Functional Assessment of ATRX Alterations Improves Glioma Diagnosis

Loss of ATRX function, a diagnostic criterion of IDH-mutant astrocytoma, is closely associated with alternative lengthening of telomeres (ALT), a telomere maintenance mechanism (TMM). As immunohistochemical (IHC) assessment of ATRX is error-prone, sequencing has been integrated into clinical workflows. While frameshift and nonsense variants can be classified as loss-of-function (LOF) mutations, missense variants remain difficult to interpret.To address this, we analyzed ATRX -altered gliomas from TCGA (N = 539 tumors, 591 alterations) and a local cohort of 100 diffuse gliomas. Aside from IDH-mutant astrocytomas and H3.3-mutant gliomas, glioblastoma (12–18%), oligodendroglioma (2%), and MAPK-altered tumors (1–6%) were consistently represented in both cohorts. Missense mutations accounted for 18% (TCGA) and 29% (local) of variants. Functional annotation of TCGA missense mutations using IHC, NGS-based TMM status, transcript levels, and RNA-seq–derived telomerase signatures (EXTEND) allowed us to functionally annotate only 3 of 106 variants. Therefore, we directly assessed the TMM status in the local cohort, using the TeloDIAG assay and retrieved 80% of ALT-positive samples, corresponding to 96% (N = 53) of IDH-mutant astrocytomas and 79% (N = 19) of histone-mutant gliomas, as expected. Importantly, ALT activity was also observed in 39% (N = 18) of glioblastomas and 83% (N = 6) of MAPK-altered gliomas, indicating potential implications for clinical behavior and therapeutic targeting.Overall, 68% (N = 28) of missense mutations were functionally classified as LOF. Notably, 83% (N = 24) of them were located within the major functional domains of the ATRX protein, compared to only 59% when considering all missense mutations, highlighting the functional relevance of the assay. As compared to TeloDIAG, IHC showed limited sensitivity (74%), dropping to 44% (N = 18) in cases with missense mutation.Collectively, these findings emphasize the clinical value of functional TMM assessment in refining glioma diagnosis. Last, targeting of ALT-associated DNA-damage deficiency and immunogenic context is under clinical investigation, highlighting the promising theranostic potential of TMM assessment.