LAUSR

Glioblastoma (GBM) is the most aggressive primary brain cancer. Identifying novel markers which stratify patients and predict response to targeted therapies is a priority. Telomeres are caps on chromosome ends protecting them from aberrant DNA damage response. Telomere length (TL) alterations and fusions are key markers for genomic instability and high-resolution telomere analysis provides powerful prognostic and predictive information in several tumour types. We therefore studied telomeric profiles and fusions in GBM patients and related this to clinical outcome. Tumours from 151 adults with isocitrate dehydrogenase-wildtype GBMs were analysed for TL using the precision High-Throughput Single Telomere Length Analysis technique. 67 patients underwent further multiplex long-range polymerase chain reaction to identify fusions via Southern hybridisation using telomere-adjacent 33P radioisotope-labelled probes. Telomere fusions from 10 tumours that exhibited the highest frequency of fusions were characterised with long-read Nanopore sequencing. GBMs display short telomeres with a median length of 4.0kb. Shorter TLs in patients with unmethylated O6-methylguanine DNA methyltransferase status (commonest current biomarker) conferred worse survival (HR 2.13, p = 0.002). 66/67 (98.5%) patients analysed revealed telomeric fusions. Nanopore sequencing validated these events, while confirming the presence of microhomology and telomeric deletion. This mutational spectrum is consistent with utilisation of specific DNA repair pathways in mediating telomere fusion. This study provides the first mechanistic evidence of dysfunctional telomeres in adult GBMs and suggests TL analysis may provide independent prognostic information. The presence of telomere fusions indicates significant genomic instability and may favour targeted therapies for patients exhibiting this signature.