Beyond ATRX: The heterogeneous basis of Alternative Lengthening of Telomeres in osteosarcoma

The alternative lengthening of telomeres (ALT) pathway is a telomerase-independent telomere maintenance mechanism, which is frequently observed in osteosarcoma. In this study, we performed a systematic review of studies assessing ALT in patient-derived osteosarcoma samples using direct experimental assays, alongside bioinformatic analysis of large public genomic datasets to evaluate the prevalence of canonical ALT-associated alterations. Across seven eligible studies comprising 342 tumours, ALT activity was identified in 62.9% of cases. In contrast, analysis of the Target-OS and PeCan datasets demonstrated that mutations in ATRX or DAXX, and amplification of TOP3A, occur at substantially lower frequencies, collectively accounting for only a minority of tumours. Statistical comparison confirmed a significant discrepancy between experimentally-observed ALT prevalence and the frequency of these canonical ALT-associated genetic alterations. These findings indicate that canonical genomic events are insufficient to explain ALT activation in osteosarcoma and support a model of marked biological heterogeneity. Potential alternative mechanisms include non-mutational disruption of chromatin remodelling pathways, replication stress-associated processes, and epigenetic dysregulation. The data highlights the limitations of relying on sequencing-based approaches alone to infer telomere maintenance mechanisms, which could have important clinical consequences for potential ALT-pathway targeting therapies. In conclusion, ALT is highly prevalent in osteosarcoma but is not adequately captured by known genetic correlates. Functional assays remain essential for accurate classification, and improved understanding of non-canonical ALT drivers will be critical for biomarker development and the design of targeted therapeutic strategies.