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International audience; Objective: The aim of this study was the comprehensive characterisation of longitudinal clinical, electrophysiological and neuroimaging measures in type III and IV adult spinal muscular atrophy (SMA) with a view to propose objective monitoring markers for future clinical trials. Methods: Fourteen type III or IV SMA patients underwent standardised assessments including muscle strength testing, functional evaluation (SMAFRS and MFM), MUNIX (abductor pollicis brevis, APB; abductor digiti minimi, ADM; deltoid; tibialis anterior, TA; trapezius) and quantitative cervical spinal cord MRI to appraise segmental grey and white matter atrophy. Patients underwent a follow-up assessment with the same protocol 24 months later. Longitudinal comparisons were conducted using the Wilcoxon-test for matched data. Responsiveness was estimated using standardized response means (SRM) and a composite score was generated based on the three most significant variables. Results: Significant functional decline was observed based on SMAFRS (p = 0.019), pinch and knee flexion strength (p = 0.030 and 0.027), MUNIX and MUSIX value in the ADM (p = 0.0006 and 0.043) and in TA muscle (p = 0.025). No significant differences were observed based on cervical MRI measures. A significant reduction was detected in the composite score (p = 0.0005, SRM = −1.52), which was the most responsive variable and required a smaller number of patients than single variables in the estimation of sample size for clinical trials. Conclusions: Quantitative strength testing, SMAFRS and MUNIX readily capture disease progression in adult SMA patients. Composite multimodal scores increase predictive value and may reduce sample size requirements in clinical trials.

Purpose: This study aims to investigate acoustic change over time as biomarkers to differentiate among spastic–flaccid dysarthria associated with amyotrophic lateral sclerosis (ALS), spastic dysarthria associated with primary lateral sclerosis (PLS), flaccid dysarthria associated with spinal and bulbar muscular atrophy (SBMA), and to explore how these acoustic parameters are affected by dysarthria severity. Method: Thirty-three ALS patients with mixed flaccid–spastic dysarthria, 17 PLS patients with pure spastic dysarthria, 18 SBMA patients with pure flaccid dysarthria, and 70 controls, all French speakers, were included in the study. Speakers produced vowel–glide sequences targeting different vocal tract shape changes. The mean and coefficient of variation of the total squared change of mel frequency cepstral coefficients were used to capture the degree and variability of acoustic changes linked to vocal tract modifications over time. Differences in duration of acoustic events were also measured. Results: All pathological groups showed significantly less acoustic change compared to controls, reflecting less acoustic contrast in sequences. Spastic and mixed spastic–flaccid dysarthric speakers showed smaller acoustic changes and slower sequence production compared to flaccid dysarthria. For dysarthria subtypes associated with a spastic component, reduced degree of acoustic change was also associated with dysarthria severity. Conclusions: The acoustic parameters partially differentiated among the dysarthria subtypes in relation to motor neuron diseases. While similar acoustic patterns were found in spastic–flaccid and spastic dysarthria, crucial differences were found between these two subtypes relating to variability. The acoustic patterns were much more variable in ALS. This method forms a promising clinical tool as a diagnostic marker of articulatory impairment, even at mild stage of dysarthria progression in all subtypes.

Purpose Amyotrophic lateral sclerosis (ALS) entails a risk of acute respiratory failure (ARF). The decision to admit such patients to the intensive care unit (ICU) is difficult given the inexorable prognosis of ALS. To fuel this discussion, this study describes the ICU and post-ICU survival of ALS-related ARF. Material and methods Retrospective cohort analysis over 10 years (university hospital setting, ALS reference center). Results Of 90 patients (66 men, median age: 67 [IQR 59–71], median interval since ALS diagnosis: 26.5 months [14–53], ALSFRS-R: 19 [12−30], bulbar signs 73%), 48 were managed by noninvasive ventilation (NIV) only, 7 were already tracheotomized upon admission, 12 were tracheotomized during the ICU stay (advance care planning project), 18 were already intubated before admission, 5 received oxygen and physiotherapy only. Median ICU stay was 4 days [ [2] , [3] , [4] , [5] , [6] , [7] , [8] , [9] ] with 20% mortality. Median hospital stay was 10 days [5–22] with 33% mortality. The 3-month and one year mortality wer 46% and 71%. Hospital mortality was higher in patients with more severe respiratory acidosis and higher simplified acute physiology scores on admission. Conclusions The prognosis of ALS-related ARF requiring ICU admission resembles that of ARF complicating other conditions with high short-term mortality (e.g. lung cancer).

BackgroundThe cause of the motor neuron (MN) death that drives terminal pathology in Amyotrophic Lateral Sclerosis (ALS) remains unknown, and it is thought that the cellular environment of the MN may play a key role in MN survival. Several lines of evidence implicate vesicles in ALS, including that extracellular vesicles may carry toxic elements from astrocytes towards motor neurons, and that pathological proteins have been identified in circulating extracellular vesicles of sporadic ALS patients. Since MN degeneration at the neuromuscular junction is a feature of ALS, and muscle is a vesicle-secretory tissue, we hypothesized that muscle vesicles may be involved in ALS pathology.MethodsSporadic ALS patients were confirmed to be ALS according to El Escorial criteria, were genotyped to test for classic gene mutations associated with ALS, and physical function was assessed using the ALSFRS-R score. Muscle biopsies of either mildly affected deltoids of ALS patients (n=27) or deltoids of aged-matched healthy subjects (n=30) were used for extraction of muscle stem cells, to perform immunohistology, or for electron microscopy. Muscle stem cells were characterized by immunostaining, RTqPCR and transcriptomic analysis. Secreted muscle vesicles were characterized by proteomic analysis, Western blot, NanoSight, and electron microscopy. The effects of muscle vesicles isolated from the culture medium of ALS and healthy myotubes were tested on healthy human-derived iPSC motor neurons and on healthy human myotubes, with untreated cells used as controls.ResultsAn accumulation of multivesicular bodies was observed in muscle biopsies of sporadic ALS patients by immunostaining and electron microscopy. Study of muscle biopsies and biopsy-derived denervation-naïve differentiated muscle stem cells (myotubes) revealed a consistent disease signature in ALS myotubes, including intracellular accumulation of exosome-like vesicles and disruption of RNA-processing. Compared to vesicles from healthy control myotubes, when administered to healthy motor neurons the vesicles of ALS myotubes induced shortened, less branched neurites, cell death, and disrupted localization of RNA and RNA-processing proteins. The RNA-processing protein FUS and a majority of its binding partners were present in ALS muscle vesicles, and toxicity was dependent on the expression level of FUS in recipient cells. Toxicity to recipient motor neurons was abolished by anti-CD63 immuno-blocking of vesicle uptake.ConclusionALS muscle vesicles are shown to be toxic to motor neurons, which establishes the skeletal muscle as a potential source of vesicle-mediated toxicity in ALS.One Sentence SummaryMuscle cells of ALS patients secrete vesicles that are toxic to motor neurons

PURPOSE/OBJECTIVE(S) To evaluate the efficacy and safety of radiotherapy (RT) of the primary salivary glands in a large series of patients with amyotrophic lateral sclerosis (ALS) and Parkinson's disease with sialorrhea. MATERIALS/METHODS Patients treated in our institution between 2010 and 2020 were retrospectively analyzed, including the first 50 patients prospectively analyzed. All patients included were refractory to conventional sialorrhea drug treatments. RT was delivered by a conventional LINAC, with 2 opposite beams including the two submandibular glands and two thirds of the two parotid glands before 2014, and from 2015 2 lateral beams including the submaxillary glands, and 2 opposite oblique beams directed to each parotid gland, with reduced oral cavity irradiation. The total dose delivered was 10 Gy in 2 fractions or 20 Gy in 4 fractions. RT efficacy was assessed using the 9-item Sialorrhea Scoring Scale (SSS), prospectively validated as the most effective and sensitive tool for measuring sialorrhea in ALS patients. RESULTS A total of 235 patients were included, and 281 treatments (45 reirradiations, 1 patient received 3 irradiations). Median age was 69 years (range: 38 - 95), 127 women (54%) and 126 men (46%), 212 had ALS (90%), 17 had Parkinson's disease (7%), and 5 patients (3%) had another neurological disorder. Median interval between the first and second radiotherapy course was 7.5 months (range: 1 - 37). The median SSS score before radiation therapy was 8 (range: 6 - 9), and the median SSS score at 1 month of irradiation was 2 (range: 1 - 6). There was no toxicity of > grade 1. At the end of RT, all but one patient had an improvement in SSS score: 272 had a complete response (CR) (97%, SSS 1-3) and 8 had a partial response (PR) (3%, SSS 4-5); one patient had a stable SSS score (< 1%). A 1-month CR after RT was achieved in 96% of the 235 patients who underwent a primary RT, and 100% of the 45 patients who received re-irradiation. Patients treated with 20Gy versus 10Gy during the first RT treatment were more likely to have a CR at 1 month (99% vs. 91%, P = 0.007), and had a greater decrease in SSS score (mean difference -6 vs. -5 points, P < 0.001). Also, the 45 patients who received reirradiation were more frequently previously treated with the RT 10Gy protocol (25/45 patients, 55%) versus 60/191 (31%) in patients who did not require reirradiation (P < 0.001). The 3-beam irradiation technique, used from 2014 and beyond, did not significantly improve treatment efficacy (reduction of the SSS score by -5.8 points on average versus -5.6 points previously, P = 0.3). Nevertheless, it reduced 1 month toxicity, in particular decreased saliva thickening discomfort (P < 0.001). CONCLUSION A 20 Gy irradiation in 4 fractions is an effective treatment for ALS patients with sialorrhea, with minimal toxicity, particularly by separately targeting parotid and submaxillary glands. A shorter RT (10 Gy in 2 fractions) may be proposed in patients with poor medical condition, subject to its lower efficacy and the greater need for reirradiation.

Due to the expanding use of non-invasive ventilation (NIV) in amyotrophic lateral sclerosis (ALS), the question of enteral nutrition is increasingly raised in NIV users ALS patients. Here, we aimed to determine the prognostic factors for survival after gastrostomy placement in routine NIV users, taking into consideration ventilator dependence. Ninety-two routine NIV users ALS patients, who underwent gastrostomy insertion for severe dysphagia and/or weight loss, were included. We used a Cox proportional hazards model to identify factors affecting survival and compared time from gastrostomy to death and 30-day mortality rate between dependent (daily use ≥ 16 h) and non-dependent NIV users. The hazard of death after gastrostomy was significantly affected by 3 factors: age at onset (HR 1.047, p = 0.006), body mass index 2 at the time of gastrostomy placement (HR 2.012, p = 0.016) and recurrent accumulation of airway secretions (HR 2.614, p = 0.001). Mean time from gastrostomy to death was significantly shorter in the dependent than in the non-dependent NIV users group (133 vs. 250 days, p = 0.04). The 30-day mortality rate was significantly higher in dependent NIV users (21.4% vs. 2.8%, p = 0.03). Pre-operative ventilator dependence and airway secretion accumulation are associated with worse prognosis and should be key decision-making criteria when considering gastrostomy tube placement in NIV users ALS patients.

The aim of this paper is to describe the clinical features of COVID-19-related encephalopathy and their metabolic correlates using brain 2-desoxy-2-fluoro-D-glucose (FDG)-positron-emission tomography (PET)/computed tomography (CT) imaging.A variety of neurological manifestations have been reported in association with COVID-19. COVID-19-related encephalopathy has seldom been reported and studied.We report four cases of COVID-19-related encephalopathy. The diagnosis was made in patients with confirmed COVID-19 who presented with new-onset cognitive disturbances, central focal neurological signs, or seizures. All patients underwent cognitive screening, brain magnetic resonance imaging (MRI), lumbar puncture, and brain 2-desoxy-2-fluoro-D-glucose (FDG)-positron-emission tomography (PET)/computed tomography (CT) (FDG-PET/CT).The four patients were aged 60 years or older, and presented with various degrees of cognitive impairment, with predominant frontal lobe impairment. Two patients presented with cerebellar syndrome, one patient had myoclonus, one had psychiatric manifestations, and one had status epilepticus. The delay between first COVID-19 symptoms and onset of neurological symptoms was between 0 and 12 days. None of the patients had MRI features of encephalitis nor significant cerebrospinal fluid (CSF) abnormalities. SARS-CoV-2 RT-PCR in the CSF was negative for all patients. All patients presented with a consistent brain FDG-PET/CT pattern of abnormalities, namely frontal hypometabolism and cerebellar hypermetabolism. All patients improved after immunotherapy.Despite varied clinical presentations, all patients presented with a consistent FDG-PET pattern, which may reflect an immune mechanism.

Fifteen ALS patients, with troublesome symptoms linked to masseter spasticity, benefited from BoNT-A injections in each masseter. Based on the medical records of patients, the effect of the first injection was assessed one month later. We retrospectively collected information for 12 patients. Eight of them reported a beneficial effect after the injection for the following symptoms: trismus, tongue, lip and cheek biting, and jaw clonus. Five patients indicated that dental care was easier after injection. Our study showed that injections of BoNT-A unequivocally reduced masseter spasticity in ALS patients who subsequently enjoyed greater comfort in their daily living.

International audience; Non-invasive ventilation (NIV) has become an essential part of the treatment of amyotrophic lateral sclerosis (ALS) since 2006. NIV very significantly improves survival, quality of life and cognitive performances. The initial NIV settings are simple, but progression of the disease, ventilator dependence and upper airway involvement sometimes make long-term adjustment of NIV more difficult, with a major impact on survival. Unique data concerning the long-term adjustment of NIV in ALS show that correction of leaks, management of obstructive apnoea and adaptation to the patient's degree of ventilator dependence improve the prognosis. Non-ventilatory factors also impact the efficacy of NIV and various solutions have been described and must be applied, including cough assist techniques, control of excess salivation and renutrition. NIV in ALS has been considerably improved as a result of application of all of these measures, avoiding the need for tracheostomy in the very great majority of cases. More advanced use of NIV also requires pulmonologists to master the associated end-of-life palliative care, as well as the modalities of discontinuing ventilation when it becomes unreasonable.

Amyotrophic lateral sclerosis (ALS) patients show progressive respiratory muscle weakness leading to death from respiratory failure. However, there are no data on diaphragm histological changes in ALS patients and how they correlate with routine respiratory measurements.We collected 39 diaphragm biopsies concomitantly with laparoscopic insertion of intradiaphragmatic electrodes during a randomised controlled trial evaluating early diaphragm pacing in ALS (https://clinicaltrials.gov; NCT01583088). Myofibre type, size and distribution were evaluated by immunofluorescence microscopy and correlated with spirometry, respiratory muscle strength and phrenic nerve conduction parameters. The relationship between these variables and diaphragm atrophy was assessed using multivariate regression models.All patients exhibited significant slow- and fast-twitch diaphragmatic atrophy. Vital capacity (VC), maximal inspiratory pressure, sniff nasal inspiratory pressure (SNIP) and twitch transdiaphragmatic pressure did not correlate with the severity of diaphragm atrophy. Inspiratory capacity (IC) correlated modestly with slow-twitch myofibre atrophy. Supine fall in VC correlated weakly with fast-twitch myofibre atrophy. Multivariate analysis showed that IC, SNIP and functional residual capacity were independent predictors of slow-twitch diaphragmatic atrophy, but not fast-twitch atrophy.Routine respiratory tests are poor predictors of diaphragm structural changes. Improved detection of diaphragm atrophy is essential for clinical practice and for management of trials specifically targeting diaphragm muscle function.

Spinal muscular atrophy (SMA) type III and IV are autosomal recessive, slowly progressive lower motor neuron syndromes. Nevertheless, wider cerebral involvement has been consistently reported in mouse models. The objective of this study is the characterisation of spinal and cerebral pathology in adult forms of SMA using multimodal quantitative imaging. Methods Twenty-five type III and IV adult SMA patients and 25 age-matched healthy controls were enrolled in a spinal cord and brain imaging study. Structural measures of grey and white matter involvement and diffusion parameters of white matter integrity were evaluated at each cervical spinal level. Whole-brain and region-of-interest analyses were also conducted in the brain to explore cortical thickness, grey matter density and tract-based white matter alterations. Results In the spinal cord, considerable grey matter atrophy was detected between C2-C6 vertebral levels. In the brain, increased grey matter density was detected in motor and extra-motor regions of SMA patients. No white matter pathology was identified neither at brain and spinal level. Conclusions Adult forms of SMA are associated with selective grey matter degeneration in the spinal cord with preserved white matter integrity. The observed increased grey matter density in the motor cortex may represent adaptive reorganisation., Highlights • (SMA) type 3 and 4 is a lower motor neuron syndrome. Nevertheless, wider involvement of the nervous system might be possible. • 25 adults type 3 and 4 SMA patients were studied using brain and cervical spinal cord neuroimaging techniques. • Grey matter atrophy was observed in the spinal cord. No white matter degeneration was present at brain and spinal level. • Increased grey matter density was detected in cerebral motor regions and explained as compensatory mechanism.

Objective Amyotrophic lateral sclerosis (ALS) is a fatal and progressive neurodegenerative disorder involving motor neurons of the cerebral cortex, brain stem and spinal cord. Besides the motor signs, cognitive disorders and apathy may be present and may impact the survival time. These elements are therefore to be taken into consideration for medical care because they can influence the disease evolution. The literature shows low psychopathological disorders in this population despite its poor prognosis. The main objective of this study is to explore the emotional feeling in apathetic and non-apathetic patients in relation to their anxiety and depressive symptoms. Methods We included 152 patients at the day hospital for the follow-up of their illness, with an average age of 61±12.2 years. All filled the following self-administered questionnaires: EPN-31 (emotional feeling), HADS (for anxiety and depressive symptoms) and the Marin's apathy evaluation scale. Most of the patients (n=110) had also a cognitive assessment with the ALS-CBS scale. Results 42% of patients could be considered as apathetic and they felt both positive and negative emotions whose frequency was related to the presence and intensity of anxiety and depressive symptoms. The only significant differences were that apathetic and anxious patients experienced more negative emotions including sadness, shame and anger than non-apathetic and anxious patients. Apathy was negatively correlated with cognitive functioning and survival time. Conclusions These results highlighted the negative impact that apathy seemed to have on the evolution of this disease. However, apathetic patients didn't show emotional blunting and were able to name and feel positive and negative emotions; and even feel more negative emotions than non-apathetic patients when they were anxious. A better understanding of apathetic and no apathetic patients' emotional feelings should lead to a more personalized care for the ALS patients.

International audience; Myasthenia gravis is an autoimmune disorder affecting neuromuscular junctions that has been associated with a small increased risk of amyotrophic lateral sclerosis (ALS). Here, we describe a retrospective series of seven cases with a concomitant diagnosis of ALS and myasthenia gravis, collected among the 18 French reference centers for ALS in a twelve years period. After careful review, only six patients strictly met the diagnostic criteria for both ALS and myasthenia gravis. In these patients, limb onset of ALS was reported in five (83%) cases. Localization of myasthenia gravis initial symptoms was ocular in three (50%) cases, generalized in two (33%) and bulbar in one (17%). Median delay between onset of the two conditions was 19 months (6-319 months). Anti-acetylcholine receptor antibodies testing was positive in all cases. All patients were treated with riluzole and one had an associated immune-mediated disease. In the one last ALS case, the final diagnosis was false-positivity for anti-acetylcholine receptor antibodies. The co-occurrence of ALS and myasthenia gravis is rare and requires strict diagnostic criteria. Its demonstration needs thoughtful interpretation of electrophysiological results and exclusion of false positivity for myasthenia gravis antibody testing in some ALS cases. This association may be triggered by a dysfunction of adaptive immunity.

International audience; Objective : Objective of this study is the comprehensive characterisation of motor unit (MU) loss in type III and IV Spinal Muscular Atrophy (SMA) using motor unit number index (MUNIX), and evaluation of compensatory mechanisms based on MU size indices (MUSIX).Methods : Nineteen type III and IV SMA patients and 16 gender- and age-matched healthy controls were recruited. Neuromuscular performance was evaluated by muscle strength testing and functional scales. Compound motor action potential (CMAP), MUNIX and MUSIX were studied in the abductor pollicis brevis (APB), abductor digiti minimi (ADM), deltoid, tibialis anterior and trapezius muscles. A composite MUNIX score was also calculated.Results : SMA patients exhibited significantly reduced MUNIX values (p

International audience; Background: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder leading to chronic respiratory failure. Few studies have investigated ALS-related dyspnoea, and none have characterised the emotional distress it inflicts. We hypothesised that ALS-related dyspnoea has a strong affective component that relates to quality of life.Methods: This prospective, observational study was conducted in 41 ALS patients >18 with chronic respiratory failure and an indication for noninvasive ventilation (NIV). Dyspnoea was assessed using the Multidimensional Dyspnea Profile (MDP) at baseline and 1 month after NIV initiation. Correlations between scores evaluating the sensory and affective dimensions of dyspnoea and other patient-reported outcomes and pulmonary function tests were analysed.Results: Dyspnoea was described as intense (median [IQR] score on a 0-10 scale: 6.5 [4.0-7.5]). The sensory dimension of dyspnoea was polymorphic, but «air hunger» was the most common (48.8%) and the most intense (6 [4-8]) sensory descriptor. In the affective domain, most patients rated «anxious» (85.4%) and «afraid» (60.9%) above 0. The MDP affective dimension correlated significantly with other patient-reported outcomes, with the strongest correlation being between MDP «anxious» and the anxiety component of the Hospital Anxiety Depression Scale (Pearson's R = 0.70). One month after initiation of NIV, dyspnoea during unassisted breathing was described in virtually the same terms, particularly the affective dimension.Discussion: ALS-related dyspnoea is intense and fear-provoking, persists during unassisted breathing between NIV sessions, and significantly impacts health-related quality of life. This study highlights the need for increased awareness of and research into ALS-related dyspnoea.

International audience; Introduction Extrapyramidal deficits are poorly characterised in amyotrophic lateral sclerosis (ALS) despite their contribu-tion to functional disability, increased fall risk and their quality-of-life implications. Given the concomitant pyramidal and cerebellar degeneration in ALS, the clinical assessment of extrapyramidal features is particularly challenging.Objective The comprehensive characterisation of postural instability in ALS using standardised clinical assessments, gait analyses and computational neuroimaging tools in a prospective study design.Methods Parameters of gait initiation in the anticipatory postural adjustment phase (APA) and execution phase (EP) were evaluated in ALS patients with and without postural instability and healthy controls. Clinical and gait analysis parameters were interpreted in the context of brain imaging findings.Results ALS patients with postural instability exhibit impaired gait initiation with an altered APA phase, poor dynamic postural control and significantly decreased braking index. Consistent with their clinical profile, “unsteady” ALS patients have reduced caudate and brain stem volumes compared to “steady” ALS patients.Interpretation Our findings highlight that the ALS functional rating scale (ALSFRS-r) does not account for extrapyramidal deficits, which are major contributors to gait impairment in a subset of ALS patients. Basal ganglia degeneration in ALS does not only contribute to cognitive and behavioural deficits, but also adds to the heterogeneity of motor disability

Summary Background Neurite outgrowth inhibitor A (Nogo-A) is thought to have a role in the pathophysiology of amyotrophic lateral sclerosis (ALS). A monoclonal antibody against Nogo-A showed a positive effect in the SOD1 G93A mouse model of ALS, and a humanised form of this antibody (ozanezumab) was well tolerated in a first-in-human trial. Therefore, we aimed to assess the safety and efficacy of ozanezumab in patients with ALS. Methods This randomised, double-blind, placebo-controlled, phase 2 trial was done in 34 centres in 11 countries. Patients aged 18–80 years with a diagnosis of familial or sporadic ALS were randomly assigned (1:1), centrally according to a computer-generated allocation schedule, to receive ozanezumab (15 mg/kg) or placebo as intravenous infusions over 1 h every 2 weeks for 46 weeks, followed by assessments at week 48 and week 60. Patients and study personnel were masked to treatment assignment. The primary outcome was a joint-rank analysis of function (ALS Functional Rating Scale-Revised) and overall survival, analysed at 48 weeks in all patients who received at least one dose of study drug. This study is registered with ClinicalTrials.gov, number NCT01753076, and with GSK-ClinicalStudyRegister.com, NOG112264, and is completed. Findings Between Dec 20, 2012, and Nov 1, 2013, we recruited 307 patients, of whom 303 were randomly assigned to receive placebo (n=151) or ozanezumab (n=152). The adjusted mean of the joint-rank score was −14·9 (SE 13·5) for the ozanezumab group and 15·0 (13·6) for the placebo group, with a least squares mean difference of −30·0 (95% CI −67·9 to 7·9; p=0·12). Overall, reported adverse events, serious adverse events, and adverse events leading to permanent discontinuation of study drug or withdrawal from study were similar between the treatment groups, except for dyspepsia (ten [7%] in the ozanezumab group vs four [3%] in the placebo group), depression (11 [7%] vs five [3%]), and diarrhoea (25 [16%] vs 12 [8%]). Respiratory failure was the most common serious adverse event (12 [8%] vs seven [5%]). At week 60, the number of deaths was higher in the ozanezumab group (20 [13%]) than in the placebo group (16 [11%]), mainly as a result of respiratory failure (ten [7%] vs five [3%]). Two deaths were considered related to the study drug (bladder transitional cell carcinoma in the ozanezumab group and cerebrovascular accident in the placebo group). Interpretation Ozanezumab did not show efficacy compared with placebo in patients with ALS. Therefore, Nogo-A does not seem to be an effective therapeutic target in ALS. Funding GlaxoSmithKline.

Purpose This study aimed to evaluate the efficiency and the tolerance of radiation therapy (RT) on salivary glands in a large series of amyotrophic lateral sclerosis (ALS) patients with hypersalivation. Methods and Materials Fifty ALS patients that had medically failure pretreatment were included in this prospective study. RT was delivered through a conventional linear accelerator with 6-MV photons and 2 opposed beams fields including both submandibular glands and two-thirds of both parotid glands. Total RT dose was 10 Gy in 2 fractions (n=30) or 20 Gy in 4 fractions (n=20). RT efficacy was assessed with the 9-grade Sialorrhea Scoring Scale (SSS), recently prospectively validated as the most effective and sensitive tool to measure sialorrhea in ALS patients. Results At the end of RT, all patients had improved: 46 had a complete response (92% CR, SSS 1-3) and 4 had a partial response (8% PR, SSS 4-5). A significant lasting salivary reduction was observed 6 months after RT completion: there was 71% CR and 26% PR, and there was a significant SSS reduction versus baseline ( P −6 ). There was no grade 3 to 4 toxicity, and most side effects (34%) occurred during RT. Nine patients (18%) underwent a second salivary gland RT course, with a 3-months mean delay from the first RT, resulting in a SSS decrease (−77%). Both RT dose regimens induced a significant SSS decrease with no significant toxicity. There were, however, more patients with CR/PR in the 20-Gy protocol ( P =.02), and 8 of 9 patients (89%) receiving a second RT course had previously been treated within the 10-Gy protocol. Conclusion Radiation therapy of 20 Gy in 4 fractions is an efficient and safe treatment for ALS patients with sialorrhea. A shorter RT course (10 Gy in 2 fractions) may be proposed in patients in poor medical condition.

Summary Background Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder associated with respiratory muscle weakness and respiratory failure. Non-invasive ventilation alleviates respiratory symptoms and prolongs life, but is a palliative intervention. Slowing the deterioration of diaphragm function before respiratory failure would be desirable. We aimed to assess whether early diaphragm pacing could slow down diaphragm deterioration and would therefore delay the need for non-invasive ventilation. Methods We did a multicentre, randomised, controlled, triple-blind trial in patients with probable or definite ALS in 12 ALS centres in France. The main inclusion criterion was moderate respiratory involvement (forced vital capacity 60–80% predicted). Other key eligibility criteria were age older than 18 years and bilateral responses of the diaphragm to diagnostic phrenic stimulation. All patients were operated laparoscopically and received phrenic stimulators. Clinicians randomly assigned patients (1:1) to receive either active or sham stimulation with a central web-based randomisation system (computer-generated list). Investigators, patients, and an external outcome allocation committee were masked to treatment. The primary outcome was non-invasive ventilation-free survival, analysed in the intention-to-treat population. Safety outcomes were also assessed in the intention-to-treat population. This trial is registered with ClinicalTrials.gov, number NCT01583088. Findings Between Sept 27, 2012, and July 8, 2015, 74 participants were randomly assigned to receive either active (n=37) or sham (n=37) stimulation. On July 16, 2015, an unplanned masked analysis was done after another trial showed excess mortality with diaphragm pacing in patients with hypoventilation (DiPALS, ISRCTN 53817913). In view of this finding, we analysed mortality in our study and found excess mortality (death from any cause) in our active stimulation group. We therefore terminated the study on July, 16, 2015. Median non-invasive ventilation-free survival was 6·0 months (95% CI 3·6–8·7) in the active stimulation group versus 8·8 months (4·2–not reached) in the control (sham stimulation) group (hazard ratio 1·96 [95% CI 1·08–3·56], p=0·02). Serious adverse events (mainly capnothorax or pneumothorax, acute respiratory failure, venous thromboembolism, and gastrostomy) were frequent (24 [65%] patients in the active stimulation group vs 22 [59%] patients in the control group). No treatment-related death was reported. Interpretation Early diaphragm pacing in patients with ALS and incipient respiratory involvement did not delay non-invasive ventilation and was associated with decreased survival. Diaphragm pacing is not indicated at the early stage of the ALS-related respiratory involvement. Funding Hospital Program for Clinical Research, French Ministry of Health; French Patients' Association for ALS Research (Association pour la Recherche sur la Sclerose Laterale Amyotrophique); and Thierry de Latran Foundation.

International audience; PurposeThe mechanisms underlying the topography of motor deficits in spinal muscular atrophy (SMA) remain unknown. We investigated the profile of spinal cord atrophy (SCA) in SMN1-linked SMA, and its correlation with the topography of muscle weakness.Materials and MethodsEighteen SMN1-linked SMA patients type III/V and 18 age/gender-matched healthy volunteers were included. Patients were scored on manual muscle testing and functional scales. Spinal cord was imaged using 3T MRI system. Radial distance (RD) and cord cross-sectional area (CSA) measurements in SMA patients were compared to those in controls and correlated with strength and disability scores.ResultsCSA measurements revealed a significant cord atrophy gradient mainly located between C3 and C6 vertebral levels with a SCA rate ranging from 5.4% to 23% in SMA patients compared to controls. RD was significantly lower in SMA patients compared to controls in the anterior-posterior direction with a maximum along C4 and C5 vertebral levels (p-values < 10−5). There were no correlations between atrophy measurements, strength and disability scores.ConclusionsSpinal cord atrophy in adult SMN1-linked SMA predominates in the segments innervating the proximal muscles. Additional factors such as neuromuscular junction or intrinsic skeletal muscle defects may play a role in more complex mechanisms underlying weakness in these patients.

There is an unmet need for validated tools to measure sialorrhea in amyotrophic lateral sclerosis, especially to evaluate treatments. We assessed the inter-/intra-rate reviewer reliability of two scales: the Oral Secretion Scale (OSS), specifically developed for ALS patients, and the Sialorrhea Scoring Scale (SSS), initially developed for Parkinson's disease patients. Sialorrhea was rated in 69 ALS consecutive patients by four evaluators: two neurologists, one nurse and one speech therapist. Inter-rater reliability was evaluated by the light kappa coefficient and intra-rater reliability by the weighted kappa coefficient. We also compared patients' and caregivers' answers. Results demonstrated that the two scales present a high inter-/intra-rater reliability: weighted kappas were 0.85 for both scales and light kappas 0.89 for the OSS and 0.88 for the SSS. Both scales also showed a good intra-profession reliability (OSS kappa = 0.84; SSS kappa = 0.79) and agreement between patients' and caregivers' answers. The SSS showed a higher responsiveness compared to OSS. In conclusion, both Oral Secretion Scale and Sialorrhea Scoring Scale are reliable tools to measure sialorrhea in ALS patients. Because of the wide range of salivation degrees, SSS may be more sensitive as a tool to evaluate treatments in patients with severe hypersialorrhea.

Objective: To describe the clinical features at first evaluation that best predict survival of the amyotrophic lateral sclerosis (ALS) population from the Salpêtrière Hospital between 1995 and 2009. Methods: Data are collected and entered into a clinical database from all patients seen at the Paris ALS Center. Variables analyzed were demographic and baseline information, strength testing (manual muscle testing; 1995-2009), the revised ALS Functional Rating Scale (ALSFRS-R; 2002-2009) and survival status. The χ2 test and ANOVA assessed differences in variables by region and across time period. Univariate and multivariate Cox proportional hazards models determined which variables best predicted survival. Flexible modeling of continuous predictors (splines) assessed trends in survival for different variables. Results: 3,885 patients with ALS were seen in 1995-2009, of whom 2,037 had ALSFRS-R scores. Age, weight, strength, and site of onset varied by region of residence. The proportion of patients living outside Paris, the time to first visit, patient age, and motor function differed across time periods. In Cox models, site of onset, time to first visit greater than 18 months, strength and the year of visit after 2006 predicted survival (all p values Conclusions: Older age, bulbar onset, shorter delay to first visit and poor motor function at first visit predicted shorter survival rates in this large center-based sample from France, showing marked consistency across time and region of residence. Survival improved after 2006, concurrent with increasing rates of noninvasive ventilation use. Clinicopathologic correlation could better define subgroups, but identification of etiologies may be needed to elucidate individual forms of ALS with unique survival patterns.

The aim of this work was to determine whether survival changed during 2002–2009 at a French amyotrophic lateral sclerosis (ALS) center. We included all patients with ALS who were seen consecutively at the center from January 2002–May 2009. Participants were followed from date of first visit through death, date of censoring, or December 31, 2009, whichever occurred first. Cox proportional hazard models computed hazard ratios (HR; 95% confidence interval CI) of death, and flexible modeling of continuous predictors (splines) assessed trends in survival. We analyzed a total of 2,037 ALS patients, of whom 1,471 died before the end of follow-up. Median survival was 2.83 years from onset and 1.65 years from first visit. Compared to patients first seen before 2004, the HR of death was 0.97 (95% CI = 0.85–1.11, p = 0.6721) for patients first seen in 2004–2005, 0.96 (95% CI = 0.83–1.10, p = 0.5125) for 2006–2007, and 0.56 (95% CI = 0.46–0.69, p

Introduction Dans la Sclerose Laterale Amyotrophique (SLA), des anomalies de la transmission neuromusculaire (TNM) peuvent etre enregistrees en ENMG de surface, et compliquer la demarche diagnostique. Objectifs Nos objectifs etaient de determiner la frequence de ces anomalies, leur topographie, leur evolution au cours du temps, ainsi que leur correlation eventuelle avec les parametres cliniques et electrophysiologiques. Patients et methodes Onze patients atteints de SLA (âge 36–63 ans) ont ete etudies prospectivement. L’absence d’anticorps anti-RACh, anti-MuSK et anti-canaux calciques voltages dependants etait systematiquement verifiee. Deux explorations ENMG ont ete realisees : a l’inclusion (M0) et apres 3 mois d’evolution. L’evaluation de la TNM comprenait 2 parties : etude en stimulation nerveuse repetitive (SNR) a 3 Hz sur 10 couples nerf-muscle et test d’effort maximal bref sur les nerfs cubital et sciatique poplite interne. Resultats Apres SNR, un decrement ≥10 % (11–26 %, moyenne = 18 %) etait retrouve chez 45 % des patients a M0 et 33 % a M3, le plus souvent sur les couples radial-ancone (5/11) et fibulaire-tibial (4/11). L’amplitude du decrement etait correlee negativement a l’amplitude distale motrice enregistree du muscle concerne. Il n’y avait aucune correlation entre amplitude du decrement et duree d’evolution, severite ou taux de progression de l’atteinte fonctionnelle motrice. Aucune facilitation post exercice n’a ete observe. Discussion Une alteration de TNM significative peut etre detectee dans pres de 50 % des cas, sans argument en faveur d’une origine pre synaptique. L’amplitude du decrement est d’autant plus importante que l’amplitude motrice initiale (donc le nombre de fibres musculaires activees) est faible. Nous n’avons pas mis en evidence de lien entre l’amplitude du decrement et les differents parametres d’evolutivite clinique. Conclusion Un decrement significatif modere (generalement

Our objectives were to analyse carbohydrate metabolism in a series of ALS patients and to examine potential association with parameters of lipid metabolism and clinical features. Glucose tolerance was assessed by the oral glucose tolerance test in 21 non-diabetic ALS patients and compared with 21 age- and sex-matched normal subjects. Lipids and lactate/pyruvate ratio, levels of pro-inflammatory cytokines (tumour necrosis factor-alpha and interleukin-6) and adipocytokines (leptin and adiponectin) were also measured in ALS patients. Mann-Whitney U-tests analysed continuous data and Fisher's exact tests assessed categorical data. Blood glucose determined 120 min after the glucose bolus was significantly higher in patients with ALS (7.41 mmol/l+/-1.68) compared to controls (6.05+/-1.44, p=0.006). ALS patients with impaired glucose tolerance (IGT) according to WHO criteria (n=7, 33%) were more likely to have elevated free fatty acids (FFA) levels compared to patients with normal glucose tolerance (0.77 nmol/l+/-0.30 vs. 0.57+/-0.19, p=0.04). IGT was not associated with disease duration or severity. In conclusion, patients with ALS show abnormal glucose tolerance that could be associated with increased FFA levels, a key determinant of insulin resistance. The origin of glucose homeostasis abnormalities in ALS may be multifactorial and deserves further investigation.

There is accumulating evidence that ALS is a multisystem degenerative disease, raising the question of whether some symptoms are of extrapyramidal origin. The objective was to better characterize the type of stiffness in a series of ALS patients and to study correlations with balance and postural impairment. We studied ALS patients who exhibited stiffness without noticeable motor deficit in the lower limbs. We analyzed the response to manual stretch velocity to differentiate between extrapyramidal (rigidity) or pyramidal (spasticity) stiffness, shortening reaction in the tibialis anterior muscles (abolished in pyramidal diseases and exaggerated in extrapyramidal diseases) and balance and posture impairment (UPDRS and Berg's scale). After a preliminary inter-rater reliability study, 39 consecutive ALS patients were prospectively recruited. Each patient was randomly assigned to one or other of two investigators who evaluated either stiffness or balance impairment. In the lower limbs, rigidity was observed in at least one muscle in 27/39 patients (69%), and shortening reaction was exaggerated in 20/39 patients (51%). Both were correlated with balance and posture impairment (UPDRS and Berg's scale, P < 0.001). This study suggests that extrapyramidal involvement plays a role in stiffness and balance impairment in a subset of ALS patients.

Death represents the main hallmark of amyotrophic lateral sclerosis (ALS). Despite its importance in clinical care and phase III trials, many uncertainties remain on the cause of death due to the lack of post-mortem verifications. To provide a more robust approach to these causes, we performed a retrospective pathological study on a large cohort of patients. 100 ALS patients referred for a deterioration of their clinical condition and who died in the ALS clinic of Salpétrière had a complete macroscopic and microscopic post-mortem analysis. The clinical causes of death reported on medical records were compared to the results of autopsy. The concordance between clinical and pathological conclusions was insufficient (20%) to consider clinical assessment as a reliable marker of causes of death. At autopsy, broncho-pneumonia and pneumonia were the main causes of death. Heart failure, representing 10% of deaths, was two times more frequent in bulbar than in spinal ALS. Pulmonary embolism representing 6% of death was exclusively found in spinal onset patients and is related to lower limbs disability. An effort has to be made for a better understanding of the causes of deterioration of ALS patients. A more proactive attitude to treat respiratory infections could have a significant impact on survival.

Objective A proportion of patients with pure lower motor neuron syndrome (LMNS) progress to amyotrophic lateral sclerosis (ALS). Early detection of this progression is impossible, which delays the patient's access to treatment. Muscle expression of Nogo-A is a new candidate marker of ALS. We tested whether detection of Nogo-A in a muscle biopsy from patients with LMNS predicts progression to ALS. Methods Thirty-three patients who had undergone a muscle biopsy during the diagnostic workup of spinal LMNS were observed for 12 months. Nogo-A expression was measured by Western blot in muscle biopsy samples and compared with the final diagnosis. Results Nogo-A expression was detected in 17 patients and was absent in 16 patients. The detection of Nogo-A in muscle biopsy samples from LMNS patients correctly identified patients who further progressed to ALS with 91% accuracy, 94% sensitivity, and 88% specificity. In patients who later developed typical ALS, Nogo-A may be detected as early as 3 months after the onset of symptoms. Interpretation Nogo-A test is able to identify ALS early in the course of the disease when diagnosis is difficult, requiring further progression. Use of the test in clinical practice may shorten the delay before introduction of neuroprotective drugs or inclusion in clinical trials. Ann Neurol 2007

Resume La decouverte en 1993 d’une mutation du gene codant pour la superoxyde dismutase cuivre/zinc (SOD1) a permis de faire des progres importants dans la connaissance des mecanismes physiopathologiques de la maladie. Differents mecanismes sont actuellement impliques dans la degenerescence des motoneurones. L’excitotoxicite et les anomalies du metabolisme calcique est une des hypotheses les plus retenues. Elle a conduit au developpement du riluzole qui reste le seul traitement ayant montre une efficacite dans la maladie. Le role des facteurs de croissance reste controverse d’autant que tous les essais therapeutiques utilisant ces molecules sont restes negatifs. Les anomalies du stress oxydant sont objectivees par de nombreuses etudes mais leur application therapeutique reste encore hypothetique. L’apoptose, et le role probable des mitochondries, est definitivement confirmee et elle constitue une piste therapeutique importante des annees a venir.

International audience; The diagnostic of Amyotrophic lateral sclerosis (ALS) remains based on clinical and neurophysiological observations. The actual delay between the onset of the symptoms and diagnosis is about 1 year, preventing early inclusion of patients into clinical trials and early care of the disease. Therefore, finding biomarkers with high sensitivity and specificity remains urgent. In our study, we looked for peptide biomarkers in plasma samples using reverse phase magnetic beads (C18 and C8) and MALDI-TOF mass spectrometry analysis. From a set of ALS patients (n=30) and healthy age-matched controls (n=30), C18-or C8-SVM-based models for ALS diagnostic were constructed on the base of the minimum of the most discriminant peaks. These two SVM-based models end up in excellent separations between the 2 groups of patients (recognition capability overall classes > 97%) and classify blinded samples (10 ALS and 10 healthy age-matched controls) with very high sensitivities and specificities (>90%). Some of these discriminant peaks have been identified by Mass Spectrometry (MS) analyses and correspond to (or are fragments of) major plasma proteins, partly linked to the blood coagulation.

Neuropathy in Waldenstrom's macroglobulinemia (WM) is very heterogeneous. We retrospectively studied 40 patients with WM and neuropathy to analyze the different presentations and mechanisms encountered and to propose a diagnostic strategy. Twenty-five patients (62.5%) had axonal neuropathy, related to the following mechanisms: amyloid neuropathy (n = 5), cryoglobulinemic neuropathy (n = 5), neuropathy associated with tumoral infiltration (n = 2), vasculitic neuropathy (n = 2), a clinical motor neuropathy possibly of dysimmune origin (n = 6), or an unclassified mechanism (n = 5). A demyelinating pattern was observed in 15 patients, 10 having anti-myelin-associated glycoprotein (anti-MAG) antibodies and 5 having neuropathy related to chronic inflammatory demyelinating polyradiculoneuropathy. On the basis of these results, we propose a diagnostic strategy combining: (1) an EMG to distinguish between a demyelinating and an axonal pattern; (2) measurement of anti-MAG and anti-ganglioside antibodies; (3) screening for "red flag" features to orientate further investigations. This strategy may help clinicians to identify the mechanism of neuropathy in order to adapt the therapeutic strategy.

Background: Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset degenerative disease characterized by the loss of upper and lower motor neurons leading to progressive muscle atrophy and paralysis. The lack of molecular markers of the progression of disease is detrimental to clinical practice and therapeutic trials. Objective: This study was designed to identify gene expression changes in skeletal muscle that could reliably define the degree of disease severity. Methods: Gene expression profiles were obtained from the deltoid muscles of ALS patients and healthy subjects. Changes in differentially expressed genes were compared to the status of deltoid muscle disability, as determined by manual muscle testing, electrophysiology and the degree of myofiber atrophy. Functionally related genes were grouped by annotation analysis, and deltoid muscle injury was predicted using binary tree classifiers. Results: Two sets of 25 and 70 transcripts appeared differentially regulated exclusively in early and advanced states of deltoid muscle impairment, respectively. The expression of another set of 198 transcripts correlated with a composite score of muscle injury combining manual muscle testing and histological examination. From the totality of these expression changes, 155 transcripts distinguished advanced from early deltoid muscle impairment with 80% sensitivity and 100% specificity. Nine of these transcripts, known also to be regulated in ALS mouse and surgically denervated muscle, predicted the advanced disease status with 100% sensitivity and specificity. Conclusion: We provide robust gene expression changes that can be of practical use when monitoring ALS status and the effects of disease-modifying drugs.

Background Expanded GGGGCC hexanucleotide repeats in the promoter of the C9ORF72 gene have recently been identified in frontotemporal dementia (FTD), Amyotrophic Lateral Sclerosis (ALS) and ALS-FTD and appear as the most common genetic cause of familial (FALS) and sporadic (SALS) forms of ALS. Methods We searched for the C9ORF72 repeat expansion in 950 French ALS patients (225 FALS and 725 SALS) and 580 control subjects and performed genotype-phenotype correlations. Results The repeat expansion was present in 46% of FALS, 8% of SALS and 0% of controls. Phenotype comparisons were made between FALS patients with expanded C9ORF72 repeats and patients carrying another ALS-related gene ( SOD1, TARDBP, FUS ) or a yet unidentified genetic defect. SALS patients with and without C9ORF72 repeat expansions were also compared. The C9ORF72 group presented more frequent bulbar onset both in FALS (p

Our objective was to assess the spectrum and clinical associations of cognitive impairment in French patients with ALS, and determine the effect of cognitive impairment on survival in this population. One hundred and thirty-one patients were enrolled in a cross-sectional cohort study of neuropsychological test performance. ANOVA and χ(2) tests assessed differences in clinical characteristics between impaired and unimpaired patients; multiple regression determined which features contributed most strongly to cognitive status, and Cox models compared survival. Fifty-three patients (40%) were categorized as cognitively impaired based on test performance. Thirteen (10%) patients had frontotemporal dementia (FTD) clinically; all scored in the moderate to severely impaired range on testing. Impaired patients had less education (p = 0.001), and severely impaired patients were more likely to have bulbar onset than unimpaired patients (p < 0.001). Severe cognitive impairment predicted shorter survival (p = 0.007), even when controlled for motor severity (p = 0.001). In summary, 10% of a consecutive series of French ALS patients had overt dementia and 40% were cognitively impaired by neuropsychological testing. We conclude that lower education level and possibly bulbar-onset ALS were associated with impairment. As in other causes of dementia, higher education attainment may protect against clinical cognitive deterioration in ALS. French patients with severe cognitive impairment have shorter survival time.

Amyotrophic lateral sclerosis (ALS) is characterized by progressive denervation leading to muscle atrophy prevented, during the early phase, by compensatory reinnervation. Little is known about muscle fibre regeneration capacity in ALS. We have carried out in vivo and in vitro investigation of skeletal muscle in ALS. Seven ALS patients underwent a deltoid muscle biopsy. Immunohistochemical analysis revealed various degrees of denervation- and reinnervation-related changes in the ALS muscle biopsies including satellite cells (SCs) activation and regenerating fibres. Only 3/7 primary cultures of ALS muscle cells were successfully established and had sufficient myogenicity, as assessed by desmin positivity, to be used without further purification. This was in contrast with the cultures derived from control muscles, predominantly desmin-positive cells. Although capable to proliferate in vitro, ALS-derived SCs presented an abnormal senescent-like morphology. Markers of senescence, including senescent-associated (SA)-βGal activity and p16 expression, were increased. Furthermore, ALS-derived SCs were also unable to fully differentiate in vitro as shown by abnormal myotubes morphology and reduced MHC isoform expression, compared to control myotubes. Our study suggests that SC function is altered in ALS. This could limit the efficacy of compensatory processes and therefore could contribute to the progression of muscle atrophy and weakness.

We carried out a retrospective multicentre study to assess the safety of home parenteral nutrition (HPN) in patients with ALS. We reviewed the case records of patients from French ALS centres treated with HPN by central venous catheter (CVC) using an implantable port between January 2005 and October 2009. Seventy-three patients received HPN for a total of 11,908 catheter days. Twenty-seven patients experienced a total of 37 CVC related complications resulting in an incidence rate of 3.11 CVC complications/1000 catheter days, including 1.93 septic complications and 1.09 mechanical complications/1000 catheter days. Metabolic complications were frequent but without serious consequences on mortality. The use of the catheter for intravenous therapies in addition to HPN was identified as a septicaemia's risk factor (relative risk (RR) = 2.54, confidence interval (CI) 1.56-4.14, p = 0.04). In conclusion, HPN is an alternative procedure to PEG in advanced ALS patients. The incidence of complications appears to be comparable to data from the literature on HPN in other diseases. A prospective study comparing HPN and radiologic inserted gastrostomy (RIG) would allow comparison of the relative risk-benefit and survival of these procedures. The relation of CVC and RIG placement timing and the complications' occurrence should also be investigated.