PURPOSEA subset of brainstem gliomas harbor an isocitrate dehydrogenase 1/2 (IDH1/2) mutation, which has important prognostic and treatment implications. We evaluated the radiographic features and the sensitivity of magnetic resonance spectroscopy (MRS) and cerebrospinal fluid cell-free DNA (CSF cfDNA) sequencing at detecting IDH1/2 mutations in a cohort of these tumors.METHODSWe identified IDH-mutant brainstem gliomas by retrospective chart review. IDH1/2 mutation was established by biopsy, CSF cfDNA sequencing, and/or the presence of a 2-hydroxyglutarate (2HG) peak by 3-Tesla MRS.RESULTSTwenty-one patients with IDH-mutant brainstem gliomas were identified, 57% male with a median age of 26 (6-59) years. All tumors involved the pons and/or abutted the brachium pontis. Although 18 (86%) of 21 were nonenhancing, only one of 21 demonstrated T2-FLAIR mismatch. An IDH1/2 mutation was identified by biopsy in 17 and by CSF cfDNA sequencing in three; in the final patient, an IDH1/2 mutation was inferred by the presence of a 2HG peak. The sensitivity of MRS at identifying 2HG in patients with confirmed IDH1/2 mutations was 64.3% (95% CI, 35.1 to 87.2), which increased to 85.7% (95% CI, 42.1 to 99.6) when the study was performed before treatment. The 2HG peak was absent in all four patients with a repeat MRS after completion of radiation. IDH1/2 mutation was identified in four of seven patients who underwent sequencing of CSF cfDNA. In this cohort, median progression-free survival and overall survival were 57.6 and 90.4 months, respectively. An objective response to radiotherapy was observed in 76%.CONCLUSIONIDH-mutant brainstem gliomas have a characteristic clinical and radiographic phenotype. MRS is more sensitive than CSF cfDNA sequencing for noninvasively identifying the presence of an IDH1/2 mutation when performed before radiotherapy.

CONTEXT:Cushing's syndrome (CS) impairs quality of life (QoL) and mood. Prospective real-life data on posttreatment recovery and predictors of improvement are limited. OBJECTIVES:Evaluate changes in QoL, depression, and anxiety in patients with CS, before and after biochemical control, and identify predictors of clinically meaningful improvement. DESIGN AND SETTING:Prospective observational study at a tertiary center. PATIENTS:Sixty-seven patients with endogenous CS (60 pituitary, 7 adrenal) were assessed with active disease and again after achieving biochemical control through surgery and/or medication. OUTCOMES:Patient-reported outcomes included CushingQoL, Beck Depression Inventory-II (BDI-II), and State-Trait Anxiety Inventory (STAI). RESULTS:Mean and longest follow-up was 2.3 and 11.5 years, respectively. Treatment led to improvements in mean scores across all domains (QoL: +18.2 ± 20.9, BDI-II: -6.8 ± 8.6, STAI-State: -9.6 ± 12.5, STAI-Trait: -8.6 ± 12.6; all P < .001). However, a minimal important difference was achieved in 64.6% for QoL, 67.9% for BDI-II, and 53.2% and 52.8% for STAI subscales. After multivariable analysis, QoL improvements were predicted by lower baseline body mass index, pretreatment symptoms <3 years, postoperative hydrocortisone replacement >6 months, and normal follow-up late-night salivary cortisol (LNSC). Depression improvements were predicted by symptoms <3 years, normal follow-up LNSC, and surgical treatment. Anxiety improvements were predicted by younger age and >6 months postoperative hydrocortisone. Depression improved more gradually than QoL and anxiety. CONCLUSION:Although effective treatment improves mood and QoL in CS, clinically meaningful recovery is variable and incomplete for some patients. Our findings highlight the need to limit diagnostic delay and provide comprehensive posttreatment care that includes normalization of cortisol circadian rhythm.

CONTEXT:Patients with Cushing syndrome (CS) often experience impaired quality of life (QoL) and mood despite biochemical control. The relationship between late-night salivary cortisol (LNSC) and psychological outcomes remains poorly characterized. OBJECTIVE:Assess QoL and mood outcomes in biochemically controlled CS patients based on circadian rhythm restoration. DESIGN:Cross-sectional. SETTING:Tertiary-care center. PARTICIPANTS:Ninety treated, biochemically controlled patients with CS (84 Cushing disease (CD); 6 adrenal CS), stratified into three groups: normal LNSC (Group A), abnormal LNSC (Group B), on long-term GC replacement (Group C). MAIN OUTCOME MEASURES:Hospital Anxiety and Depression Scale (HADS), CushingQoL questionnaire, Nottingham Health Profile (NHP). RESULTS:Group A had lower HADS-Anxiety (4 vs 7, A vs B; p=0.006) and (HADS-Depression 2.5 vs 6 vs 9, A vs B; p=0.006, A vs C; p<0.001). QoL was better in group A vs C in psychosocial (67.5 vs 39.5; p<0.001) and physical (63.9 vs 44.3; p=0.005) domains. Group A had better Emotional Reaction (0 vs 24; p=0.002), A vs B; Energy Level (0 vs 63; p=0.001) and Sleep (13 vs 56; p<0.001), A vs C. In multivariable analyses excluding group C, LNSC normalization was consistently associated with better outcomes in all HADS domains, CushingQoL psychosocial, NHP Emotional Reaction, Social Isolation, Physical Abilities and Home Relationships. Group B had the highest diabetes rate. Among patients with surgically remitted CD, 18.6% had abnormal LNSC, characterizing a previously unrecognized clinical phenotype. CONCLUSIONS:In biochemically controlled CS, LNSC normalization correlates with QoL, mood, and metabolic outcomes, and could represent a therapeutic target.

While clinical trials of human pluripotent stem cell-derived midbrain dopamine (mDA) neuron precursor grafts for Parkinson's disease (PD) are ongoing, current protocols remain suboptimal. In particular, the yield of TH+ mDA neurons after in vivo grafting and the expression of certain mDA neuron and subtype-specific markers require improvement. Single-cell transcriptomic analyses of grafts have revealed low proportions of mDA neurons and substantial off-target contamination. Here, we present an optimized mDA neuron differentiation strategy that builds on our clinical-grade ("Boost") protocol by adding FGF18 and IWP2 treatment ("Boost+") at the neurogenesis stage. Boost+ mDA neurons show higher expression of EN1, PITX3, and ALDH1A1. Improvements in mDA neuron yield and transcriptional similarity to primary mDA neurons are observed in vitro and following transplantation. Single-nucleus RNA sequencing demonstrates enrichment of A9 mDA neurons within Boost+ grafts. Functional studies in vitro demonstrate increased dopamine production and release and improved electrophysiological properties. In vivo analyses show higher percentages of TH+ mDA neurons, resulting in efficient rescue of amphetamine-induced rotation behavior in the 6-OHDA rat model and rescue of deficits in some nondrug-induced assays, including the ladder rung assay, which are not improved by Boost mDA neurons. The Boost+ conditions present an optimized differentiation protocol with advantages for disease modeling and mDA neuron grafting paradigms.

High-level copy-number (CN) amplification (HLAMP) is a major mechanism of oncogene activation in human cancer. Despite progress in therapeutically targeting amplified oncogenes, the processes underlying amplicon evolution remain incompletely understood, leaving critical knowledge gaps in their etiology and mechanisms of therapeutic response. To address this, we analyzed the evolutionary trajectories of HLAMPs using single-cell whole-genome sequencing data from 86,239 cancer cells across 93 patients and 9 experimental systems. We found that cell-to-cell CN variability provides a quantifiable readout of HLAMP mechanism, clearly distinguishing extrachromosomal circular DNA (ecDNA) from intrachromosomal amplification (ICamp) through characteristic CN distributions that reflect distinct modes of segregation and correspond to clonal architecture. Notably, ICamp events frequently showed multiple amplitude peaks specific to subclones, indicating punctuated shifts in oncogene dosage through numeric or structural modulatory mechanisms with transcriptional impact. In contrast, ecDNAs exhibited broad, continuous CN distribution with extreme high-copy outliers, consistent with asymmetric segregation. The CN and structural diversity of ecDNA regions enabled systematic deconvolution of ecDNA subspecies and estimation of their per-cell abundance, revealing the history of ecDNA-mediated oncogenesis at single-nucleotide resolution. We observed ecDNA diversification through internal rearrangements across cases and, notably, convergent evolution in glioblastoma cases marked by multiple, recurrent acquisition of EGFR-targeting ecDNAs. Finally, single-cell genome-based identification of ecDNAs showed substantial discrepancy with bulk genome graph-based predictions and reliably distinguished actively maintained ecDNAs from historical genomic footprints after chromosomal re-integration. These findings reveal marked tissue-type specificity of ecDNAs, suggesting that ecDNA-mediated oncogenesis may depend on a permissive tissue context.

Growing evidence indicates that childhood cancer is a developmental disease and the oncogenic impact of mutations depends on spatiotemporal developmental contexts. This dependency leads to distinct molecular, genetic, and clinical characteristics across various cancer (sub)types. However, the underlying molecular mechanisms of tumorigenesis are not fully understood, and the development of precision medicine for childhood cancers is still an ongoing effort, partially due to their relative rarity. Therefore, it is crucial to develop and use "developmental models" that replicate both mutations and specific developmental contexts that determine their impact. In this review, we summarize recent advances in the growing field of developmental modeling of childhood cancers, which enhance our understanding of the pathogenic mechanisms and pave the way for the development of new therapeutic approaches.

A clinical trial attempts to standardize fetal cell transplantation and highlights challenges to be addressed in upcoming studies that use stem cell-derived neuron progenitors.

BACKGROUND:Neoadjuvant systemic therapy combined with response-adapted surgery holds significant promise for improving oncologic and functional outcomes in head and neck cancer. Consistent and rigorous documentation is essential to evaluate this approach across disease sites. A system accounting for the complex anatomy of sinonasal tumors is currently lacking. We developed a framework to elucidate how neoadjuvant therapy impacts the extent and morbidity of surgery in sinonasal squamous cell carcinoma (SCC). METHODS AND RESULTS:A review of the medical literature highlighted a gap in research into response-adapted surgery for sinonasal malignancies. We developed a novel classification system to document resection at initial presentation and following neoadjuvant treatment. The framework is grounded in the surgical anatomy of the sinonasal region and weighs completeness with broad reproducibility to categorize adaptations according to (1) structures resected (2) exposure (3) reconstruction and (4) lymph node management. The surgical morbidity score was developed through a structured, iterative consensus process with a multidisciplinary expert panel. The final classification system was formally reviewed and endorsed by the institutional sinonasal tumor disease management team, establishing it as the standardized framework for documenting surgical outcomes in patients with SCC undergoing neoadjuvant therapy. CONCLUSIONS:The proposed system introduces the first response-adapted surgery classification to encompass patients with locoregionally advanced sinonasal SCC treated with neoadjuvant therapy prior to definitive surgical resection. By extending standardized terminology, this framework enhances communication within multidisciplinary teams, enables meaningful comparisons across published studies, and supports consistency in developing clinical trial endpoints seeking to balancing oncologic control and functional preservation.

PURPOSE:Incidentally found brain metastases often lead to emergency room referrals even in asymptomatic patients-a pathway of care that could be unnecessary. We sought to compare time and financial toxicity, and treatment outcomes between a multidisciplinary outpatient (MP) and acute care pathways (AP). METHODS:Patients referred for de novo asymptomatic brain metastases at an NCI-designated Cancer Center with a Multidisciplinary Brain Metastasis Program were identified via retrospective review. Scans, encounters, time to local interventions, and survival data were collected and compared. RESULTS:Seventy-eight patients were identified, 47 referred to MP and 31 AP. Both groups had similar disease-specific prognostic scores and received similar treatments. Patients managed via AP had larger dominant lesions (2.9 cm vs. 2.0 cm, p < 0.002) and shorter time to therapy (13.7 days vs. 9.2 days; p = 0.047). AP patients also had more medical-encounter (7.1 vs. 2.5; p < 0.001) and admitted days (5.9 vs. 1.1; p < 0.001), with increased median gross charge amount ($185,961 vs. $126,831; p = 0.003) despite similar 6-month survival (83% MP vs. 81% AP, p > 0.999) and local tumor control (95% MP vs. 96% AP, p = 0.849). CONCLUSION:Patients with asymptomatic brain metastases managed through an outpatient pathway attained similar disease outcomes with lower time and financial toxicity compared to patients managed through inpatient pathways. Characteristics of such patients that qualify them for outpatient pathway should be confirmed prospectively.

ABSTRACT Parkinson’s disease (PD) is characterized by the progressive loss of midbrain dopaminergic (mDA) neurons 1 . Stem cell–derived mDA neurons hold promise for disease modelling 2,3 and are currently in clinical trials for cell replacement therapy 4–6 . However, systematic benchmarking has been limited by the lack of a unified high-resolution reference and methods that quantify incomplete or mixed lineage specification in vitro 7 . We establish a single-cell and spatial atlas of the human developing diencephalon–midbrain–hindbrain axis resolving 93 cell subtypes, including 39 lacking prior single-cell characterization and 4 entirely novel populations. Using this atlas as a reference, we integrate 19 hPSC-derived mDA datasets, both published 2,8–25 and unpublished, to build the Human Dopaminergic Neural Atlas (HDNA) spanning 2D, 3D, and graft models, including those used in clinical trials. To classify cells and quantify lineage fidelity, we develop CapybaraBrain, a marker-driven non-negative decomposition framework that assigns each cell continuous identity scores across all 93 developmental programs, enabling systematic discrimination of discrete, transitioning, and cross-lineage hybrid states 26 . We uncover a pervasive landscape of off-target populations reflecting relaxed transcriptional boundaries in vitro, including a previously unrecognized TH–PITX2 midbrain neuronal population, and we validate atlas-predicted latent lineage plasticity through inducible genetic fate mapping in mouse models. We further define maturation-associated transcriptional programs by harmonizing adult mDA subtype atlases, revealing that dopaminergic identity and maturation are partially decoupled across protocols. Finally, projecting PD patient-derived tri-cultures onto the HDNA uncovers genotype- and cell-type-specific transcriptional dysregulation. Together, these integrated atlases and computational framework establish a unified standard for benchmarking differentiation fidelity, exposing off-target states, and guiding next-generation PD models and cell therapies.

OBJECTIVE:Seizures cause significant morbidity in patients with brain metastasis (BrM). Local therapies for BrM, including resection with adjuvant stereotactic radiosurgery and stereotactic radiosurgery (SRS) alone, have undefined seizure outcomes. The authors sought to compare seizure control in patients with BrM-induced seizures treated with either modality. METHODS:Patients who received resection surgery plus adjuvant SRS (S+SRS) or SRS for BrM at a National Cancer Institute (NCI)-designated Comprehensive Cancer Center between January 2015 and December 2023 were retrospectively reviewed. Patients with pretreatment seizure and semiology attributable to an untreated metastasis were included. The cumulative incidence rates of first posttreatment seizure were estimated for both treatment groups. Multivariable analyses identified risk of posttreatment seizure under subdistribution hazards modeling in the competing risk setting to account for death. The authors secondarily assessed overall survival (OS) and local and distal progression rates between treatments. RESULTS:Two hundred fifty-five of 5284 patients treated for BrM had pretreatment seizures, and 190 patients met the inclusion criteria: 76 (40%) treated with S+SRS and 114 (60%) treated with SRS. One hundred eighty-five (97.4%) patients were taking an antiseizure medication at the end of treatment. The 6-month rate (95% CI) of seizure freedom was 86.71% (77.97%-93.23%) for patients treated with S+SRS and 69.18% (60.54%-77.46%) for patients treated with SRS (p = 0.003). The 12-month rates of seizure freedom were 74.40% (64.01%-83.74%) and 65.59% (56.80%-74.24%) for the S+SRS and SRS groups, respectively (p = 0.091). In the multivariable models, S+SRS was associated with 56% and 27% reductions in seizure risk relative to SRS at 6 and 12 months, respectively, though this was only statistically significant at 6 months. Median OS (95% CI) was 2.62 (1.52-6.52) years for S+SRS patients and 0.83 (0.62-1.54) years for SRS patients (p < 0.001). Treatments did not differ in terms of the rates of local failure (p = 0.52) or distal intracranial progression (p = 0.26) among patients with radiographic follow-up. In subgroups stratified by maximum tumor diameter, patients treated with S+SRS had a lower 6-month cumulative incidence rate of recurrent seizure than patients treated with SRS, though this was only statistically significant for patients with tumors > 3 cm. CONCLUSIONS:Patients treated with S+SRS experienced fewer posttreatment seizures at 6 months than patients treated with SRS, despite larger index lesions in patients treated with S+SRS and prevalent antiseizure medication use in the entire study population. Although multivariable models showed no significant associations at 12 months or beyond, the role of resection in controlling BrM-induced seizures warrants further investigation.

Primary de novo high grade gliomas, such as glioblastoma and lower grade gliomas both converge on a common aggressive phenotype, and the basis for this progression is unknown. Glioma associated macrophages (GAM) have been strongly implicated in supporting tumor growth, however, robust isolation of functional subpopulations has been elusive. We hypothesize that functional populations of GAMs can be resolved through gene regulatory network (GRN) inference and show that a subpopulation of human GAMs, defined by a GRN centered around the activator protein-1 transcription factor FOSL2 is preferentially enriched in high grade gliomas. We nominate ANXA1 and HMOX1 as surrogate cell surface markers for a subpopulation we term malignancy associated GAMs (mGAMs) which possess distinct pro-tumorigenic properties, share partial ontogeny with peripheral blood monocytes, and are enriched in newly transformed regions of glioma. mGAMs potentially play a pivotal role in glioma progression and represent a plausible therapeutic target.

Abstract Introduction: Few existing models accurately recapitulate and sustain the phenotype of IDH-mutant low-grade gliomas (LGGs). Maintaining a stable IDH-mutant glioma line remains a major challenge, as patient-derived cultures rapidly lose mutant IDH expression due to selective pressure favoring more aggressive, IDH wild-type cells. To address this limitation, we developed an inducible human embryonic stem cell (hESC)-derived model for longitudinal studies of IDH-mutant Low-Grade Astrocytoma (LGA) pathogenesis, progression, and therapeutic response. Methods: We engineered hESCs to carry a doxycycline-inducible IDH1-R132H mutation, with or without CRISPR/Cas9-mediated knockouts of TP53 and ATRX. These combinations generated three isogenic lines: IDH-mutant alone, IDH-mutant/TP53-null, and IDH-mutant/TP53-ATRX-null. This allows for the dissection of how common co-mutations influence glioma initiation and evolution. Cells underwent neural induction to the progenitor stage to model early gliomagenesis, with induction of mutant IDH expression being verified via Western blot. Tumorigenic potential and progression are currently being assessed both in vitro and in vivo. Results: Upon differentiation to the neural progenitor stage, IDH1-R132H induction produced hallmark features of LGG biology, including elevated γH2AX, a marker of DNA damage response associated with IDH-mutant gliomas. Induced cells exhibited slower proliferation and limited invasiveness compared to non-induced controls, mirroring the slower growth of patient-derived LGGs. In vivo, IDH-mutant xenografts demonstrated delayed tumor expansion relative to IDH-wild-type counterparts. Ongoing studies are comparing therapeutic responses between our hESC-derived model and patient-derived lines to evaluate its translational utility for preclinical drug testing and longitudinal progression studies. Conclusions: We established an inducible hESC-based model with the capability to recapitulate the behavior and characteristics of IDH-mutant LGA. This platform could overcome the instability of patient-derived lines, enabling controlled, long-term studies of glioma evolution and treatment response. Our model provides a powerful system to dissect how IDH and cooperating mutations drive gliomagenesis and to test targeted therapies across disease stages. Citation Format: Greta Henriette Ghita, Yanhong Yang, Viviane Tabar. Modeling IDH-mutant low-grade astrocytoma using human embryonic stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4861.

Glioblastoma (GBM) is an aggressive primary brain cancer with few effective therapies. Stereotactic needle biopsies are routinely used for diagnosis; however, the feasibility and utility of investigative biopsies to monitor treatment response remains ill-defined. Here, we demonstrate the depth of data generation possible from routine stereotactic needle core biopsies and perform highly resolved multi-omics analyses, including single-cell RNA sequencing, spatial transcriptomics, metabolomics, proteomics, phosphoproteomics, T-cell clonotype analysis, and MHC Class I immunopeptidomics on standard biopsy tissue obtained intra-operatively. We also examine biopsies taken from different locations and provide a framework for measuring spatial and genomic heterogeneity. Finally, we investigate the utility of stereotactic biopsies as a method for generating patient-derived xenograft (PDX) models. Multimodal dataset integration highlights spatially mapped immune cell-associated metabolic pathways and validates inferred cell-cell ligand-receptor interactions. In conclusion, investigative biopsies provide data-rich insight into disease processes and may be useful in evaluating treatment responses.

While clinical trials are ongoing using human pluripotent stem cell-derived midbrain dopamine (mDA) neuron precursor grafts in Parkinson's disease (PD), current protocols to derive mDA neurons remain suboptimal. In particular, the yield of TH+ mDA neurons after in vivo grafting and the expression of some mDA neuron and subtype-specific markers can be further improved. For example, characterization of mDA grafts by single cell transcriptomics has yielded only a small proportion of mDA neurons and a considerable fraction of contaminating cell populations. Here we present an optimized mDA neuron differentiation strategy that builds on our clinical grade ("Boost") protocol but includes the addition of FGF18 and IWP2 treatment ("Boost+") at the mDA neurogenesis stage. We demonstrate that Boost+ mDA neurons show higher expression of EN1, PITX3 and ALDH1A1. Improvements in both mDA neurons yield and transcriptional similarity to primary mDA neurons is observed both in vitro and in grafts. Furthermore, grafts are enriched in authentic A9 mDA neurons by single nucSeq. Functional studies in vitro demonstrate increased dopamine production and release and improved electrophysiological properties. In vivo analyses show increased percentages of TH+ mDA neurons resulting in efficient rescue of amphetamine induced rotation behavior in the 6-OHDA rat model and rescue of some motor deficits in non-drug induced assays, including the ladder rung assay that is not improved by Boost mDA neurons. The Boost+ conditions present an optimized protocol with advantages for disease modeling and mDA neuron grafting paradigms.

Single-cell genomics technologies have accelerated our understanding of cell-state heterogeneity in diverse contexts. Although single-cell RNA sequencing identifies rare populations that express specific marker transcript combinations, traditional flow sorting requires cell surface markers with high-fidelity antibodies, limiting our ability to interrogate these populations. In addition, many single-cell studies require the isolation of nuclei from tissue, eliminating the ability to enrich learned rare cell states based on extranuclear protein markers. In the present report, we addressed these limitations by developing Programmable Enrichment via RNA FlowFISH by sequencing (PERFF-seq), a scalable assay that enables scRNA-seq profiling of subpopulations defined by the abundance of specific RNA transcripts. Across immune populations (n = 184,126 cells) and fresh-frozen and formalin-fixed, paraffin-embedded brain tissue (n = 33,145 nuclei), we demonstrated that programmable sorting logic via RNA-based cytometry can isolate rare cell populations and uncover phenotypic heterogeneity via downstream, high-throughput, single-cell genomics analyses. Programmable Enrichment via RNA FlowFISH by sequencing (PERFF-seq) isolates rare cells based on RNA marker transcripts for single-cell RNA sequencing profiling of complex tissues, with applicability to a broad variety of samples and cell types.

Interventional neuro-oncology is an evolving subspecialty that leverages minimally invasive endovascular and percutaneous techniques to improve outcomes for patients with spine, brain, and head-neck tumours. While conventional interventions have historically focused on vascular pathologies such as stroke and aneurysms, interventional techniques in oncology are gaining prominence. This review explores the role of image-guided interventions in preoperative tumour embolization, middle meningeal artery embolization in thrombocytopenic cancer patients, management of head and neck hemorrhage, intra-arterial drug delivery, and spinal interventions. Through a synthesis of current evidence, we highlighted the growing importance of interventional techniques in neuro-oncology and discuss future advancements in image guidance, robotics, and targeted drug delivery.