ALT in Pediatric Brain Tumors Can Occur without ATRX Mutation and is Enriched in Patients with Pathogenic Germline MMR Variants

Background To achieve replicative immortality, most cancers develop a telomere maintenance mechanism, such as reactivation of telomerase or alternative lengthening of telomeres (ALT). There are limited data on the prevalence and clinical significance of ALT in pediatric brain tumors, and ALT-directed therapy is not available. Methods We performed C-circle analysis (CCA) on 586 pediatric brain tumors that had corresponding tumor/normal whole genome sequencing through the Open Pediatric Brain Tumor Atlas (OpenPBTA). We detected ALT in 6.8% (n=40/586) of these tumors and completed additional validation by ultrabright telomeric foci in situ on a subset of these tumors. We used CCA to validate TelomereHunter for computational prediction of ALT status and focus subsequent analyses on high-grade astrocytic tumors (HGAT). Finally, we examined whether ALT is associated with recurrent somatic or germline alterations. Results ALT is common in pediatric HGAT (n=24/63, 38.1%), but occurs infrequently in other pediatric brain tumors (<3%). Somatic ATRX mutations occur in 50% of ALT+ HGAT and in 30% of ALT-HGAT. Rare pathogenic germline variants in mismatch repair (MMR) genes are significantly associated with an increased occurrence of ALT. Conclusions We demonstrate that ATRX is mutated in only a subset of ALT+ HGAT, suggesting other mechanisms of ATRX loss of function or alterations in other genes may be associated with the development of ALT in these patients. We show that germline variants in MMR are associated with development of ALT in patients with HGAT. Key Points ATRX alterations are frequent, but not required, for an ALT phenotype in HGATs HGAT patients with germline mismatch repair variants have higher rate of ALT tumors TelomereHunter is validated to predict ALT in high-grade astrocytic tumors Importance of the Study We performed orthogonal molecular and computational analyses to detect the presence of alternative lengthening of telomeres in a highly characterized cohort of pediatric brain tumors. We demonstrate that many high-grade astrocytic tumors utilize ALT without a mutation in ATRX , suggesting either loss of function of ATRX via an alternative mechanism or an alternate means of development of ALT. We show that germline variants in MMR genes are significantly associated with ALT in HGAT. Our work adds to the biological understanding of the development of ALT and provides an approach to stratify patients who may benefit from future ALT-directed therapies in this patient population. ### Competing Interest Statement Dr. Angela J. Waanders is a member of the Scientific Advisory boards for Alexion and DayOne Biopharmaceuticals.