CSF with a single molecule array (SIMOA) HD-1 analyser (Quanterix). Twelve months later EDSS and cognitive testing were repeated. BDNF concentrations of patients with vs. without disability or cognitive improvement (disability improvement: decrease in EDSS ≥ 0.5; cognitive improvement: average z-score increase in neuropsychological performance ≥ 0.5) were compared using univariate ANOVAs adjusting for covariates. RESULTS: Compared to subjects without, patients with disability improvement had higher sBDNF at baseline (q = 0.04). Subjects with cognitive improvement had higher cBDNF at baseline than those without cognitive improvement (q = 0.004). Secondary analysis demonstrated significant correlations between sBDNF and EDSS change (q = 0.036), cBDNF and average z-score change (q = 0.04) and cBDNF and number of cognitive tests with improvement (q = 0.04), while controlling for covariates. CONCLUSIONS: Our findings suggest a possible role for BDNF in neurological and cognitive improvement in MS. These findings have to be confirmed in a larger sample but they already highlight the potential of BDNF as a biomarker for disability improvement and neuroplasticity in MS.

The Acute Superficial Siderosis Syndrome - Clinical Entity, Imaging Findings, and Histopathology.

Superficial siderosis is a consequence of repetitive bleeding into the subarachnoid space, leading to toxic iron and hemosiderin deposits on the surface of the brain and spine. The clinical and radiological phenotypes of superficial siderosis are known to manifest over long time intervals. In contrast, this study defines the "acute superficial siderosis syndrome" and illustrates typical imaging and histopathological findings of this entity. We describe the case of a 61-year-old male patient who was diagnosed with a melanoma metastasis in the right frontal cortex in February 2019. Within a few weeks he developed a progressive syndrome characterized by cerebellar ataxia, gait disturbance, signs of myelopathy, and radiculopathy. MRI revealed ongoing hemorrhage from the metastasis into the lateral ventricle and the subarachnoid space. A semiquantitative assessment of three subsequent MRI within an 8-week period documented the rapid development of superficial siderosis along the surface of the cerebellum, the brain stem, and the lower parts of the supratentorial regions on T2*-weighted sequences. The diagnosis of a superficial siderosis was histopathologically confirmed by identifying iron and hemosiderin deposits on the cortex along with astrogliosis. The recognition of this "acute superficial siderosis syndrome" triggered surgical removal of the hemorrhagic metastasis. Based on a single case presentation, we define the "acute superficial siderosis syndrome" as a clinical entity and describe the radiological and histopathological characteristics of this entity. Early recognition of this syndrome may allow timely elimination of the bleeding source, in order to prevent further clinical deterioration.

Effect of Estimated Blood Volume and Body Mass Index on GFAP and NfL Levels in the Serum and CSF of Patients With Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: To increase the validity of biomarker measures in multiple sclerosis (MS), factors affecting their concentration need to be identified. Here, we test whether the volume of distribution approximated by the patients' estimated blood volume (BV) and body mass index (BMI) affect the serum concentrations of glial fibrillary acidic protein (GFAP). As a control, we also determine the relationship between BV/BMI and GFAP concentrations in CSF. To confirm earlier findings, we test the same hypotheses for neurofilament light chain (NfL). METHODS: NfL and GFAP concentrations were measured in serum and CSF (sNFL/sGFAP and cNFL/cGFAP) in 157 patients (n = 106 with MS phenotype and n = 51 with other neurologic/somatoform diseases). Using multivariate linear regressions, BV was tested in the MS cohort as a predictor for each of the biomarkers while controlling for age, sex, MS phenotype, Expanded Disability Status Scale score, gadolinium-enhancing lesions, and acute relapse. In addition, overweight/obese patients (BMI ≥25 kg/m2) were compared with patients with BMI <25 kg/m2 using the general linear model. The analyses were repeated including the neurologic/somatoform controls. RESULTS: In the MS cohort, BV predicted sGFAP (ß = -0.301, p = 0.014). Overweight/obese patients with MS had lower sGFAP concentrations compared with patients with MS and BMI <25 kg/m2 (F = 4.732, p = 0.032). Repeating the analysis after adding patients with other neurologic/somatoform diseases did not change these findings (ß = -0.276, p = 0.009; F = 7.631, p = 0.006). Although sNfL was inversely correlated with BV (r = -0.275, p = 0.006) and body weight (r = -0.258, p = 0.010), those results did not remain significant after adjusting for covariates. BV and BMI were not associated with cGFAP or cNfL concentrations. DISCUSSION: These findings support the notion that the volume of distribution of sGFAP approximated by BV and BMI is a relevant variable and should therefore be controlled for when measuring sGFAP in MS, while this might not be necessary when measuring cGFAP concentrations.

Brain-derived neurotrophic factor and neurofilament light chain in cerebrospinal fluid are inversely correlated with cognition in Multiple Sclerosis at the time of diagnosis.

BACKGROUND: Cognitive performance may be impaired in MS even at the earliest stages of disease. We tested whether brain-derived neurotrophic factor and neurofilament light chain levels in serum and cerebrospinal fluid (CSF) samples (sNfL/cNfL/sBDNF/cBDNF) collected at the time of diagnosis are associated with cognitive performance. METHODS: We measured sNfL/cNfL/sBDNF/cBDNF using single-molecule array (Simoa) in 47 newly diagnosed patients (32 relapsing-remitting MS/6 primary progressive MS/9 clinically isolated syndrome). Partial correlations between average z-score on neuropsychological tests and sNfL/sBDNF/cNfL/cBDNF were computed after adjusting for covariates. Multivariate analysis of covariance determined the effect of cognitive status on biomarker levels. A composite measure of NfL and BDNF was submitted to similar exploratory analysis. RESULTS: Cognitive performance correlated inversely with cNfL (r=-0.451/q=0.032) and cBDNF (r=-0.406/q=0.034). Impairment in at least two different tests was linked to higher cNfL (p=0.011) and cBDNF (p=0.035) levels compared to impairment in only one test and for cNfL also compared to no impairment at all (p=0.01). Composite CSF biomarker measure accounting for both cNfL and cBDNF correlated more strongly with tests of information processing (p=0.048) and verbal learning/memory consolidation (p = 0.02) as compared to the single CSF biomarkers. CONCLUSIONS: CSF BDNF and NfL levels measured at the time of diagnosis are inversely associated with cognitive performance in MS. Our findings suggest that CSF biomarkers linked to different pathophysiological processes reflect neuropsychological impairment in the earliest stages of the disease. Combining different CSF measures might facilitate the developing of a better biomarker of cognition in MS.

Analysis of CSF D-Dimer to Identify Intrathecal Fibrin-Driven Autoimmunity in Patients With Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: Proteins of the coagulation system contribute to autoimmune inflammation in patients with multiple sclerosis (MS). On blood-brain barrier (BBB) disruption, fibrinogen enters the CNS and is rapidly converted to fibrin, unfolding pleiotropic autoimmune mechanisms. Fibrin accumulation leads to subsequent proteolytic degradation that results in D-dimer generation. The primary objective of this study was to determine intrathecal levels of D-dimer in CSF as a measure of intrathecal coagulation cascade activation and to evaluate its diagnostic utility in patients with MS in contrast to healthy subjects. Key secondary objectives included analysis of CSF D-dimer in differential diagnoses of MS and its relation to routine clinical markers of disease activity. METHODS: Patients admitted for the assessment of suspected MS were prospectively recruited from October 2017 to December 2020. Blood plasma and citrated CSF samples were analyzed using a highly sensitive luminescent oxygen channeling immunoassay. Intrathecal generation of D-dimer was analyzed by adjusting for CSF/serum albumin (Qalb) and CSF/plasma D-dimer quotients (QD-dimer), and corresponding CSF fibrinogen levels were determined. Final diagnoses after full evaluation and clinical data were recorded. RESULTS: Of 187 patients, 113 patients received a diagnosis of MS or clinically/radiologically isolated syndrome. We found increased intrathecal CSF D-dimer generation levels (QD-dimer/Qalb-index) for patients with relapsing-remitting MS (RRMS; n = 71, median 4.7, interquartile range [IQR] 2.5-8.0) when compared with those for disease controls (n = 22, median 2.6, IQR 2.1-4.8, p = 0.031). Absolute CSF D-dimer values correlated with CSF fibrinogen levels (r = 0.463; p < 0 .001) and CSF leukocytes (r = 0.273; p = 0.003) and were elevated in MS patients with contrast enhancement (CE) compared with MS patients without CE on MRI (n = 48, median 6 ng/mL, and IQR 3-15.25 vs n = 41, median 4 ng/mL, and IQR 2-7; p = 0.026). Exploratory subgroup analyses indicated a correlation of intrathecal inflammatory activity and CSF D-dimer levels. DISCUSSION: D-dimer in CSF can be reliably determined and correlates with markers of CNS inflammation and CSF fibrinogen levels. Adjusted for BBB dysfunction, CSF D-dimer may allow the identification of intrathecal coagulation cascade activation in patients with MS. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that CSF D-dimer levels are elevated in patients with RRMS.

Measurement of prothrombin fragment 1+2 in cerebrospinal fluid to identify thrombin generation in inflammatory central nervous system diseases.

BACKGROUND: The interaction of central nervous system inflammation and coagulation system activation in multiple sclerosis (MS) receives increasing attention for its diagnostic and therapeutic potential. During blood-brain barrier (BBB) disruption, fibrinogen migrates into the CNS and contributes to inflammation. In the coagulation cascade, fibrinogen is converted into fibrin by thrombin, which itself is cleaved from prothrombin by activated factor XII. We hypothesized that the conversion of prothrombin to thrombin can be quantified by prothrombin fragment 1+2 (PF1.2) in cerebrospinal fluid (CSF). Primary endpoint was the correlation between PF1.2, D-dimer and fibrinogen in CSF of patients with neuroinflammatory diseases. Secondary endpoints were PF1.2 levels depending on presence of contrast enhancement (CE) on MRI, and correlation between PF1.2 with serum-CSF albumin quotient (Qalb). Additionally, an exploratory analysis of CSF PF1.2 levels to distinguish between MS-patients and controls without neurological disease was performed. METHODS: Patients admitted for a suspected inflammatory CNS disease were prospectively recruited from October 2017 to December 2020. Citrated CSF samples were obtained and analyzed for PF1.2, fibrinogen and D-dimer using a highly sensitive luminescent oxygen channeling immunoassay. Patient clinical data and final diagnoses were retrospectively collected and analyzed. RESULTS: 187 patients were included, of whom 116 received diagnoses of relapsing-remitting (RRMS), primary-progressive MS, clinically or radiologically isolated syndrome, or anti-aquaporin-4-/anti-myelin-oligodendrocyte-glycoprotein-antibody-related diseases. CSF analysis of those 116 patients revealed a correlation between PF1.2 and CSF fibrinogen (ρ=.315; p<.001) as well as between PF1.2 and CSF D-dimer (ρ=.531; p<.001). Among all 187 patients, CSF PF1.2 was increased in patients with CE on MRI (n=71; 147.38 pmol/l; IQR 83.68-215.36) compared to patients without CE (n=86; 100.03 pmol/l; IQR 33.87-162.80; p=.008). CSF PF1.2 correlated significantly with Qalb (ρ=.445; p<.001). No differences of CSF PF1.2 levels were observed between RRMS (131.48 pmol/l, IQR 42.75-204.10) and disease controls (102.28 pmol/l; IQR 55.60-159.94; p=.606). CONCLUSION: In patients with autoimmune inflammatory CNS diseases PF1.2 correlated strongly with fibrinogen and D-dimer in CSF, indicating coagulation system activation. The findings suggest that thrombin generation might require acute BBB dysfunction to exert autoimmune effects in the CNS.

Spinal dural leaks in patients with infratentorial superficial siderosis of the central nervous system-Refinement of a diagnostic algorithm.

BACKGROUND AND PURPOSE: Superficial siderosis of the central nervous system is a sporadic finding in magnetic resonance imaging, resulting from recurrent bleedings into the subarachnoid space. This study aimed to determine the frequency of spinal dural cerebrospinal fluid (CSF) leaks amongst patients with a symmetric infratentorial siderosis pattern. METHODS: In all, 97,733 magnetic resonance images performed between 2007 and 2018 in our neurocenter were screened by a keyword search for "hemosiderosis" and "superficial siderosis." Siderosis patterns on brain imaging were classified according to a previously published algorithm. Potential causative intracranial bleeding events were also assessed. Patients with a symmetric infratentorial siderosis pattern but without causative intracranial bleeding events in history were prospectively evaluated for spinal pathologies. RESULTS: Forty-two patients with isolated supratentorial siderosis, 30 with symmetric infratentorial siderosis and 21 with limited (non-symmetric) infratentorial siderosis were identified. Amyloid angiopathy and subarachnoid hemorrhage were causes for isolated supratentorial siderosis. In all four patients with a symmetric infratentorial siderosis pattern but without a causative intracranial bleeding event in history, spinal dural abnormalities were detected. Dural leaks were searched for in patients with symmetric infratentorial siderosis and a history of intracranial bleeding event without known bleeding etiology, considering that spinal dural CSF leaks themselves may also cause intracranial hemorrhage, for example by inducing venous thrombosis due to low CSF pressure. Thereby, one additional spinal dural leak was detected. CONCLUSIONS: Persisting spinal dural CSF leaks can frequently be identified in patients with a symmetric infratentorial siderosis pattern. Diagnostic workup in these cases should include magnetic resonance imaging of the whole spine.

C-Reactive Protein Levels and Gadolinium-Enhancing Lesions Are Associated With the Degree of Depressive Symptoms in Newly Diagnosed Multiple Sclerosis.

Background: Inflammation is essential for the pathogenesis of multiple sclerosis (MS). While the immune system contribution to the development of neurological symptoms has been intensively studied, inflammatory biomarkers for mental symptoms such as depression are poorly understood in the context of MS. Here, we test if depression correlates with peripheral and central inflammation markers in MS patients as soon as the diagnosis is established. Methods: Forty-four patients were newly diagnosed with relapsing-remitting MS, primary progressive MS or clinically isolated syndrome. Age, gender, EDSS, C-reactive protein (CRP), albumin, white blood cells count in cerebrospinal fluid (CSF WBC), presence of gadolinium enhanced lesions (GE) on T1-weighted images and total number of typical MS lesion locations were included in linear regression models to predict Beck Depression Inventory (BDI) score and the depression dimension of the Symptoms Checklist 90-Revised (SCL90RD). Results: CRP elevation and GE predicted significantly BDI (CRP: p = 0.007; GE: p = 0.019) and SCL90RD (CRP: p = 0.004; GE: p = 0.049). The combination of both factors resulted in more pronounced depressive symptoms (p = 0.04). CSF WBC and EDSS as well as the other variables were not correlated with depressive symptoms. Conclusions: CRP elevation and GE are associated with depressive symptoms in newly diagnosed MS patients. These markers can be used to identify MS patients exhibiting a high risk for the development of depressive symptoms in early phases of the disease.

Severe COVID-19 infection in a patient with multiple sclerosis treated with fingolimod.

BACKGROUND: Fingolimod is used for immune therapy in patients with multiple sclerosis. Long-term treatment is associated with a small increase in the risk of herpes virus reactivation and respiratory tract infections. Patients with coronavirus disease 2019 (COVID-19) under Fingolimod treatment have not been described. METHODS AND RESULTS: We report a 57-year old female patient with a relapsing remitting multiple sclerosis under fingolimod treatment who experienced a severe COVID-19 infection in March 2020 (Extended Disability Status Scale: 2.0). Having peripheral lymphopenia typical for fingolimod treatment (total lymphocytes 0.39/nL [reference range 1.22-3.56]), the patient developed bilateral interstitial pneumonia with multiple ground-glass opacities on chest CT. Fingolimod medication was stopped. On the intensive care unit, non-invasive ventilation was used to provide oxygen and ventilation support regularly. Over the following two days, oxygenation improved, and the patient was transferred to a normal ward five days after admission. CONCLUSION: The implications fingolimod has on COVID-19 are complex. As an S1P analogue, fingolimod might enhance lung endothelial cell integrity. In addition, in case of a so-called cytokine storm, immunomodulation might be beneficial to reduce mortality. Future studies are needed to explore the risks and therapeutic effects of fingolimod in COVID-19 patients.