Statistical analysis
death, ICH, ALS, Treg%, ALSFRS-R
REGRESSION, DISEASE
Sample size was calculated using data from an Italian study showing that ALS patients have a decreased number of circulating Treg% (mean ± SD: 2.1 ± 0.7) if compared to healthy controls (2.6 ± 0.6), except for slow progressorsSafety analyses were performed including all patients who received at least one tablet of rapamycin or placebo. All AEs, SAEs, and AEs leading to treatment discontinuation were recorded according to ICH Guidelines, listed, and compared in the treatment arms at any follow-up visit and at the end of the study.The primary population for analyses was the ITT population, which included all the participants who received at least one tablet of the investigation drug. PP analysis was performed after excluding patients as per major protocol deviations (i.e., patients who took <80% therapy) from the above-mentioned population. Descriptive statistics comparing the two groups of rapamycin treatment and placebo was performed using mean and standard deviation for continuous variables, counts and percentages for categorical variables. Immune response to rapamycin was analyzed as the difference in positive response to rapamycin (mean Tregs increase exceeding 30%) between the placebo group and the rapamycin groups Results were expressed as the relative risk (RR) comparing treatment arms. The comparison was carried out with a chi-square test without any correction. Mean absolute differences from baseline to week 18 and other time points among treatment arms for S6RP phosphorylation, of different T, B, NK cell subpopulations, of biomarkers, inflammasome, cytokines, were calculated and compared using linear regression models that include indicator variables for treatment arms as the independent variables. Results were expressed as the mean difference (MD) comparing treatment arms. Correlations between numerical variables were calculated with the Pearson’s linear correlation coefficient. The time to death, tracheostomy or permanent ventilation, were compared by using the log-rank test. Adjustment for main prognostic or unbalanced factors (namely sex, months from symptoms onset, ALSFRS-R slope at baseline and edaravone treatment) has also been performed using logistic or linear regression models. Results of logistic models were expressed as the OR comparing treatment arms.A segmented repeated measures linear mixed model analysis was carried out to assess whether the average monthly variation from baseline in selected numerical outcomes was different amongst treatment arms in two segments of time: during the treatment (after baseline and up to week 18), or after the treatment (after week 18). The dependent variables were the raw measurements of the outcomes, whereas the independent variables were: arm, time (months from baseline) × period (during or after treatment) interaction, and arm × time × period interaction. A random intercept term was also used to account for repeated measurements over the same individual, as well as a random slope term was used to account for individual linear variations over time. Random intercept and random slope terms were kept if they improved the overall goodness-of-fit of the models (assessed using the Akaike information criterion). Results of this analysis were expressed as: the monthly variation of outcomes in the placebo group; the MD in the monthly variations, comparing treatments arms vs placebo. Both these quantities were reported for the two segments of time (during and after treatment). In the analysis of average monthly variation of ALSFRS-R, three segments of time were analyzed: before, during and after treatment, by considering two additional timepoints such as the prebaseline screening visit and the disease onset. At disease onset, the ALSFRS-R was assumed to be equal to 48 for all patients. The repeated measures linear mixed model for ALSFRS-R included arm, time (months from baseline), period (before, during or after treatment), and all their pairwise and three-way interactions. The intra-group differences comparing ALSFRS-R monthly variations in the periods during and after treatment with those occurred before treatment were also reported for each arm, as well as the comparison between treatment arms and placebo in these quantities. A post hoc analysis on tracheostomy-free survival from baseline to 31st December 2021 was performed.For the comparisons between Rapamycin 1 mg/mAnalyses were performed using STATA software, version 15 (StataCorp. 2017. Stata Statistical Software: Release 15. College Station, TX: StataCorp LLC) and R software, version 3.6.3 (The R Foundation for Statistical Computing, Wien).