Dottorato in MORFOGENESI E INGEGNERIA TISSUTALE

Titolo della tesi: UNVEILING ALS PHENOTYPIC CHARACTERISTICS: TOWARDS A NOVEL MODEL FOR UTILIZING SYNTHETIC PLACEBO POPULATIONS IN RANDOMIZED CLINICAL TRIALS

BACKGROUND We have investigated the idea of creating a population of virtual patients to serve as a comparison group in clinical trials. To do this effectively, it is essential to develop a realistic model that captures the key characteristics of patients with ALS. Recent advancements in genetics technology have allowed to analyze specific gene mutations. Additionally, certain environmental factors, such soccer, have been implicated as potential risk factors for ALS. Understanding and accurately modelling these characteristics would allow for more accurate trial design, and potentially reduce the need for a placebo arm. METHODS - Use of synthetic placebo populations in ALS randomized clinical trials Sample subsets were extracted from the MND register using an evolutionary algorithm to match baseline variables with a target trials group, with additional adjustments to mimic real recruitment practices. A survival prediction model was used to estimate survival for the target population. The survival of the synthetic placebo population was compared to that of real trial placebo arms using Kaplan-Meier analysis and log-rank tests. Two target populations were studied: participants enrolled in the LiCALS trial and individuals included in the PRO-ACT clinical trials database. -Analysis of genetic characteristics A total of 194 patients were enrolled. All exons of the SOD1 gene were sequenced. Hexanucleotide intronic repeat expansions (G4C2) in the C9orf72 gene were tested. Whole Exome NGS sequencing was performed. An in silico multigene panel targeted for neuromuscular diseases, and other diseases was used to filter the mutations found after WES sequencing. Nonparametric tests, Kaplan-Meier survival curves and log-rank tests were employed to evaluate differences between groups. - Prevalence of ALS cases among soccer players All professional soccer players practicing in the period 1946-2015 were identified. ALS cases diagnosed among football players during the specified period were considered to calculate disease incidence. The expected incidence rate was calculated as the number of ALS cases per 100,000 person-years expected in the cohort. The standardized incidence ratio (SIR) was then calculated as the ratio between observed and expected cases. RESULTS - Use of synthetic placebo populations in ALS randomized clinical trials The synthetic placebo groups generated from the MND register closely matched the target trials outcomes. The ENCALS model produced synthetic placebo groups that differed significantly from real placebo groups. However, when participants were censored at 6-month intervals, the ENCALS synthetic group closely matched the target group between 24 and 48 months, indicating a potential timeframe for utilizing this method. - Analysis of genetic characteristics Clinically significant pathogenetic variants were detected in 14.43% of cases. The highest prevalence of pathogenetic variants was observed in fALS patients, but a substantial proportion of sALS patients also displayed at least one mutation, either pathogenetic or of uncertain significance (VUS). The most observed pathogenetic mutation was the expansion of the C9orf72 gene, which was associated with a shorter survival. SOD1 mutations were found in 1.6% of fALS and 2.5% of sALS patients. - Prevalence of ALS cases among soccer players A total of 14 cases of ALS were identified. A significant decrease in the risk of ALS diagnosis was observed, with an SIR of 0.407 (95% CI 0.163-0.838). Individuals who initiated their careers prior to 1950 exhibited a higher reduction in risk. The period between 1990 and 2015 showed the highest increase in risk. The risk reduction did not reach statistical significance within the different age groups considered. Former soccer players experienced the onset of the disease approximately 10.8 years earlier than expected. DISCUSSION - Use of synthetic placebo populations in ALS randomized clinical trials It is possible to use filters and an evolutionary algorithm to match the demographics of people in a dataset collected at a population level to people in the placebo arm of a trial. Using a prognostic model to predict survival generates comparable estimates to people in the active arm of trials. The implementation of a prognostic model for survival prediction yields comparable results to individuals in the active arm of trials. Notably, both the register-based approach and the ENCALS prognostic model have successfully generated synthetic placebo groups that closely resemble those observed in historical trials - Analysis of genetic characteristics The study reveals a significant number of ALS patients carrying pathogenic mutations. The expansion of the C9orf72 gene is the most common, affecting familial and sporadic cases. SOD1 mutations are detected at an unexpectedly higher rate, even in patients without a sALS, underscoring the crucial role of genetic testing in treatment decisions and potential participation in clinical trials. We also investigated mutations in other genes, shedding light on their potential involvement in ALS. These findings underscore the complexity of interpreting variants of uncertain significance (VUS) and their ethical implications in patient communication and genetic counseling for patients' relatives. - Prevalence of ALS cases among soccer players The observed pattern, indicating a lower risk for players who began their careers before 1950 and a slightly higher risk for those who started between 1990 and 2015, may be influenced by certain biases. As the intensity and demands of the sport have evolved over time, it is possible that these changes have had an impact on the health outcomes of players. The former soccer players were diagnosed with ALS at an earlier stage compared to the general population. This finding suggests that the combination of high-intensity exercise and the overall strain on the body may potentially accelerate the onset of ALS in individuals who are predisposed to the disease. CONCLUSION The study demonstrated the potential of using ALS as a model disease to address challenges in clinical trials. We have shown that two different strategies can be used to create synthetic placebo populations: matching people from population registers and predicting non-treatment survival of people enrolled in trials. However, these results need to be replicated with new trial data It is of utmost importance to underscore the diverse genetic foundation of ALS and advocate for the integration of comprehensive genetic testing into diagnostic protocols. The presence of VUS serves as a reminder of the multifaceted nature of ALS genetics, thereby prompting further exploration into the intricate interactions among genetic variations, environmental factors, and the development of the disease. The discovery that soccer players have a lower risk of developing ALS in comparison to the general population highlights the significance of acknowledging the limitations inherent in these findings during interpretation. To obtain a more precise representation of ALS incidence among soccer players, it is crucial to involve validated medical records and consider the data's origin in subsequent investigations.