Molecular Characterization of Adult-Type Lower-Grade Glioma (WHO Grade 1–3) with Targeted Next-Generation Sequencing: A Retrospective, Single-Institution Experience

<b>Background/Objectives</b>: The 2021 WHO Classification of Central Nervous System (CNS) tumors emphasizes the integration of molecular data with histopathological features. Lower-grade gliomas (LGGs) represent a heterogeneous group of neoplasms with variable clinical behavior. This study aimed to explore the molecular landscape of a single-institution series of LGGs using targeted next-generation sequencing (NGS). <b>Methods</b>: Eleven adult patients diagnosed with LGG between 2015 and 2024 at Cattinara University Hospital (Trieste, Italy) were retrospectively analyzed. DNA and RNA were extracted from formalin-fixed, paraffin-embedded (FFPE) tissue and analyzed using the TruSight Oncology 500 panel (Illumina). Mutational, amplification, and transcriptomic profiles were evaluated. <b>Results</b>: IDH1 mutations were the most frequent alteration (75%), commonly co-occurring with TP53 and ATRX mutations, consistent with the canonical IDH-mutant astrocytoma profile. CDK4 amplification was found in four cases, while MYCN amplification and MET amplification were each identified in isolated cases. Two diffuse IDH-wild-type gliomas displayed aggressive clinical courses and shorter survival, and one was reclassified as glioblastoma (grade 4) based on EGFR amplification. The transcriptome analysis revealed heterogeneous expression signatures and distinct clustering of IDH1/ATRX-mutant tumors. <b>Conclusions</b>: Targeted NGS confirmed the key molecular features of diffuse gliomas and enabled precise WHO 2021 classification even in archival FFPE samples. Despite the exploratory nature of the analysis on a small population, the study underscores the biological and transcriptional heterogeneity of LGGs and highlights the limitations of tumor-only sequencing approaches. Broader genomic profiling and matched normal controls are warranted to refine the interpretation of rare or non-canonical variants.