Gas6/TAM system as potential biomarker for multiple sclerosis prognosis.

Introduction: The protein growth arrest-specific 6 (Gas6) and its tyrosine kinase receptors Tyro-3, Axl, and Mer (TAM) are ubiquitous proteins involved in regulating inflammation and apoptotic body clearance. Multiple sclerosis (MS) is the most common inflammatory demyelinating disease of the central nervous system leading to progressive and irreversible disability if not diagnosed and treated promptly. Gas6 and TAM receptors have been associated with neuronal remyelination and stimulation of oligodendrocyte survival. However, few data are available regarding clinical correlation in MS patients. We aimed to evaluate soluble levels of these molecules in the cerebrospinal fluid (CSF) and serum at MS diagnosis and correlate them with short-term disease severity. Methods: In a prospective cohort study, we enrolled 64 patients with a diagnosis of clinical isolated syndrome (CIS), radiological isolated syndrome (RIS) and relapsing-remitting (RR) MS according to the McDonald 2017 Criteria. Before any treatment initiation, we sampled the serum and CSF, and collected clinical data: disease course, presence of gadolinium-enhancing lesions, and expanded disability status score (EDSS). At the last clinical follow-up, we assessed EDSS and calculated MS severity score (MSSS) and age-related MS severity (ARMSS). Gas6 and TAM receptors were determined using an ELISA kit (R&D Systems) and compared to neurofilament (NFLs) levels evaluated with SimplePlex™ fluorescence-based immunoassay. Results: At diagnosis, serum sAxl was higher in patients receiving none or low-efficacy disease-modifying treatments (DMTs) versus patients with high-efficacy DMTs (p = 0.04). Higher CSF Gas6 and serum sAXL were associated with an EDSS <3 at diagnosis (p = 0.04; p = 0.037). Serum Gas6 correlates to a lower MSSS (r2 = -0.32, p = 0.01). Serum and CSF NFLs were confirmed as disability biomarkers in our cohort according to EDSS (p = 0.005; p = 0.002) and MSSS (r2 = 0.27, p = 0.03; r2 = 0.39, p = 0.001). Results were corroborated using multivariate analysis. Conclusions: Our data suggest a protective role of Gas6 and its receptors in patients with MS and suitable severity disease biomarkers.

Orthologous proteins of experimental de- and remyelination are differentially regulated in the CSF proteome of multiple sclerosis subtypes.

OBJECTIVE: Here, we applied a multi-omics approach (i) to examine molecular pathways related to de- and remyelination in multiple sclerosis (MS) lesions; and (ii) to translate these findings to the CSF proteome in order to identify molecules that are differentially expressed among MS subtypes. METHODS: To relate differentially expressed genes in MS lesions to de- and remyelination, we compared transcriptome of MS lesions to transcriptome of cuprizone (CPZ)-induced de- and remyelination. Protein products of the overlapping orthologous genes were measured within the CSF by quantitative proteomics, parallel reaction monitoring (PRM). Differentially regulated proteins were correlated with molecular markers of inflammation by using MesoScale multiplex immunoassay. Expression kinetics of differentially regulated orthologous genes and proteins were examined in the CPZ model. RESULTS: In the demyelinated and remyelinated corpus callosum, we detected 1239 differentially expressed genes; 91 orthologues were also differentially expressed in MS lesions. Pathway analysis of these orthologues suggested that the TYROBP (DAP12)-TREM2 pathway, TNF-receptor 1, CYBA and the proteasome subunit PSMB9 were related to de- and remyelination. We designed 129 peptides representing 51 orthologous proteins, measured them by PRM in 97 individual CSF, and compared their levels between relapsing (n = 40) and progressive MS (n = 57). Four proteins were differentially regulated among relapsing and progressive MS: tyrosine protein kinase receptor UFO (UFO), TIMP-1, apolipoprotein C-II (APOC2), and beta-2-microglobulin (B2M). The orthologous genes/proteins in the mouse brain peaked during acute remyelination. UFO, TIMP-1 and B2M levels correlated inversely with inflammation in the CSF (IL-6, MCP-1/CCL2, TARC/CCL17). APOC2 showed positive correlation with IL-2, IL-16 and eotaxin-3/CCL26. CONCLUSIONS: Pathology-based multi-omics identified four CSF markers that were differentially expressed in MS subtypes. Upregulated TIMP-1, UFO and B2M orthologues in relapsing MS were associated with reduced inflammation and reflected reparatory processes, in contrast to the upregulated orthologue APOC2 in progressive MS that reflected changes in lipid metabolism associated with increased inflammation.

Alectinib's activity against CNS metastases from ALK-positive non-small cell lung cancer: a single institution case series.

In the present study we assessed the activity of the next-generation anaplastic lymphoma kinase (ALK)-tyrosine kinase inhibitor (-TKI) alectinib, in patients with ALK-postive, advanced non-small cell lung cancer (NSCLC) and central nervous system (CNS) metastases. NSCLCs with ALK-positive disease, as assessed by fluorescence in situ hybridization, and CNS metastases were treated with alectinib 600 mg BID. Included patients were followed prospectively in order to evaluate the efficacy of the drug, with particular emphasis on activity in the CNS. Eleven consecutive patients were enrolled. The majority of them were pretreated with crizotinib (n = 10, 90.9 %), and cranial radiotherapy (n = 8, 72.7 %). Six of the seven patients with measurable CNS disease experienced a CNS response, including three patients who were naïve for cranial radiation. Median duration of response was 8 months. For the whole population, median CNS-progression-free survival (-PFS), systemic-PFS, overall-PFS, overall survival, and 1-year survival were 8, 11, 8, 13 months, and 31.1 %, respectively. Two patients experiencing a CNS response were assessed for alectinib's concentrations in serum and cerebro-spinal fluid (CSF), and showed a CSF-to-serum ratio ranging from 0.001 to 0.003 ng/mL. Alectinib is highly active against CNS metastases from ALK-positive NSCLCs, irrespective of prior treatment(s) with ALK-TKI(s) and/or cranial radiotherapy. The low CSF-to-serum ratio of alectinib suggests that measuring the concentrations of the drug in the CSF may not be a reliable surrogate of its distribution into the CNS.

CSF protein biomarkers predicting longitudinal reduction of CSF ß-amyloid42 in cognitively healthy elders.

ß-amyloid (Aß) plaque accumulation is a hallmark of Alzheimer's disease (AD). It is believed to start many years prior to symptoms and is reflected by reduced cerebrospinal fluid (CSF) levels of the peptide Aß1-42 (Aß42). Here we tested the hypothesis that baseline levels of CSF proteins involved in microglia activity, synaptic function and Aß metabolism predict the development of Aß plaques, assessed by longitudinal CSF Aß42 decrease in cognitively healthy people. Forty-six healthy people with three to four serial CSF samples were included (mean follow-up 3 years, range 2-4 years). There was an overall reduction in Aß42 from a mean concentration of 211-195 pg ml(-1) after 4 years. Linear mixed-effects models using longitudinal Aß42 as the response variable, and baseline proteins as explanatory variables (n=69 proteins potentially relevant for Aß metabolism, microglia or synaptic/neuronal function), identified 10 proteins with significant effects on longitudinal Aß42. The most significant proteins were angiotensin-converting enzyme (ACE, P=0.009), Chromogranin A (CgA, P=0.009) and Axl receptor tyrosine kinase (AXL, P=0.009). Receiver-operating characteristic analysis identified 11 proteins with significant effects on longitudinal Aß42 (largely overlapping with the proteins identified by linear mixed-effects models). Several proteins (including ACE, CgA and AXL) were associated with Aß42 reduction only in subjects with normal baseline Aß42, and not in subjects with reduced baseline Aß42. We conclude that baseline CSF proteins related to Aß metabolism, microglia activity or synapses predict longitudinal Aß42 reduction in cognitively healthy elders. The finding that some proteins only predict Aß42 reduction in subjects with normal baseline Aß42 suggest that they predict future development of the brain Aß pathology at the earliest stages of AD, prior to widespread development of Aß plaques.

Separation and determination of peptide hormones by capillary electrophoresis with laser-induced fluorescence coupled with transient pseudo-isotachophoresis preconcentration.

A new method for the determination of the peptide hormones and their fragments by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection and transient pseudo-isotachophoresis (pseudo-tITP) preconcentration was established in this study. The LIF detector used an argon ion laser with excitation wavelength at 488 nm and emission wavelength at 535 nm. Fluorescein isothiocyanate (FITC) was used as precolumn derivatization reagent to label cholecystokinin tetrapeptide (CCK-4), neurotensin (NT), neurotensin hexapeptide (NT(8-13)), and neurokinin B (NKB). Borate (10 mmol/L, pH 9.0) was selected as derivatization medium to get the high efficiency. When the addition of 70% (v/v) methanol and 1% (m/v) sodium chloride (NaCl) to the sample matrix, and with borate buffer (110 mM, pH 9.5) and 20% (v/v) methanol as running buffer, a preconcentration based on the pseudo-tITP afforded 100-fold improvement in peak heights compared with the traditional hydrodynamic injection (2.3% capillary volume). The detection limits (signal/noise=3) based on peak height were found to be 0.04, 0.1, 0.2, and 0.08 nmol/L for NT(8-13), NT, NKB, and CCK-4, respectively. The method was validated and applied to qualitative analysis of NT and NT(8-13) in human cerebrospinal fluid sample.