Identification of NTRK missense mutations in glioblastoma multiforme: Insights from AACR Project GENIE and structural-functional analyses

Background: Neurotrophic tyrosine receptor kinase ( NTRK ) alterations are oncogenic drivers in diverse malignancies; however, their relevance in glioblastoma multiforme (GBM) is incompletely defined. While NTRK fusions have been well characterized, the prevalence, pathogenicity, and potential clinical impact of NTRK1/2/3 missense mutations in GBM remain largely unexplored. Using the AACR Project GENIE registry (v18.0), we profiled NTRK missense variants in GBM and integrated survival and structural analyses to evaluate their functional significance. Methods: Data from 7048 GBM samples (6523 patients) were curated. Post-sequencing overall survival (OS) was assessed by exploratory Kaplan-Meier (KM) and univariate Cox regression. Pathogenicity and structural consequences were evaluated through AlphaMissense, REVEL, LoGoFunc, PROVEAN, MutPred2, and DynaMut on AlphaFold-predicted structures. Results: Missense mutations in NTRK1 (1.8%), NTRK2 (1.3%), and NTRK3 (1.3%) were rare. Four variants were consistently detected in the extracellular domain: NTRK2 p.Y359D (0.23%), NTRK2 p.G344D (0.21%), NTRK1 p.H298Q (0.25%), and NTRK1 p.P302S (0.23%). In NTRK1, 15/16 patients carried both p.H298Q and p.P302S together, while isolated p.H298Q (n = 1) or p.P302S (n = 0) events were rare. A similar pattern was observed in NTRK2 p.Y359D/p.G344D mutants, where p.Y359D and p.G344D co-occurred in 14/15 patients, indicating co-mutation enrichment. KM analysis suggested a trend toward improved OS for p.Y359D (p = 0.047) and H298Q (p = 0.037). Cox regression pointed to protective effect of p.H298Q (HR = 0.32, 95% CI: 0.103-0.992). AlphaMissense classified p.Y359D and p.G344D as ‘likely pathogenic' and p.H298Q/p.P302S as ‘likely benign', whereas PROVEAN predicted all four as deleterious. DynaMut suggested destabilization for p.Y359D (ΔΔG −2.48 kcal/mol) and increased flexibility, while it demonstrated stabilization for p.H298Q (ΔΔG +0.261 kcal/mol), consistent with its gain-of-function classification by LoGoFunc, and low REVEL score of 0.104. MutPred2 analysis signaled functional impact for p.Y359D, which showed a high pathogenicity score (0.923; REVEL score = 0.910) with coordinated disruption of regulatory motifs and mechanistic changes, including gain of loop structure, loss of a sulfation site at p.Y359, altered ordered interfaces, and perturbed metal binding. Conclusions: This analysis suggests that previously underrecognized NTRK missense mutations may be present in GBM and could have functional relevance; however, these findings should be interpreted cautiously given the study's retrospective registry design, reliance on tumor-only sequencing (with potential germline or passenger variants), incomplete clinical and molecular covariates, lack of multiple-testing adjustment, and the absence of experimental validation.