Data-driven subtyping and staging of ALS: A multicenter, longitudinal, deformation-based morphometry study

Amyotrophic lateral sclerosis (ALS) is clinically and biologically heterogeneous, yet data-driven imaging subtyping approaches have rarely been validated longitudinally or linked to clinical and survival outcomes. We aimed to identify and validate distinct ALS subtypes and disease stages using deformation-based morphometry (DBM) and the Subtype and Stage Inference (SuStaIn) model, and to characterize their cross-sectional and longitudinal imaging, clinical, cognitive, and survival profiles. Data from 198 ALS patients and 144 healthy controls in the Canadian ALS Neuroimaging Consortium (CALSNIC) multicenter cohort were analyzed. Baseline regional DBM w-scores from 14 ALS-relevant regions served as input to SuStaIn to infer subtypes and stages. Longitudinal consistency of subtype and stage assignments (e.g. adherence to the expected disease evolution) was assessed using follow-up visits. Imaging and clinical trajectories were compared across subtypes using linear mixed-effects models incorporating stage and elapsed time. Associations between longitudinal variables and SuStaIn stage were estimated using mixed models, while baseline clinical and cognitive differences were assessed with ordinary least squares regression. Survival differences were evaluated using Kaplan-Meier curves and log-rank tests. SuStaIn identified one normal-appearing group (S0) and three ALS atrophy subtypes. S0 showed no baseline atrophy but exhibited longitudinal motor decline and the most favorable survival (log-rank p < 0.05 to p < 0.01). S1 exhibited classical motor/corticospinal tract-dominant degeneration, greater lower motor neuron burden, and intermediate survival. S2 showed limbic-onset atrophy progressing toward motor pathways, with preserved cognition and a milder course. S3 demonstrated extensive fronto-parietal and striatal atrophy, longitudinal motor-thalamic degeneration, and the shortest survival. Subtype and stage assignments demonstrated high longitudinal consistency (>90%). SuStaIn stage was strongly associated with widespread brain atrophy (and ventricular expansion), with the strongest effects in limbic-subcortical regions. Stage also correlated with ALS Functional Rating Scale-Revised (ALSFRS-R) decline and forced vital capacity (FVC) reduction, indicating that stage reflects disease-linked progression. This study establishes a robust, longitudinally validated model of ALS heterogeneity, showing that SuStaIn-derived subtypes define distinct disease trajectories, whereas the normal-appearing group reflects an early, structurally preserved state with a more favorable survival profile. By integrating probabilistic staging with longitudinal modeling, these findings clarify dynamic subtype-specific progression patterns and support the use of SuStaIn for biologically informed patient stratification, prognostication, and clinical trial enrichment in ALS.