Clinical and preclinical insights into a novel MDM2::PDGFRA fusion in recurrent glioblastoma

Abstract

Glioblastoma is an aggressive and treatment-refractory primary brain tumor with limited therapeutic options and high recurrence. The molecular heterogeneity of glioblastoma poses a significant challenge to therapeutic development, as targeted therapies have mostly failed in small-scale clinical trials, underscoring the need for comprehensive next-generation sequencing (NGS) characterization to identify mechanisms of resistance. In this study, we identify and functionally characterize a novel amplified fusion, MDM2 (exon 1)::PDGFRA (exon 8), mediating resistance to cetuximab in an EGFR-amplified glioblastoma. The fusion results in a truncated PDGFRA isoform, in vitro assays demonstrate that MDM2::PDGFRA acts as a constitutively active oncogenic driver with a distinct sensitivity profile to tyrosine kinase inhibitors. Analysis of a glioblastoma cohort indicates PDGFRA structural variants often co-occur with amplification and may serve as biomarkers. These findings highlight the importance of repeat NGS profiling in clinical management and provide a translational framework for identifying and targeting emergent fusion-driven alterations.

Fig. 1. Patient’s clinical course with radiographic findings.

A Initial presentation, axial and coronal T1 MRI images demonstrate T2 expansile lesion in the left posterior superior temporal gyrus with small area of enhancement near the superior temporal sulcus. B Post-operative axial and coronal T1 scan following initial resection demonstrating gross total resection. C Post-gadolinium axial and coronal T1 images reveal disease progression with a new cystic mass with peripheral enhancement in the left temporal lobe. D Post-operative axial and coronal T1 scan following second resection demonstrating gross total resection. E Axial T1 images demonstrate correct intraoperative placement of GammaTile brachytherapy.

Fig. 2. Detection of MDM2::PDGFRA as a mechanism of resistance to cetuximab in glioblastoma.

A Molecular pathology of glioblastoma before treatment. H&E reveals a malignant glial neoplasm characterized by hypercellularity, marked nuclear atypia, brisk mitotic activity, microvascular proliferation, and GFAP positivity. TP53, TERT promoter, MDM2, and CDK4 alterations were detected in all samples and can be regarded as founder events. Amplification of EGFR, but not of PDFGRA, was detected on initial sequencing. By IHC, EGFR showed dominant overexpression (intense in ~90%); however, a small subset was PDGFRA-positive (moderate-to-intense in ~5%), suggestive of a minor PDGFRA-amplified subclone undetected by bulk sequencing. B Following treatment with cetuximab, MDM2::PDGFRA and PDGFRA amplification were detected, and EGFR amplification disappeared, indicating that a major clonal sweep developed in the recurrent tumor. PDGFRA IHC was heterogeneous, ranging from negative (i) to strong (ii) immunoreactivity. By correlation of IHC Ab binding sites and fusion structure, PDGFRA-negative areas with decreased MDM2 were interpreted as MDM2::PDFGRA fusion expression, while intense PDGFRA labeling was interpreted as expression of amplified wild-type allele. EGFR IHC was markedly decreased; nonetheless, a small population retained immunoreactivity (moderate in ~10%) that may represent residual EGFR-amplified subclones. C Diagram of the genomic structure of MDM2 (NM_001145339) located on chromosome 12q15, with exons and functional domains annotated. The fusion breakpoint, occurring in exon 1 after amino acid 5 (red line). MDM2 antibody binding epitope located between amino acids 26–169 (red band). D Schematic of PDGFRA (NM_006206) located on chromosome 4q12, showing exons and key functional domains. The fusion breakpoint, occurring in exon 8 following amino acid 374 (blue line). PDGFRA antibody binding epitope located between amino acids 32-324 (red band). E Diagram of the resulting MDM2::PDGFRA in-frame chimeric fusion gene. The translocation juxtaposes MDM2 exon 1 to PDGFRA exon 8.

Fig. 3. Landscape of PDGFRA structural variants and fusions in glioblastoma.

A Workflow of PDGFRA structural variant (SV) selection, available in the GENIE data registry (v17.0-public), and a circos plot indicating the partner genes (bottom). A total of 32 PDGFRA SVs detected in 25 gliomas were suitable for further analysis. B Oncoprint showing demographics, reporting center, and genomic co-alterations from 25 gliomas (24 glioblastomas and 1 diffuse glioma) with PDGFRA SVs. C Copy number segments at the 4q12 locus for 5 cases with available copy number profiles. D Detailed structural variant annotation and predicted chimeric proteins of PDGFRA SVs available for 5 cases. AA: amino acid, LOF: loss-of-function (tyrosine kinase domain truncation).

Fig. 4. Functional characterization of MDM2::PDGFRA oncogenicity and TKI sensitivity.

Dose-response cell viability comparing onco-addicted Ba/F3 cells expressing MDM2::PDGFRA (A) or PDGFRA D842V (positive control) (B) after treatment with avapritinib, crenolanib, dasatinib, lenvatinib, or ripretinib for 72 h. Representative data are average ± standard error of means (SEM) from three independent experiments, with each experiment containing three internal replicates per inhibitor. C Heat map summarizing inhibitory concentration (nM) required to induce 50% inhibition (IC₅₀) as determined from non-linear regression analysis of dose-response assays. Average value from three replicate experiments displayed. Immunoblot analysis from Ba/F3 cells expressing MDM2::PDGFRA (D) or PDGFRA D842V (E) of phosphorylated and total PDGFRA following 2-hour treatment with DMSO (vehicle) or 1, 10, 100 nM concentrations of avapritinib, dasatinib and ripretinib. GFP, expressed from the same vector via IRES, serves as a total protein control. Data are representative of four independent experiments. F Densitometry graphs quantitatively compare extent of inhibition on PDGFRA after treatment with indicated inhibitors from four independent replicate experiments. Pixel density of phosphorylated PDGFRA signal was divided by total PDGFRA signal and normalized to DMSO. Significance vs. vehicle: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.