QLTI-06. NEXT GENERATION SEQUENCING SIGNIFICANTLY IMPROVES THE DIAGNOSTIC ACCURACY IN GLIOBLASTOMAS

Abstract The 2021 WHO Classification of CNS Tumours recognizes the presence of TERT promoter (pTERT) mutations, combined +7/-10 chromosome copy-number alterations, or EGFR amplification as a defining molecular feature(s) of glioblastoma, independent of histology. We evaluated these biomarkers in a cohort of Indian patients using a single-run next-generation sequencing (NGS) platform at a relatively low depth of 100. Seventy-four adult diffuse gliomas were selected from the Exsegen Genomics Research Pvt Ltd Biobank established during the trial CTRI/2021/09/036861 after reviewing their clinical and pathological data. Paired surgical tissue and blood samples underwent whole-exome sequencing on an Illumina NovaSeq-6000 (Illumina, San Diego, USA). Single-nucleotide variations (SNVs) were processed using the DRAGEN pipeline to generate Mutation Annotation Format (MAF) files. Copy number variations (CNVs) analysis was performed using GATK and GISTIC2.0. Thirty-nine glioma samples with IDH1R132H mutations were excluded. A total of 32/35 (91.4%) IDH-wildtype tumours met the 2021 WHO criteria for glioblastoma, IDH-wildtype and were included. Twenty-four glioblastomas also underwent whole-transcriptome analysis. Microvascular proliferation (n=24, 75%) and necrosis (n=30, 93.9%) were present in the majority. pTERT mutations were identified in 23 (71.9%) samples, with C228T mutations (n=15/23, 65.2%) being more common than C250T mutations (n=8/23, 34.8%). EGFR amplification, defined as ≥5 copies by NGS CNV, was seen in 15 (46.9%) samples, with a strong correlation between EGFR CNV and logEGFR expression (r=0.79; p=0.017). Whole-arm +7/-10 genotype was evident in 17 (53.1%) tumours, and another 5 (15.6%) showed partial copy-number alterations. Notably, two gliomas without necrosis and proliferation initially diagnosed as oligodendrogliomas due to misinterpretation of the IDH immunostain, were reclassified as glioblastoma, IDH-wildtype. The data was validated using Sanger Sequencing and targeted panel sequencing to a depth of 500. Even low depth NGS sequencing, that is cost effective and time efficient offers significant improvements in diagnostic molecular neuropathology.