Statistics.
INS, RECURRENCES, RECURRENCE, SECONDARY, REMISSION, REGRESSION
The sample size was calculated based on the following assumptions: the primary purpose of this study was to demonstrate the safety and tolerability of intravenous MSC infusion and to ascertain preliminary efficacy parameters. The efficacy in reducing INS recurrence was the main outcome for sample size estimation, i.e., the first secondary efficacy endpoint. Patients entering the study were expected to have a 100% rate of INS recurrence after steroid/immunosuppressive therapy tapering/withdrawal. Our working hypothesis was that MSC infusion could decrease the expected rate of recurrence by at least 30%. On the basis of this assumption, the inclusion of 18 patients would confer to the analyses a 90% power to demonstrate a statistically significant reduction (α = 0.05, 1-tailed test) in the incidence of recurrences observed after MSC infusion (70% or less) compared with the 100% expected during treatment tapering/withdrawal without previous MSC infusion. To account for a 10% dropout rate, 20 patients should be included in the study. Due to the COVID-19 pandemic, a variety of logistical difficulties emerged in February 2020, particularly in Bergamo where all face-to-face visits and nonemergency medical procedures were interrupted between February and June 2020. At the onset of the pandemic, all 10 pediatric patients had been treated in Rome, while in Bergamo, 8 young adults had been enrolled and 5 treated. When non-emergency medical procedures were restarted in June 2020, 2 enrolled patients had lost inclusion criteria or acquired an exclusion criterion, while 1 adult patient was treated according to protocol. Therefore, due to the unforeseeable emergency, the study treated 16 patients overall. All of them relapsed during the subsequent observation period. This was discussed internally with our statisticians. Based on our initial sample size calculation, we calculated that the inclusion of 15 patients would confer an 80% power to demonstrate a statistically significant reduction (α = 0.05, 1-tailed test) in the incidence of recurrences observed after MSC infusion (70% or less) compared with the 100% expected during treatment tapering/withdrawal without previous MSC infusion. Therefore, with 16 patients we would have greater than 80% power to demonstrate a statistically significant reduction in the incidence of recurrences after MSC infusion. Considering that this parameter was a secondary end-point of this phase I study, whose primary endpoint comprised safety and feasibility, we concluded enrolment and considered the sample size obtained adequate.Analyses are presented for the intention-to-treat population, defined as all patients treated with autologous BM-MSCs in the study (The Kaplan-Meier method was used to plot the probability of experiencing a relapse. Survival times for each patient were calculated starting from first MSC infusion to the occurrence of the first relapse of NS. In an exploratory analysis, an unmatched Cox’s regression model and a stratified Cox’s regression for matched pairs between the year before versus the year after of first MSC infusion by PROC PHREG (SAS Institute Inc.), using the matching variable “patient” as the STRATA statement was carried out. Within-group comparisons were made using nonparametric Wilcoxon’s signed-rank test or Fisher’s exact test, as appropriate. The therapeutic efficacy of BM-MSC treatment was also scored by evaluating the sum of number of relapses and number of immunosuppressive drugs in the 12 months preceding and 12 months after BM-MSC treatment for each patient. Mean natural course of systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), total cholesterol, triglycerides, body weight, height and glucose are presented in table format over time (To assess efficacy in a post hoc analysis, we created a scoring system to integrate in a single measurement the cumulative effects of reducing the number of relapses in the previous and subsequent 12-month periods and the number of immunosuppressive drugs needed to maintain remission at time of BM-MSC infusions and at +12 months. To this end, we simply cumulated these 2 values for each patient at time 0 and at +12 months from BM-MSC infusions.Analyses and graphical representations of results were carried out using SAS version 9.4 (SAS Institute Inc.), STATA version 15 (StataCorp), and GraphPad Prism 9.0.We used an electronic case report form (eCRF) to collect data in this study, providing a platform for data insertion in a password-encrypted fashion only to authorized study personnel. After study completion, following written authorization by the study principal investigator, the eCRF database was locked. Extracted data was sent in a password-encrypted fashion to the principal investigator, converted into SAS-compliant format for statistic evaluations, and saved onto a pen drive kept among the study material.