Roeland G. W. Verhaak

Here we present AmpliconSuite, a toolkit for focal amplification analysis from cancer whole-genome sequencing that identifies extrachromosomal DNA (ecDNA), breakage–fusion–bridges and other amplification modes. Its ecContext module categorizes ecDNA structures to understand formation mechanisms. Validation across 175 metaphase fluorescence in situ hybridization-probed genes in 72 models demonstrated accurate predictions. Analysis of 6,506 patient pan-cancer samples revealed ecDNA-mediated oncogene co-amplification preferences. To democratize data sharing, we created AmpliconRepository, a community-contributable platform, enabling investigation of results across >15,000 publicly hosted samples.

Extrachromosomal DNA (ecDNA) contributes to cancer genome instability by enabling high-copy oncogene amplification and genetic intratumoral heterogeneity. Micronuclei (MNs) are common in chromosomally unstable cancers, yet their role in ecDNA-driven tumors remains unclear. Here we investigate how ecDNA damage, mitotic segregation and MN formation are linked. We find that ecDNAs are a major source of MNs following their damage, particularly under replication stress. Replication stress promotes mitotic ecDNA clustering, asymmetric inheritance, mis-segregation into MNs and reduced transcriptional output. Single-MN sequencing reveals that multiple distinct ecDNA species coalesce within individual MNs. Cells inheriting ecDNA⁺ MNs display impaired proliferative capacity and are more likely to undergo cell death. In patients with MYCN-amplified neuroblastoma, higher frequencies of ecDNA⁺ MNs at diagnosis are associated with improved overall survival. Together, these findings identify ecDNA mis-segregation as a mechanism that limits tumor fitness, highlighting mitotic ecDNA retention as a potential therapeutic vulnerability.

Extrachromosomal DNA (ecDNA) can arise early in the development of aggressive tumors or later upon progression, but the impact of chronology on patient outcome is not known. Currently, it is not possible to determine when an ecDNA arose in the course of tumor development and progression, based on genome sequencing data. Here, we develop an analytic method, ecEvol, to infer ecDNA emergence time, selection strength, mutation rate, and clonal dominance from allele-frequency spectra in bulk whole-genome sequencing. EcEvol outperforms other methods and predicts parameters with high accuracy across a wide range of parameter values. Applying ecEvol to primary, treatment-naive tumors, we find that ecDNA typically emerges early and undergoes strong positive selection, albeit with substantial variation across cancer types. EcDNAs carrying known oncogenes emerge earlier and reach greater clonal dominance than ecDNAs lacking known oncogenes. In advanced and predominantly treated tumors, ecDNA emerges substantially later than in matched primary tumors, consistent with the generation and selection of new ecDNA during tumor progression and therapy. Longitudinal samples further support continued expansion of ecDNA-bearing lineages. Finally, earlier ecDNA emergence is associated with poorer overall survival. These results provide insight into the chronology of ecDNA formation from a single tumor biopsy taken at any point during the course of the disease, revealing that allele-frequency spectra encode the evolutionary history of ecDNA and provide a quantitative framework for reconstructing its emergence and expansion.

Isocitrate dehydrogenase (IDH)-mutant gliomas represent most lower-grade diffuse gliomas in young adults. Although IDH-mutant gliomas initially behave indolently relative to IDH-wildtype counterparts, they undergo inevitable malignant transformation. The mechanisms driving this progression remain poorly defined. We analyzed a unique longitudinal cohort of two patients with IDH-mutant diffuse gliomas, each with matched WHO grade 2, 3, and 4 tumours. Spatial transcriptomics (NanoString GeoMx and 10X Visium), combined with differential expression, spatially variable gene analysis, and gene ontology enrichment, were used to define transcriptional programs underlying progression. Unexpectedly, across both patients, higher-grade tumours demonstrated reduced proliferation at the infiltrative margin as measured by Ki67. Spatial transcriptomic analysis identified an enrichment of invasion-associated genes associated with increasing tumour grade. In an independent bulk RNA-seq cohort, expression of this spatially derived program did not outperform grade-only PFS or OS analysis. These observations underscore the need to develop new metrics that account for spatial evolution that occurs across progression, but escapes detection in bulk analyses and provide the rationale for prioritizing migration-, cytoskeletal- and stress-associated programs for further investigation.

Background: Although radiation therapy (RT) remains one of the most effective treatments for patients with the lethal brain tumor glioblastoma (GBM), quite a number of patients show resistance or relapse shortly after RT. There is an urgent need to uncover the temporal and spatial dynamics of RT resistance during tumor progression and treatment. Methods: The radio-resistance signature score (RRSS) was calculated by utilizing glioma sphere-forming cells (GSCs), which recapitulate the transcriptomic landscape of GBM. Results: RRSS can assess the RT response on both cell lines and GBM patients. Both transdifferentiated and irradiated glioma cells show increased radiation resistance, demonstrating that the recurrent GBM may acquire resistance along with cell differentiation and selective advantage during treatment. Moreover, RRSS of the marginal tumors is found to be higher than that of core tumors (Wilcoxon’s rank-sum test, P-value <.01). The infiltration of macrophages was significantly associated with radiation resistance (P-value <.01), which was further validated by single-cell RNAseq-based experiments and clonogenic assays. Moreover, we demonstrated that IL1B, a pro-inflammatory cytokine secreted by macrophages, could enhance the radiation resistance of tumor cells. Conclusions: Our study revealed the spatiotemporal evolution of radiation resistance of GBM and shed light on the development of novel adjuvant therapeutic strategies.

Glioblastomas remain the most lethal primary brain tumour in adults, with targeted therapies delivering only limited benefit despite deep molecular characterization. Several targeted drugs have received regulatory approval for low-grade gliomas, although progress in glioblastomas remains constrained by, among other aspects, extensive intratumoural heterogeneity, pathway redundancy, cellular plasticity and limited drug delivery to the central nervous system. Some of these challenges might be mitigated through strategies that enhance blood–brain penetration, including focused ultrasonography, convection-enhanced delivery, efflux avoidance and chemical modifications. Improved tumour profiling through multiregional sampling, prioritization of truncal dependencies and the development of novel therapeutic modalities, such as antibody–drug conjugates and theranostics, might also further improve outcomes. In this Review, we summarize the therapeutic landscape of targeted therapies in glioblastomas, spanning major target classes including receptor tyrosine kinases, intracellular signalling proteins, cell-cycle dysregulation and synthetic-lethal vulnerabilities. We also examine emerging strategies targeting genome integrity and telomeres, epigenetic modulators, and tumour–neural circuitry. Furthermore, we highlight tumour heterogeneity and extrachromosomal DNA dynamics as key drivers of oncogene amplification and therapeutic resistance as well as the roles of novel clinical trial designs and liquid biopsy-based monitoring strategies. Lastly, we discuss pathway-based glioblastoma classification and master kinase mapping as methods for aligning drugs with functional tumour states.

Gliomas with mutant isocitrate dehydrogenase (IDH) are malignant brain tumours that typically arise in early to mid-adulthood and nearly always recur following treatment ^1,2 . However, the genetic and cellular-state changes that drive IDH-mutant glioma progression under treatment remain incompletely understood. Here we integrated single-nucleus transcriptomic profiles, chromatin accessibility profiles and bulk DNA and RNA sequencing from 75 temporally separated gliomas across 35 patients comprising both the oligodendroglioma and astrocytoma IDH-mutant glioma tumour types. We show that malignant cell states transcriptionally resemble stages of normal glial–neuronal lineage development or a reactive mesenchymal-like state, mirroring states previously described in IDH wild-type glioblastoma ^3,4 . Malignant cell states displayed distinct chromatin accessibility profiles that were comparable between both IDH-mutant glioma types. The abundance of less differentiated malignant cells increased with grade and with genetic alterations such as PDGFRA amplification. Longitudinal analysis highlighted two major malignant cell-state transition patterns. First, reduced lineage differentiation and increased proliferative malignant cells at recurrence were enriched in gliomas that acquired recurrence-associated genetic events. These included treatment-associated hypermutation, increased copy number changes and cell cycle alterations. Second, increased mesenchymal-like-state abundance occurred independently of acquired genetic alterations and instead coincided with elevated macrophage expression. Overall, our findings provide an integrative model that traces the cell intrinsic and extrinsic factors that shape cellular states during IDH-mutant glioma disease progression.

Lay Summary: The United States (US) Food and Drug Administration (FDA) recently granted accelerated approval to dordaviprone (ONC201, ModeysoTM) for patients with H3 K27M–altered diffuse midline glioma, a rare and aggressive brain tumor with very few treatment options. This Point/Counterpoint editorial discusses what this decision means for science, regulation, and patient care. Accelerated approval allows the FDA to make promising treatments available sooner for serious diseases based on early signs of benefit, such as tumor shrinkage or how long patients respond to treatment. However, additional studies are still required to confirm that the drug truly improves outcomes. In this case, the approval was based on early studies and expanded-access programs that showed the drug may help a subset of patients. Important questions remain. Most of the data used for approval came from non-randomized studies, and it is unclear how well the drug works in newly diagnosed patients or in related tumors such as diffuse intrinsic pontine glioma (DIPG) and spinal cord tumors. We argue that this approval should be seen as conditional, an important step that may speed up research on drug combinations, biomarkers, and better clinical trials, while still requiring strong evidence. The ongoing ACTION trial (NCT05580562) will be critical for confirming the benefit of the drug and guiding how it should be used in practice.