GENO-29MUTATIONAL DIVERSITY UNDERLYING TELOMERE MAINTENANCE MECHANISMS IN AN APPRECIABLE FRACTION OF ADULT GLIOMAS

Abstract

Somatic alterations in the TERT promoter and in ATRX facilitate telomere maintenance and permit glial cells to escape replicative senescence. We assessed the mechanism of telomere maintenance in 452 infiltrative adult gliomas (WHO Grades II-IV) from the UCSF Adult Glioma Study. Hotspot mutations in the TERT promoter were detected by Sanger sequencing, while ATRX mutations were identified by immunohistochemistry. In total, 267 gliomas were classified as “TERT-mutated” (59%), 127 as “ATRX-mutated” (28%), and 58 as “TERTwt/ATRXwt” (13%). To identify the mechanism of telomere maintenance in these 58 TERTwt/ATRXwt gliomas, we designed a custom-sequencing panel targeting 36 genes with known roles in telomere biology. Parallel high-depth sequencing was performed, covering introns, exons and 15kb flanking regions on 5′ and 3' ends. Ten TERTwt/ATRXwt glioma specimens and matched-normal controls have been sequenced to-date, to an average depth of 250X. In 2 cases we identified a missense mutation of ATRX (p.R2197C and p.H1767D). In-silico functional analyses predict these to be highly-damaging mutations. This was confirmed by FISH, which revealed the presence of the Alternative Lengthening of Telomeres (ALT) phenotype. These ATRX mutations are located in a helicase ATP-binding and a helicase C-terminal domain, respectively, and likely lead to the expression of a non-functional ATRX protein that appears wild-type by IHC. We also identified a subclonal TERT C250T mutation in a 47 year-old glioblastoma patient with no IDH mutation. A fourth case had several somatic alterations of potential interest, including a highly clonal mutation in the Regulator of Telomere Elongation Helicase 1 (RTEL1) gene. Results from the first ten TERTwt/ATRXwt gliomas are promising and suggest a greater diversity of mutation types than can be revealed through simple Sanger sequencing or immunohistochemistry. Extension of these preliminary findings to a larger number of TERTwt/ATRXwt glioma cases is ongoing.