Targeted NGS-Based Mutational Signature Analysis in Hypermutated Gliomas: A Case-Centered Feasibility Assessment

Introduction: Gliomas generally display a low tumor mutational burden (TMB) and an immunosuppressive microenvironment, both of which restrict the effectiveness of immune checkpoint inhibitors. However, certain gliomas acquire a high TMB, which may arise either due to temozolomide (TMZ) therapy or mismatch repair deficiency (MMRd). Mutational signature analysis identifies specific somatic mutation patterns, providing insight into underlying mutational mechanisms. This study provides a case-centered feasibility assessment of mutational signature refitting from a clinically implemented targeted NGS panel in two hypermutated gliomas identified within a real-world glioma testing cohort. Methods: Twenty-eight gliomas tested using the Oncomine Comprehensive Assay Plus NGS panel in routine clinical practice were retrospectively reviewed. Panel-derived TMB was used for coarse stratification and to identify hypermutated tumors for detailed mutational signature interpretation. Mutational signature refitting was performed using Signature Analyzer for Targeted Sequencing (SATS). Results: Two gliomas exhibited elevated panel-derived TMB estimates: one after TMZ therapy and another in association with MSH2 deficiency. The remaining 26 non-hypermutated tumors had sparse mutation counts, limiting reliable signature interpretation. COSMIC single-base substitution signature 11 (SBS11) was predominant in the TMZ-treated hypermutated glioma, whereas the MSH2-deficient glioma demonstrated increased SBS1 and SBS26. Conclusion: This real-world, case-centered study suggests that targeted-panel mutational signature refitting may provide complementary molecular context in selected hypermutated gliomas. The observed patterns were concordant with prior TMZ exposure or mismatch repair deficiency. However, in non-hypermutated tumors, sparse mutation counts limited reliable signature interpretation, and the clinical impact of this approach remains to be established.