Multi-region Whole Exome Sequencing Reveals Unique Spatial Distribution of Driver Mutations in GBM

Introduction: Glioblastoma (GBM) is an aggressive primary brain malignancy with a median survival under two years, despite aggressive treatment approaches. Currently, adjuvant treatment is informed by tissue analysis of a singular region of enhancing tumor intended to represent all residual tumor and microscopic disease, despite known high intra-tumoral heterogeneity in GBM. Here, we sought to examine intra-tumoral heterogeneity in GBM using multi-region sequencing.

Methods: 18 patients with GBM undergoing resection had tissue collected from the central core (CC), enhancing rim (ER), and non-enhancing rim (NER) of the tumor using a stereotactically-navigated handpiece and automated preservation system. Tissue samples were flash frozen and underwent whole exome sequencing at mean depth of >80-100X. OncodriveCLUST and OncoKB were used to detect disease-causing mutation variants in the regions sequenced.

Results: Among the 18 patients, median age was 67.5 years, 38.9% were male, and 22.2% had hypermethylation of the MGMT promoter. Tumor mutational burden was preserved across the three regions sampled (CC = 2.3 ± 1.9 (median ± IQR); ER = 2.8 ± 1.8; NER = 2.1 ± 0.8, all mutations per million bases). The NER was enriched for pathogenic variants in RGS10, involved in G-protein signaling (p = 0.04, Fisher’s test). Nine driver genes were preferentially mutated across different regions of the tumor, including KMT2C, a known tumor suppressor, mutated in 24% of NER samples but in only 6% of the remaining regions (q = 6.77e-3, false discovery rate).

Conclusion : Distinct genomic variants occupied different radiographically identified sub-regions of GBM. These highlight the degree of intra-tumoral heterogeneity and may indicate branched evolution of residual tumor cells. For instance, mutations in KMT2C may contribute to oncogenesis in the NER tissue left post-resection. Further profiling of an expanded cohort is needed to validate these findings.