Functional analysis of telomere maintenance mechanisms is more informative than immunohistochemistry for ATRX mutation interpretation in Gliomas

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, 587 alterations) and a local cohort of 100 diffuse gliomas. Aside from IDH-mutant astrocytoma and H3.3-mutant glioma, glioblastoma (13–19%), oligodendroglioma (2%), and MAPK-altered tumors (2–7%) were consistently represented in both cohorts. Missense mutations accounted for 18% (TCGA) and 28% (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 74% (N = 19) of histone-mutant gliomas, as expected. Importantly, ALT activity was also observed in 42% (N = 19) of glioblastomas and 86% (N = 7) of MAPK-altered gliomas, indicating potential implications for clinical behavior and therapeutic targeting. While sensitivity for LOF variant detection was similar—88% for TMM and 80% for IHC—TMM performed better for missense alterations (85 vs. 65%) and is more discriminant in overall survival prediction. Overall, 83% (N = 24) of missense mutations associated with an ALT phenotype 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. 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.