The first periods of the project, from September 2015 to June 2017, was dedicated to the clinical trial implementation, including regulatory and Ethics developments. Overall, 17 Clinical Centres were contracted, trained, and activated. All necessary tools for running the trial were developed, assembled, and distributed to all Clinical Centres. A Central laboratory (Biocytex, Marseille, France) was contracted to perform all core biomarker determinations in blood and CSF. SOPs for sampling and pre-analytical processing were drafted and centres trained to SOPs. For treatment preparation, a CMO was contracted (BathASU, Bath, UK), and SOPs for treatment logistics and handling drafted. In summary, all necessary tools to comply rigorously with regulatory constraints where set in place, and subsequently find appropriate by a contracted Independent Audit body (Qualilab, France).
From June 19th 2017 to October 29th 2019 (end of patients’ recruitment period), 17 centres included 304 patients of whom 220 were randomised (last randomised January 2020). All randomised patients were subsequently followed-up 3-monthly for 640 days until 30th October 2021 (cut-off date).
Over the trial period, at 2-weekly intervals, treatments packages were ordered by Centres, manufactured by CMO, transported to centres, and dispensed on a per-patient/per-cycle basis. By the end of the study, 3,678 individual treatment packages had been GMP manufactured, logistically managed and dispensed to patients.
Trial safety was monitored throughout the study by an Independent Data-Safety Monitoring board (DSMB). For the five DSMB Trial-safety assessments done, database management was performed, and data files ready for analysis were blindly prepared for the Independent DSMB statistician.
A biobank was constituted with (i) 30,800 plasma aliquots collected from 1,064 visits over 220 patients, (ii) 19,600 CSF aliquots from 613 visits over 220 patients, (iii) 8,200 PBMCs aliquots from 1031 visits over 220 patients.
For fresh-blood cytometry, 1161 blood samples were collected and analysed in real time. Data-bank on cytometry has been completed and database validation completed by July 2022. In addition, laboratory assays for deep immune & inflammatory phenotyping were also performed on biobank-samples. As of the October 2022, related tasks are completed (cytokine/chemokine database) or nearing completion (comprehensive cytometry).
For the analysis of Brain biomarker, we used antibodies (Abs) and immune complexes capture methods designed to test the humoral response to neurofilaments to two large ALS cohorts from the ALS biomarkers study, UK, and the Phenotype Genotype Biomarker study (CReATE), US. The results have shown that the Abs response to specific neurofilament isoforms can be used for clinical stratification of ALS. As of 30th September 2022 the data on the characterization of circulating protein aggregates in blood and their content in brain proteins including neurofilaments have been completed and statistical analysis ongoing. In addition free NFL determination was performed in blood and CSF (completed September 2022).
For genomics and transcriptomics analysis, preliminary activities were conducted to see what genes are expressed in the blood in response to treatment with riluzole and a low dose of interleukin-2 (ld-IL2) over a period of time, and to screen the patients for rare variants sequences in genes associated with ALS or T-lymphocyte regulation (the process the IL2 treatment is targeting). A total of 1,144 samples were processed for RNA extraction with quality controls showing a good RNA yield from all 17 centres. As of September the 30th 2022 the gene expression profiling over the entire cohort has been determined and statistical analysis is ongoing. In parallel, DNA extraction was performed for the 220 randomised patients and extracted DNA genotyping is now completed, and statistical analysis ongoing.
Last treatment for last patient in study was administered on June 28th 2021, and last status determined on the 30Th October 2021. Clinical Database cleaning was completed by July 13th 2022, allowing database lock and statistical analysis, which is ongoing as of October 2022.
In summary, MIROCALS project has been successful in setting-up and conducting to its planned end, a large randomised clinical trial recruiting 220 de novo ALS patients. All project defined parameters, clinical or laboratory, were successfully collected in a database, and as of the 30th September 2022, are undergoing statistical analysis. This unique resource should now help respond to all defined project objectives and beyond.
