Use of the combination of spirometry, arterial blood gas analysis and overnight oximetry to predict the outcomes of patients affected by motor neuron disease: The Milan-Torin respiratory score (Mi-To-RS).

BACKGROUND AND PURPOSE: The use of multiple tests, including spirometry, arterial blood gas (ABG) analysis and overnight oximetry (OvOx), is highly recommended to monitor the respiratory function of patients with motor neuron disease (MND). In this study, we propose a composite score to simplify the respiratory management of MND patients and better stratify their prognosis. MATERIALS AND METHODS: We screened the clinical charts of 471 non-ventilated MND patients referred to the Neuro-rehabilitation Unit of the San Raffaele Scientific Institute of Milan (January 2001-December 2019), collecting spirometric, ABG and OvOx parameters. To evaluate the prognostic role of each measurement, univariate Cox regression for death/tracheostomy was performed, and the variables associated with survival were selected to design a scoring system. Univariate and multivariate Cox regression analyses were then carried out to evaluate the prognostic role of the score. Finally, results were replicated in an independent cohort from the Turin ALS Center. RESULTS: The study population included 450 patients. Six measurements were found to be significantly associated with survival and were selected to design a scoring system (maximum score = 8 points). Kaplan-Meier analysis showed significant stratification of survival and time to non-invasive mechanical ventilation adaptation according to score values, and multivariate analysis confirmed the independent effect of the respiratory score on survival of each cohort. CONCLUSION: Forced vital capacity, ABG and OvOx parameters provide complementary information for the respiratory management and prognosis of MND patients and the combination of these parameters into a single score might help neurologists predict prognosis and guide decisions on the timing of the implementation of different diagnostic or therapeutic approaches.

Predictors for progression in amyotrophic lateral sclerosis associated to SOD1 mutation: insight from two population-based registries.

BACKGROUND: Uncovering distinct features and trajectories of amyotrophic lateral sclerosis (ALS) associated with SOD1 mutations (SOD1-ALS) can provide valuable insights for patient' counseling and stratification for trials, and interventions timing. Our study aims to pinpoint distinct clinical characteristics of SOD1-ALS by delving into genotype-phenotype correlations and factors that potentially impact disease progression. METHODS: This is a retrospective observational study of a SOD1-ALS cohort from two Italian registers situated in the regions of Emilia-Romagna, Piedmont and Valle d'Aosta. RESULTS: Out of 2204 genotyped ALS patients, 2.5% carried SOD1 mutations, with a M:F ratio of 0.83. SOD1-ALS patients were younger, and more frequently reported a family history of ALS and/or FTD. SOD1-ALS had a longer survival compared to patients without ALS-associated gene mutations. However, here was considerable variability in survival across distinct SOD1 mutations, with an average survival of less than a year for the L39V, G42S, G73S, D91N mutations. Among SOD1-ALS, multivariate analysis showed that, alongside established clinical prognostic factors such as advanced age at onset and high progression rate at diagnosis, mutations located in exon 2 or within highly conserved gene positions predicted worse survival. Conversely, among comorbidities, cancer history was independently associated with longer survival. INTERPRETATION: Within the context of an overall slower disease, SOD1-ALS exhibits some degree of heterogeneity linked to the considerable genetic diversity arising from the multitude of potential mutations sites and specific clinical prognostic factors, including cancer history. Revealing the factors that modulate the phenotypic heterogeneity of SOD1-ALS could prove advantageous in improving the efficacy of upcoming therapeutic approaches.

Association of Copresence of Pathogenic Variants Related to Amyotrophic Lateral Sclerosis and Prognosis.

BACKGROUND AND OBJECTIVES: Despite recent advances, it is not clear whether the various genes/genetic variants related to amyotrophic lateral sclerosis (ALS) interact in modifying patients' phenotype. The aim of this study was to determine whether the copresence of genetic variants related to ALS has interactive effects on the course of the disease. METHODS: The study population includes 1,245 patients with ALS identified through the Piemonte Register for ALS between 2007 and 2016 and not carrying superoxide dismutase type 1, TAR DNA binding protein, and fused in sarcoma pathogenic variants. Controls were 766 Italian participants age-matched, sex-matched, and geographically matched to cases. We considered Unc-13 homolog A (UNC13A) (rs12608932), calmodulin binding transcription activator 1 (CAMTA1) (rs2412208), solute carrier family 11 member 2 (SLC11A2) (rs407135), and zinc finger protein 512B (ZNF512B) (rs2275294) variants, as well as ataxin-2 (ATXN2) polyQ intermediate repeats (≥31) and chromosome 9 open reading frame 72 (C9orf72) GGGGCC intronic expansions (≥30). RESULTS: The median survival time of the whole cohort was 2.67 years (interquartile range [IQR] 1.67-5.25). In univariate analysis, only C9orf72 (2.51 years, IQR 1.74-3.82; p = 0.016), ATXN2 (1.82 years, IQR 1.08-2.33; p < 0.001), and UNC13A C/C (2.3 years, IQR 1.3-3.9; p < 0.001) significantly reduced survival. In Cox multivariable analysis, CAMTA1 also emerged to be independently related to survival (hazard ratio 1.13, 95% CI 1.001-1.30, p = 0.048). The copresence of 2 detrimental alleles/expansions was correlated with shorter survival. In particular, the median survival of patients with CAMTA1 G/G+G/T and UNC13A C/C alleles was 1.67 years (1.16-3.08) compared with 2.75 years (1.67-5.26) of the patients not carrying these variants (p < 0.001); the survival of patients with CAMTA1 G/G+G/T alleles and ATXN2 ≥31 intermediate polyQ repeats was 1.75 years (0.84-2.18) (p < 0.001); the survival of patients with ATXN2 ≥31 polyQ repeats and UNC13A C/C allele was 1.33 years (0.84-1.75) (p < 0.001); the survival of patients with C9ORF72 ≥30 and UNC13A C/C allele was 1.66 years (1.41-2.16). Each pair of detrimental alleles/expansions was associated to specific clinical phenotypes. DISCUSSION: We showed that gene variants acting as modifiers of ALS survival or phenotype can act on their own or in unison. Overall, 54% of patients carried at least 1 detrimental common variant or repeat expansion, emphasizing the clinical impact of our findings. In addition, the identification of the interactive effects of modifier genes represents a crucial clue for explaining ALS clinical heterogeneity and should be considered when designing and interpreting clinical trials results.

Integrative genetic analysis illuminates ALS heritability and identifies risk genes.

Amyotrophic lateral sclerosis (ALS) has substantial heritability, in part shared with fronto-temporal dementia (FTD). We show that ALS heritability is enriched in splicing variants and in binding sites of 6 RNA-binding proteins including TDP-43 and FUS. A transcriptome wide association study (TWAS) identified 6 loci associated with ALS, including in NUP50 encoding for the nucleopore basket protein NUP50. Independently, rare variants in NUP50 were associated with ALS risk (P = 3.71.10-03; odds ratio = 3.29; 95%CI, 1.37 to 7.87) in a cohort of 9,390 ALS/FTD patients and 4,594 controls. Cells from one patient carrying a NUP50 frameshift mutation displayed a decreased level of NUP50. Loss of NUP50 leads to death of cultured neurons, and motor defects in Drosophila and zebrafish. Thus, our study identifies alterations in splicing in neurons as critical in ALS and provides genetic evidence linking nuclear pore defects to ALS.

Exposure to electromagnetic fields does not modify neither the age of onset nor the disease progression in ALS patients.

Being exposed to electromagnetic fields has been suggested to increase the risk of developing Amyotrophic Lateral Sclerosis (ALS). Here, we investigated the effect of exposure to electromagnetic fields on ALS onset age and progression rate (ΔALSFRS-r). A large cohort of ALS patients (n = 1098) was geolocalized at the time of their diagnosis. Concomitantly, data on the distribution of power lines and repeater antennas (extremely low frequency electromagnetic fields) during the same period were retrieved. Exposure to each repeater antenna was calculated as the sum of 1/(distance from each antenna)^2. Exposure to power lines was calculated assuming each patient's address as the center of several circles of variable radius (100, 250, 500, 1000, and 2000 m). For each radius, the exposure was calculated as the length of the power lines included in the circle. Finally, patients were divided into low- and high-exposed based on the median of the exposure and compared using the Mann-Whitney test. A regression model (one for each radius) was also performed. Neither the onset age nor the ΔALSFRS-r differed among patients' low- and high-exposed to electromagnetic fields. Similarly, we could not find any significant relationship using the regression models. Our findings suggest that electromagnetic fields do not modify the ALS phenotype or progression.

Phenotype Analysis of Fused in Sarcoma Mutations in Amyotrophic Lateral Sclerosis.

Background and Objectives: Pathogenic variations in fused in sarcoma (FUS) are among the most common genetic causes of amyotrophic lateral sclerosis (ALS) worldwide. They are supposedly characterized by a homogeneous pure motor phenotype with early-onset and short disease duration. However, a few FUS-mutated cases with a very late disease onset and slow progression have been reported. To analyze genotype-phenotype correlations and identify the prognostic factors in FUS-ALS cases. Methods: We identified and cross-sectionally analyzed 22 FUS-ALS patient histories from a single-center cohort of 2,615 genetically tested patients and reviewed 289 previously published FUS-ALS cases. Survival analysis was performed by Kaplan-Meier survival curves, followed by the log-rank test and multivariate Cox analysis. Results: Survival of FUS-ALS is age-dependent: In our cohort, early-onset cases had a rapid disease progression and short survival (p = 0.000003) while the outcome of FUS-mutated patients with mid-to-late onset did not differ from non-FUS-ALS patients (p = 0.437). Meta-analysis of literature data confirmed this trend (p = 0.00003). This survival pattern is not observed in other ALS-related genes in our series. We clustered FUS-ALS patients in 3 phenotypes: (1) axial ALS, with upper cervical and dropped-head onset in mid-to-late adulthood; (2) benign ALS, usually with a late-onset and slow disease progression; and (3) juvenile ALS, often with bulbar onset and preceded by learning disability or mild mental retardation. Those phenotypes arise from different mutations. Discussion: We observed specific genotype-phenotype correlations of FUS-ALS and identified age at onset as the most critical prognostic factor. Our results demonstrated that FUS mutations underlie a specific subtype of ALS and enable a careful stratification of newly diagnosed FUS-ALS cases for clinical course and potential therapeutic windows. This will be crucial in the light of incoming gene-specific therapy.

G507D mutation in FUS gene causes familial amyotrophic lateral sclerosis with a specific genotype-phenotype correlation.

Mutations in FUS gene have been described classically in young ALS patients with aggressive disease course. Here we report a large family carrying a missense mutation c.1520 G>A in FUS gene with a tight association with an atypical FUS-ALS phenotype. A 60-year-old man with unilateral leg involvement at onset showed very slow disease progression with selective posterior legs atrophy, tracing his aunt's disease history. His father and uncle died for ALS after a long disease course. Another patient with a 14 years history of ALS with the same phenotype, was found to belong to the same family. In all cases, genetic analysis of FUS gene revealed a missense mutation c.1520 G>A (p.G507D) inherited with a heterozygous pattern. Co-segregation of p.G507D mutation and a specific disease phenotype within the family, characterised by predominant involvement at the lower limbs, slow progression, late bulbar and respiratory failure, demonstrates pathogenicity of this mutation, establishes a well-defined genotype-phenotype correlation and expands the clinical spectrum of heterogeneity in FUS-ALS.

Causal associations of genetic factors with clinical progression in amyotrophic lateral sclerosis.

BACKGROUND AND OBJECTIVE: Recent advances in the genetic causes of ALS reveals that about 10% of ALS patients have a genetic origin and that more than 30 genes are likely to contribute to this disease. However, four genes are more frequently associated with ALS: C9ORF72, TARDBP, SOD1, and FUS. The relationship between genetic factors and ALS progression rate is not clear. In this study, we carried out a causal analysis of ALS disease with a genetics perspective in order to assess the contribution of the four mentioned genes to the progression rate of ALS. METHODS: In this work, we applied a novel causal learning model to the CRESLA dataset which is a longitudinal clinical dataset of ALS patients including genetic information of such patients. This study aims to discover the relationship between four mentioned genes and ALS progression rate from a causation perspective using machine learning and probabilistic methods. RESULTS: The results indicate a meaningful association between genetic factors and ALS progression rate with causality viewpoint. Our findings revealed that causal relationships between ALSFRS-R items associated with bulbar regions have the strongest association with genetic factors, especially C9ORF72; and other three genes have the greatest contribution to the respiratory ALSFRS-R items with a causation point of view. CONCLUSIONS: The findings revealed that genetic factors have a significant causal effect on the rate of ALS progression. Since C9ORF72 patients have higher proportion compared to those carrying other three gene mutations in the CRESLA cohort, we need a large multi-centric study to better analyze SOD1, TARDBP and FUS contribution to the ALS clinical progression. We conclude that causal associations between ALSFRS-R clinical factors is a suitable predictor for designing a prognostic model of ALS.

Identification of genetic risk loci and prioritization of genes and pathways for myasthenia gravis: a genome-wide association study.

Myasthenia gravis is a chronic autoimmune disease characterized by autoantibody-mediated interference of signal transmission across the neuromuscular junction. We performed a genome-wide association study (GWAS) involving 1,873 patients diagnosed with acetylcholine receptor antibody-positive myasthenia gravis and 36,370 healthy individuals to identify disease-associated genetic risk loci. Replication of the discovered loci was attempted in an independent cohort from the UK Biobank. We also performed a transcriptome-wide association study (TWAS) using expression data from skeletal muscle, whole blood, and tibial nerve to test the effects of disease-associated polymorphisms on gene expression. We discovered two signals in the genes encoding acetylcholine receptor subunits that are the most common antigenic target of the autoantibodies: a GWAS signal within the cholinergic receptor nicotinic alpha 1 subunit (CHRNA1) gene and a TWAS association with the cholinergic receptor nicotinic beta 1 subunit (CHRNB1) gene in normal skeletal muscle. Two other loci were discovered on 10p14 and 11q21, and the previous association signals at PTPN22, HLA-DQA1/HLA-B, and TNFRSF11A were confirmed. Subgroup analyses demonstrate that early- and late-onset cases have different genetic risk factors. Genetic correlation analysis confirmed a genetic link between myasthenia gravis and other autoimmune diseases, such as hypothyroidism, rheumatoid arthritis, multiple sclerosis, and type 1 diabetes. Finally, we applied Priority Index analysis to identify potentially druggable genes/proteins and pathways. This study provides insight into the genetic architecture underlying myasthenia gravis and demonstrates that genetic factors within the loci encoding acetylcholine receptor subunits contribute to its pathogenesis.

Effects of intracellular calcium accumulation on proteins encoded by the major genes underlying amyotrophic lateral sclerosis.

The aetiology of Amyotrophic Lateral Sclerosis (ALS) is still poorly understood. The discovery of genetic forms of ALS pointed out the mechanisms underlying this pathology, but also showed how complex these mechanisms are. Excitotoxicity is strongly suspected to play a role in ALS pathogenesis. Excitotoxicity is defined as neuron damage due to excessive intake of calcium ions (Ca2+) by the cell. This study aims to find a relationship between the proteins coded by the most relevant genes associated with ALS and intracellular Ca2+ accumulation. In detail, the profile of eight proteins (TDP-43, C9orf72, p62/sequestosome-1, matrin-3, VCP, FUS, SOD1 and profilin-1), was analysed in three different cell types induced to raise their cytoplasmic amount of Ca2+. Intracellular Ca2+ accumulation causes a decrease in the levels of TDP-43, C9orf72, matrin3, VCP, FUS, SOD1 and profilin-1 and an increase in those of p62/sequestosome-1. These events are associated with the proteolytic action of two proteases, calpains and caspases, as well as with the activation of autophagy. Interestingly, Ca2+ appears to both favour and hinder autophagy. Understanding how and why calpain-mediated proteolysis and autophagy, which are physiological processes, become pathological may elucidate the mechanisms responsible for ALS and help discover new therapeutic targets.

Phosphorylated TDP-43 aggregates in peripheral motor nerves of patients with amyotrophic lateral sclerosis.

Phosphorylated TDP-43 (pTDP-43) aggregates in the cytoplasm of motor neurons and neuroglia in the brain are one of the pathological hallmarks of amyotrophic lateral sclerosis. Although the axons exceed the total volume of motor neuron soma by several orders of magnitude, systematic studies investigating the presence and distribution of pTDP-43 aggregates within motor nerves are still lacking. The aim of this study is to define the TDP-43/pTDP-43 pathology in diagnostic motor nerve biopsies performed on a large cohort of patients presenting with a lower motor neuron syndrome and to assess whether this might be a discriminating tissue biomarker for amyotrophic lateral sclerosis and non-amyotrophic lateral sclerosis cases. We retrospectively evaluated 102 lower motor neuron syndrome patients referred to our centre for a diagnostic motor nerve biopsy. Histopathological criteria of motor neuron disease and motor neuropathy were applied by two independent evaluators, who were blind to clinical data. TDP-43 and pTDP-43 were evaluated by immunohistochemistry, and results compared to final clinical diagnosis. We detected significant differences between amyotrophic lateral sclerosis and non-amyotrophic lateral sclerosis cases in pTDP-43 expression in myelinated fibres: axonal accumulation was detected in 98.2% of patients with amyotrophic lateral sclerosis versus 30.4% of non-amyotrophic lateral sclerosis samples (P < 0.0001), while concomitant positive staining in Schwan cell cytoplasm was found in 70.2% of patients with amyotrophic lateral sclerosis versus 17.4% of patients who did not have amyotrophic lateral sclerosis (P < 0.001). Importantly, we were also able to detect pTDP-43 aggregates in amyotrophic lateral sclerosis cases displaying normal features at standard histopathological analysis. Our findings demonstrated that a specific pTDP-43 signature is present in the peripheral nervous system of patients with amyotrophic lateral sclerosis, and could be exploited as a specific, accessible tissue biomarker. The detection of pTDP-43 aggregates within motor nerves of living patients with amyotrophic lateral sclerosis, occurring before axonal degeneration, suggests that this is an early event that may contribute to amyotrophic lateral sclerosis pathogenesis.

A novel splice site FUS mutation in a familial ALS case: effects on protein expression.

Objective: To investigate the impact of a novel heterozygous FUS mutation in the acceptor splice site of intron 14 (c.1542 - 1 g > t) on protein expression in Peripheral Blood Mononuclear Cells (PBMC) from a familial ALS patient. Methods: PBMC were isolated for mRNA analysis (cDNA synthesis, sequencing and one-step RT-PCR), Western Immunoblot (WI), and Immunofluorescence (IF). Results: cDNA analysis revealed the skipping of exon 15 and a premature stop codon at c.228. RT-PCR showed reduced FUS mRNA by more than half compared to a healthy control (HC) and an ALS patient without genetic mutations (wtALS). In WI FUS band intensity in the proband was 30-50% compared to HC and wtALS. An antibody expected to detect only the wild-type protein did not reveal any reduction of FUS band intensity compared to the other antibodies. IF showed no difference among HC, wtALS, and the proband. Discussion: The reduction of FUS mRNA and protein in PBMC suggests the absence of the truncated protein, probably due to nonsense-mediated decay, leading to loss of function.

Arterial blood gas analysis: base excess and carbonate are predictive of noninvasive ventilation adaptation and survival in amyotrophic lateral sclerosis.

Objective: To investigate the role of arterial blood gas (ABG) analysis parameters (blood carbon dioxide, pCO2; oxygen, pO2; carbonate, HCO3-; standard base excess, SBE) in monitoring respiratory function and ventilation compliance after noninvasive mechanical ventilation (NIV) adaptation, predicting survival in ALS patients. Methods: We selected the first ABG performed after NIV start in ALS patients followed from 2000 to 2015 in Turin ALS Center. Correlations between ABG parameters and survival were calculated. Risk for death/tracheostomy was computed at modifying ABG parameters by using Cox regression models, adjusted for the main prognostic factors. Kaplan-Meier curves were then performed and compared. Results: A total of 186 post-NIV ABGs were included. HCO3- and SBE showed a significant correlation with survival after NIV (respectively, R = -0.183, p = 0.018 and R = -0.200, p = 0.010). Risk for death/tracheostomy after NIV was significantly higher at increasing HCO3- and SBE blood levels, especially when HCO3- was >29 mmol/L and SBE >4 mmol/L (respectively, HR 1.466, 95% CI 1.068-2.011, p = 0.018 and HR = 1.411, 95% CI 1.030-1.32, p = 0.032). Survival in NIV was higher in patients with HCO3- < 29.0 mmol/L and SBE < 4.0 mmol/L. Conclusions: HCO3- and SBE blood levels are markers of ventilation compliance, tolerance and efficacy, being able to predict survival after NIV start in ALS.

The heterozygous deletion c.1509_1510delAG in exon 14 of FUS causes an aggressive childhood-onset ALS with cognitive impairment.

We report a case of childhood-onset ALS with a FUS gene mutation presenting cognitive impairment and a rapid clinical progression. The patient, an 11-year-old girl, presented with right distal upper limb weakness and mild intellectual disability at the Griffith Mental Development Scales. The disease rapidly worsened and the patient became tetraplegic and bed-ridden 2 years after symptom onset. A c.1509_1510delAG mutation in exon 14 of the FUS gene was detected, resulting in a predicted truncated protein, p.G504Wfs*12, lacking the nuclear localization signal. The levels of FUS mRNA in the proband were not significantly different compared to controls. Western immunoblot analysis showed that one antibody (500-526) detected in the proband ~50% of the amount of FUS protein compared to controls, while 3 other antibodies (2-27, 400-450 and FUS C-terminal), which recognize both wild type and the mutated FUS, detected 60% to 75% of the amount of the protein. These findings indicate that p.G504Wfs*12 FUS is more prone to undergo post-translational modification respect to wild type FUS.

Comorbidity of Cervical Spondylogenic Myelopathy and Amyotrophic Lateral Sclerosis: When Electromyography Makes the Difference in Diagnosis.

Cervical spondylogenic myelopathy (CSM) represents a common differential diagnosis for spinal onset Amyotrophic Lateral Sclerosis (ALS). Identifying occurrence of ALS in patients with CSM may be challenging. We evaluated the accuracy of Awaji criteria in the diagnosis of ALS in a cohort of patients with CSM. We screened all patients attending Turin ALS Center during the 2006-2018 period. We selected only patients for whom cervical cord MRI showed radiological signs of CSM. All patients underwent electromyography (EMG), and Awaji criteria were used for diagnosis of clinically probable ALS. All patients were followed up clinically for at least 6 months, and ALS diagnosis was eventually confirmed according to El-Escorial revised criteria, based on disease progression. Of 2,059 patients screened, in 42 cases, MRI showed signs of CSM; CSM incidence and prevalence risks were 0.16 and 2.04%, respectively. Based on clinical progression, 72.7% of patients were diagnosed as CSM and 27.3% as CSM + ALS. At EMG 6 (18.2%) patients fulfilled the criteria for ALS, 5 of them (83.3%) during clinical follow-up were diagnosed as clinical definite ALS + CSM. Accuracy of Awaji criteria in diagnosing ALS was good (AUC = 0.757, p = 0.03). Sensitivity and specificity of Awaji criteria were, respectively, 55.6 and 95.8%. Positive predictive value was 83.3%, while negative predictive value was 85.2%. CSM-ALS comorbidity is a relatively common problem in clinical practice. To better choose patients who could benefit from surgery, EMG should be performed in CSM patients, due to its good accuracy in recognizing ALS.

The NGS technology for the identification of genes associated with the ALS. A systematic review.

BACKGROUND: More than 30 causative genes have been identified in familial and sporadic amyotrophic lateral sclerosis (ALS). The next-generation sequencing (NGS) is a powerful and groundbreaking tool to identify disease-associated variants. Despite documented advantages of NGS, its diagnostic reliability needs to be addressed in order to use this technology for specific routine diagnosis. MATERIAL AND METHODS: Literature database was explored to identify studies comparing NGS and Sanger sequencing for the detection of variants causing ALS. We collected data about patients' characteristics, disease type and duration, NGS and Sanger properties. RESULTS: More than 200 bibliographic references were identified, of which only 14 studies matching our inclusion criteria. Only 2 out of 14 studies compared results of NGS analysis with the Sanger sequencing. Twelve studies screened causative genes associated to ALS using NGS technologies and confirmed the identified variants with Sanger sequencing. Overall, data about more 2,000 patients were analysed. The number of genes that were investigated in each study ranged from 1 to 32, the most frequent being FUS, OPTN, SETX and VCP. NGS identified already known mutations in 21 genes, and new or rare variants in 27 genes. CONCLUSIONS: NGS seems to be a promising tool for the diagnosis of ALS in routine clinical practice. Its advantages are represented by an increased speed and a lowest sequencing cost, but patients' counselling could be problematic due to the discovery of frequent variants of unknown significance.

G-CSF (filgrastim) treatment for amyotrophic lateral sclerosis: protocol for a phase II randomised, double-blind, placebo-controlled, parallel group, multicentre clinical study (STEMALS-II trial).

INTRODUCTION: Amyotrophic lateral sclerosis (ALS) is a fatal progressive neurological disorder characterised by a selective degeneration of motor neurons (MNs). Stem cell transplantation is considered as a promising strategy in neurological disorders therapy and the possibility of inducing bone marrow cells (BMCs) to circulate in the peripheral blood is suggested to investigate stem cells migration in degenerated ALS nerve tissues where potentially repair MN damage. Granulocyte-colony stimulating factor (G-CSF) is a growth factor which stimulates haematopoietic progenitor cells, mobilises BMCs into injured brain and it is itself a neurotrophic factor for MN. G-CSF safety in humans has been demonstrated and many observations suggest that it may affect neural cells. Therefore, we decided to use G-CSF to mobilise BMCs into the peripheral circulation in patients with ALS, planning a clinical trial to evaluate the effect of G-CSF administration in ALS patients compared with placebo. METHODS AND ANALYSIS: STEMALS-II is a phase II multicentre, randomised double-blind, placebo-controlled, parallel group clinical trial on G-CSF (filgrastim) and mannitol in ALS patients. Specifically, we investigate safety, tolerability and efficacy of four repeated courses of intravenous G-CSF and mannitol administered in 76 ALS patients in comparison with placebo (indistinguishable glucose solution 5%). We determine increase of G-CSF levels in serum and cerebrospinal fluid as CD34+ cells and leucocyte count after treatment; reduction in ALS Functional Rating Scale-Revised Score, forced vital capacity, Scale for Testing Muscle Strength Score and quality of life; the adverse events/reactions during the treatment; changes in neuroinflammation biomarkers before and after treatment. ETHICS AND DISSEMINATION: The study protocol was approved by the Ethics Committee of Azienda Ospedaliera Universitaria 'Città della Salute e della Scienza', Torino, Italy. Results will be presented during scientific symposia or published in scientific journals. TRIAL REGISTRATION NUMBER: Eudract 2014-002228-28.