Citations

Abstract Glioma stem cells (GSCs) are key drivers of glioblastoma (GBM) progression, therapy resistance, and recurrence, making them critical therapeutic targets. While the lineage-defining transcription factor OLIG2 is critical for GSC maintenance, the molecular co-regulators and downstream pathways it engages remain poorly defined. Here, we identify a previously unrecognized, functionally significant interaction between OLIG2 and STAT5B, uncovering a novel regulatory axis in GSCs. Using proximity ligation assay (PLA), we demonstrate that OLIG2 interacts with STAT5 in human and mouse GSC models, particularly in EGFRvIII-mutant cells. We find that OLIG2 preferentially partners with STAT5B and not STAT5A. Knockdown studies in GSCs reveal that OLIG2 and STAT5B co-regulate critical oncogenes BIRC5 and NUSAP1, both of which are required for sphere formation and migration capacity of EGFRvIII positive GSCs. In orthotopic xenograft models, knockdown of either STAT5B or OLIG2 significantly prolongs survival reduces expression of stemness and invasion markers, SOX2, VIMENTIN, and NUSAP1. In contrast, STAT5A knockdown fails to recapitulate these phenotypes, underscoring the role of OLIG2-STAT5B signaling in GSCs. These findings reveal a novel OLIG2-STAT5B transcriptional circuit that plays a role in GSC stemness maintenance, invasion, and tumor growth, particularly in EGFRvIII-driven gliomas. Targeting this axis may offer a new avenue for GBM treatment.

Abstract IDH-wildtype glioblastoma (GBM) is the most malignant primary adult brain tumor, with a five-year survival rate below 5%. Despite maximal treatment, including surgical resection, radiotherapy, and temozolomide (TMZ), recurrence is universal, driven by profound intratumoral heterogeneity, diffuse invasion, and therapy resistance. A hallmark of IDH-wildtype GBM is frequent alteration of epidermal growth factor receptor (EGFR), including amplification (50%), the EGFRvIII deletion variant (25%), and extracellular missense mutations such as G598V and A289V (10–15%). These missense mutations increase ligand affinity and receptor activity, yet their downstream signaling consequences and role in shaping the tumor phenotypes remain poorly defined. Recent pathway-based subclassifications identified four subtypes in IDH-wildtype GBM. Notably, neuronal (NEU) and glycolytic/plurimetabolic (GPM) subtypes predominate the invasive tumor margins. NEU programs are enriched in synaptic and neurodifferentiation signals, while GPM programs are associated with metabolic adaptation, angiogenesis, and immune evasion. Here, we show EGFR-G598V and EGFR-A289V mutations differentially regulate these invasive subtypes identities. Transcriptional analysis reveals EGFR G598V upregulates metabolic and inflammatory pathways consistent with the GPM phenotype, while EGFR A289V activates genes involved in neuronal differentiation and synaptic function, aligning with the NEU subtype. Phosphoproteomics and kinase-substrate analysis further uncovered distinct kinases to be functionally activated/inhibited in EGFR A289V-expressing cells and G598V cells. Mechanistic studies revealed EGFR A289V induces sustained EGFR phosphorylation at Y992, activating PLCγ2 via the SRC-family kinase LCK. This cascade drives calcium release, CAMK1 activation, and STAT5 phosphorylation, defining a novel EGFR A289V–LCK/PLCγ2/CAMK1/STAT5 axis. In addition, Y1068 phosphorylation is also constitutively active in EGFR A289V, resulting in increased BRAF activation. In contrast, EGFR G598V activates MAPK6 and CAMK2 inducing AKT2 activation, reinforcing GPM-associated signaling. These findings uncover mutation-specific signaling networks that drive subtype fate in invasive GBM and highlight unique pathway(s) as tractable targets for subtype-directed therapy in EGFR-mutant GBM.

Abstract PURPOSE Age over 40 years is commonly regarded as a “high risk” feature and potential indication for adjuvant chemoradiotherapy for patients with lower grade glioma in most clinical practice guidelines. However, these recommendations are based on retrospective observations and their subsequent incorporation into legacy clinical trials. It is unclear whether age remains a relevant prognostic factor in the molecular era. METHODS The Prospective Gliomas Research [PROGRES] Database contains individual patient-level data from 11 prospective clinical trials or observational registries, including seven international trials from the RTOG, EORTC, or Alliance cooperative groups. We determined the association of age with progression-free survival (PFS) in low grade or anaplastic glioma, stratified by isocitrate dehydrogenase (IDH1/2) status. We validated our findings in a separate multi-institutional retrospective cohort (Retrospective Glioma Research [REGRES] Database). RESULTS We identified 1,619 and 1,271 eligible patients in the PROGRES and REGRES cohorts, respectively. IDH-wildtype tumors were more common in patients >40 years (38% vs, 5%, odds ratio: 11.3 [95%CI 6.5 – 19.7]). Age was associated with PFS in IDH-wildtype (5-year PFS: 6% vs, 24%, P =.003) but not in IDH-mutant glioma (60% vs, 59%, P = 0.22, P value of interaction <.001). In IDH-wildtype tumors, older age predicted aggressive molecular features, including TERT promoter mutation (65% vs, 28%), EGFR amplification (41% vs, 15%), and chromosome +7/-10 alteration (57% vs, 25%). Age was not predictive of a benefit from chemoradiotherapy versus radiotherapy alone in IDH-mutant glioma. CONCLUSION Older age is a putative surrogate for IDH-wildtype status, but was not an independent predictor of progression in IDH-mutant lower grade glioma and did not predict benefit from chemoradiotherapy. In the absence of additional clinical or molecular risk factors, age alone should not be considered an indication for administration or deferral adjuvant treatment. Practice guidelines should be revised to reflect contemporary prognostic factors in the molecular era.

Abstract Imaging-based monitoring of gliomas is hampered by treatment-related changes, as well as the insidious growth of gliomas harboring the isocitrate dehydrogenase (IDH) mutation. The IDH mutation results in elevated D-2-hydroxyglutarate (D-2-HG), which is detectable in cerebrospinal fluid (CSF) that can be accessed longitudinally from multiple compartments, including cranially via CSF access devices. As such, we evaluated the early utility of CSF D-2-HG as a serially accessible biomarker for IDH-mutant gliomas. A CLIA-approved gas chromatography mass spectrometry assay was developed for CSF D- and L-2-HG. Lumbar and cranial CSF samples were collected from patients with IDH-mutant gliomas or IDH-wild-type brain tumors and non-tumor pathologies via surgical field collection, lumbar punctures, Ommaya reservoirs, and ventriculoperitoneal shunts. First, to confirm the ability of CSF D-2-HG to detect IDH-mutant gliomas, concentrations were compared between IDH-mutant gliomas and wild-type lesions. Cranial, but not lumbar, CSF D-2-HG distinguished primary IDH-mutant gliomas from IDH-wild type lesions (cranial AUC= 0.89, 95% confidence interval (CI)= 0.80-0.97); lumbar AUC= 0.52, 95% CI=0.28-0.76). Indeed, CSF D-2-HG was significantly higher in cranial than lumbar samples from IDH-mutant glioma patients (median lumbar=0.20 μM, cranial = 1.72 μM; p<0.0001), confirming a significantly elevated abundance of CSF D-2-HG only in cranial CSF from IDH-mutant gliomas. Then, response to cytoreduction was evaluated, demonstrating that cranial CSF D-2-HG levels decreased to 0.54x of baseline with resection in seventeen patients (p=0.0129). Changes in tumor-to-CSF contact impacted the serial evaluation of CSF D-2-HG prior to versus after chemoradiation in some patients. Nevertheless, in patients with grade 4 IDH-mutant astrocytomas, serial cranial CSF D-2-HG increased with disease progression and differentiated stability from pseudoprogression when tumor-CSF contact was sufficient. In conclusion, serial cranial CSF D-2-HG shows promise as a monitoring biomarker in patients with IDH-mutant gliomas, although anatomic variables must be considered in parallel to CSF D-2-HG evaluation.

Abstract Purpose: CD4 T-cell immunity is associated with improved survival in patients with breast cancer. We previously reported a phase I trial evaluating a folate receptor alpha (FRα) vaccine administered with cyclophosphamide (CP), demonstrating excellent safety and immunogenicity. To simplify the vaccine strategy for ease of use, we report here the results of a randomized phase II trial evaluating a lower vaccine dose alongside the original dose, administered with or without CP pretreatment. Patients and Methods: Patients with triple-negative breast cancer who completed all systemic and local therapies were assigned to receive either low-dose (825 μg) or high-dose (2.5 mg) vaccine peptides plus GM-CSF in six 4-week cycles, with or without CP prior to vaccination. Safety was monitored, and immunogenicity was evaluated before and after vaccination. Results: A total of 80 patients were dosed with vaccine, and 58 patients were evaluable for immunogenicity. Vaccination was well tolerated and elicited immunity in 83% of patients. Levels of FRα-specific T cells were high, persistent, and comparable with T-cell levels against tetanus toxoid. No differences in immunity were observed between the two doses. CP pretreatment also did not affect the immune responses. Recurrences occurred in eight evaluable patients and were unrelated to the treatment arm. No patients died during the study because of their disease. Conclusions: The reduced-dose FRα vaccine is as effective as the original dose and does not require CP to generate maximal immunity. These findings may inform the design of trials testing efficacy in triple-negative breast cancer or other cancers.

Importance Transthyretin amyloid cardiomyopathy (ATTR-CM) is underdiagnosed despite expanding treatment options. Objective To develop and evaluate an artificial intelligence (AI)–augmented clinical program for ATTR-CM detection. Design, Setting, and Participants This nonrandomized clinical trial involved constructing an AI model, ATTRACTnet, using electrocardiogram waveforms, echocardiographic measurements, demographics, and diagnosis codes for orthopedic manifestations of amyloidosis. A single-system, multisite, single-arm, open-label trial was conducted to evaluate its real-world performance. The model was trained and validated at a large academic referral site for ATTR-CM with external validation at an academic site. The trial was conducted as a single-system, multisite trial. Patients with left ventricular (LV) wall thickness 12 mm or more and an ATTRACTnet score 0.5 or higher were eligible. Exclusions included prior ATTR-CM testing, hypertrophic cardiomyopathy, expected life span less than 1 year, nursing home residence, advanced dementia, or LV wall thickness less than 14 mm explained by uncontrolled hypertension or moderate/severe aortic stenosis. Intervention Eligible patients were notified and offered nuclear scintigraphy testing, monoclonal protein testing, and follow-up care on agreement from the treating physician. Main Outcomes and Measure The primary outcome was a diagnosis of ATTR-CM by consensus criteria. ATTR-CM testing positivity was compared with historical and contemporary controls. Results ATTRACTnet was developed in an internal test set of 799 patients (mean [SD] age, 75.1 [11.1] years; 516 [64.7%] male and 283 [35.3%] female) using 5-fold cross-validation with an additional external test set of 422 patients. It had good discrimination for ATTR-CM detection with an area under the receiver operator characteristic curve of 0.85 (5-fold cross-validation, 0.77-0.85) in the internal set and 0.82 (95% CI, .81-0.83) in the external test set with similar performance in Hispanic, non-Hispanic Black, and non-Hispanic White patients. A total of 1471 patients were identified with positive AI model scores 0.5 or more during the study period, with 256 eligible patients who met study criteria. Of these patients, 50 underwent amyloidosis testing after physician and patient approval; 24 (48%) were diagnosed with ATTR-CM, and 21 (88%) initiated treatment within 3 months. The positivity rate was more than 2.8 times higher than historical controls (15.3%; 95% CI, 13.1%-17.9%; P < .001), with an 18% relative increase in new diagnoses vs the prior year. Conclusions and Relevance AI-augmented screening may improve ATTR-CM detection and identify patients who are missed by usual care. Prospective randomized trials are needed to determine if outcomes are improved. Trial Registration ClinicalTrials.gov Identifier: NCT06469372

Introduction Many clinical practice guidelines (CPGs) claim to use Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach, but its implementation varies. This suggests that CPG developers, methodologists and users would benefit from an instrument to evaluate the extent to which CPGs adhere to GRADE approach. Such a structured instrument is currently unavailable. Accordingly, this study will develop an evaluation instrument for assessing guideline adherence to the GRADE approach, which we have named ‘GRADE-Check’. The goal is to target items to which CPGs fail to adhere and that potentially have serious consequences resulting in inaccuracies in certainty of evidence and inappropriate direction or strength of recommendations, thereby discriminating across CPGs in issues of importance. Methods and analysis The panel will include up to 25 individuals with specific knowledge and expertise, including experienced authors, educators and methodologists on CPGs methodology and GRADE approach from relevant organisations. The instrument will focus on the key elements of GRADE, aiming for clarity for GRADE experts and non-GRADE experts to apply. The development process for GRADE-Check will consist of the following five phases: (1) recruitment of a panel of GRADE experts; (2) development of objectives and scope for the development of GRADE-Check and criteria for item selection; (3) generation of candidate items through a literature review and panel consultation; (4) panellist discussion to construct the initial draft and extended explanation manual and (5) user testing. Ethics and dissemination This study has been approved by the Medical Ethics Committee of Zhongnan Hospital of Wuhan University (no. (2025047K)). Our research findings will be published in peer-reviewed journal articles and presented at academic conferences. Additionally, the dissemination plan will include considerations for the development of implementation manuals, a dedicated project website and training tools.

Obstructive sleep disordered breathing (oSDB), including obstructive sleep apnea (OSA), frequently coexists with cleft lip and palate, velopharyngeal insufficiency (VPI), and other craniofacial conditions. Moreover, oSDB is often a key factor in decisions for primary or revision cleft palate or velpharyngeal surgery. Due to the frequency with which cleft and craniofacial team members care for patients with oSDB, this article provides a primer for OSA evaluation and management for team members. oSDB can have a significant impact on quality of life, health outcomes, surgical planning, and global development for all children, especially those with cleft and craniofacial conditions. In this review, oSDB screening and testing methods are discussed, including interpretation of OSA testing. Considerations for specific craniofacial populations are included, such as those with 22q11.2 deletion syndrome and Pierre-Robin sequence. The impact of VPI surgery on OSA is discussed. Finally, approaches to OSA management are reviewed, with attention to how standard management approaches may shift in children with cleft and craniofacial conditions. As awareness of oSDB in children has increased, including increased access to diagnostic testing, studies have shown certain patient populations are more likely to have OSA and would benefit from screening. Children with craniofacial conditions, including cleft palate and velopharyngeal insufficiency, have altered risk profiles which impact management considerations for OSA. This article provides an introduction to key concepts in oSDB screening, diagnositics, and management, which can be valuable for any professional assisting in the care of children with a cleft or craniofacial condition.

Importance Treatment for cognitive dysfunction due to postacute sequelae of long COVID (ie, symptoms of fatigue, malaise, weakness, confusion that persist beyond 12 weeks after an initial COVID infection) remains a significant unmet need. Objective To test evidence-based rehabilitation strategies for improving cognitive symptoms in persons with long COVID. Design, Setting, and Participants This was a 5-arm, multicenter, randomized clinical trial of 3 remotely delivered interventions conducted between August 17, 2023, and June 10, 2024. The study took place at 22 trial sites and included the screening of individuals with cognitive long COVID. Interventions Participants were randomized to 1 of 5 arms: adaptive computerized cognitive training (BrainHQ [Posit Science]), cognitive-behavioral rehabilitation involving both group and individual counseling sessions (PASC-Cognitive Recovery [PASC-CoRE]) paired with BrainHQ, and transcranial direct current stimulation (tDCS) paired with BrainHQ. Two comparator arms were included as follows: unstructured computer puzzles and games (active comparator) and sham tDCS paired with BrainHQ. The interventions occurred 5 times per week over 10 weeks. Main Outcomes and Measures Cognitive and behavioral in-person assessments were performed at baseline, midintervention, at the end of intervention, and 3 months after the end of the intervention. The primary outcome measure was the modified Everyday Cognition Scale 2 (ECog2) completed at the end of the intervention compared to the baseline visit based on participant self-report looking back over the prior 7 days. Results A total of 378 individuals were screened, from which there were 328 participants (median [IQR] age, 48.0 [37.0-58.0] years; 241 female [73.5%]; race: 15 Asian [4.6%], 47 Black [14.3%], and 235 White [71.6%]; ethnicity: 52 Hispanic [15.9%]). None of the 3 active interventions demonstrated benefits on the modified ECog2 in the intention-to-treat population by the end of the intervention period. The adjusted differences in mean change were 0.0 (95% CI, −0.2 to 0.2) for BrainHQ vs active comparator, 0.1 (95% CI, −0.1 to 0.3) for PASC-CoRE + BrainHQ vs active comparator, 0.0 (95% CI, −0.2 to 0.2) for tDCS-active + BrainHQ vs tDCS-sham + BrainHQ, and 0.1 (95% CI, −0.1 to 0.3) for PASC-CoRE + BrainHQ vs BrainHQ alone. Secondary participant-reported outcomes and neuropsychological tests showed no differential benefits for any treatment arm. All 5 arms demonstrated some improvements over time on the modified ECog2 and on secondary outcomes. There were no serious adverse events attributable to the interventions. Conclusions and Relevance This phase 2 randomized clinical trial failed to demonstrate differential benefits for online cognitive training, a structured cognitive rehabilitation program, and tDCS for cognitive long COVID. Trial Registration ClinicalTrials.gov Identifier: NCT05965739 .