ARID1B as the top gene of interest in the pediatric chordoma cohort with significantly high dn/ds ratio.

BACKGROUND:Accurate molecular classification of medulloblastoma is critical for prognosis and treatment planning, but current methods rely on surgical tissue sampling and molecular profiling. This study evaluated whether in vivo proton MR spectroscopy (¹H-MRS) can provide noninvasive metabolic markers to support presurgical molecular group stratification. METHODS:In this single-center retrospective study, pretreatment ¹H-MRS data were analyzed from 95 pediatric patients with medulloblastoma (median age 7.4 years; 56 male). Single-voxel point-resolved spectroscopy (TE = 35 ms, TR = 1.5-2.0 s) were acquired during routine clinical MRI, adding approximately 5 minutes of scan time. Absolute metabolite concentrations and selected ratios were quantified using automated spectral fitting. Metabolic profiles were compared across molecular groups (group 3, n = 22; group 4, n = 35; sonic hedgehog [SHH], n = 26; wingless [WNT], n = 12) and assessed for qualitative concordance with prior ex vivo high-resolution HR-MAS NMR findings. Group differences were tested using Kruskal-Wallis with Dunn post hoc correction. RESULTS:Significant metabolic differences were observed across molecular groups, with strong group effects for taurine, creatine, choline, glutamate, and γ-aminobutyric acid (GABA) (all P < .0015). Taurine was elevated in group 3 and group 4 relative to SHH (log₂FC = 1.77 and 1.40, adjusted P < 4 × 10⁻⁵). SHH tumors exhibited lower creatine compared with group 3, group 4, and WNT (adjusted P < .05). Glutamate was higher in SHH than in WNT, while WNT tumors showed increased choline and GABA relative to other groups (adjusted P < .05). In vivo patterns were qualitatively concordant with ex vivo NMR findings. CONCLUSIONS:In vivo ¹H-MRS is a widely available, clinically feasible imaging biomarker that complements existing diagnostics and supports presurgical stratification of medulloblastoma.

List of rare ARID1B variants identified in the skull-base chordoma cohort.

Allele frequency of mtDNA variants in the skull base chordoma cohort across multiple samples. Comparisons are broken down by sample number and by variant class.

Rare nucleic variants of pediatric chordomas in COSMIC Cancer Census Genes

Rare nucleic variants of adult chordomas in COSMIC Cancer Census Genes, extracted from the original study, and lifted over from GRCh37 build to GRCh38 build.

Pediatric chordoma cohort counts of rare nonsynonymous variants determined from somatic exome sequencing in COSMIC Cancer Census Genes, broken down by variant class (VEP).

Rare mtDNA variants of pediatric chordomas

Abstract Outcomes for high-grade pediatric brain tumors are poor,butthere is optimism that chimeric antigen receptor (CAR) T cell therapycan improve prognosis. We present results from the firsttwo cohorts of a phase I clinical trial of IL13BBζ-CAR T cellsinfused weekly into the cerebral ventricles for children and young adults with recurrent or refractory high-grade neuromalignancies. Results Among the 18 patients (ependymoma =5, DIPG/DMG =9, pHGG=4) treated on trial, patients in cohort 2 (n = 15) received systemic lymphodepletion prior to first infusion; cohort 1 (n = 3) patients did not. The trial met its primary objectives of establishing feasibility, safety, and tolerability. There was one dose-limiting toxicity (Gr3 hypoxia, cohort 2). Secondary objectives included CAR T cell distribution and persistence in CSF and peripheral blood, response rates by RAPNO criteria, and overall survival. Patients received a median of 8 (range: 2-19) infusions. Common adverse events included headache, fever, and fatigue. Patients receiving lymphodepletion also experienced cytopenias. Two patients met protocol criteria for radiographic response, and half experienced radiographic decreases consistent with an anti-tumor response. Median overall survival from diagnosis for patients receiving lymphodepletion was 187m for patients with ependymoma, 20.5m for patients with midline glioma, and 30m for other patients. Median overall survival was 36m from diagnosis for patients not receiving lymphodepletion. Importantly, patients who did not receive lymphodepletion developed anti-CAR humoral and cellular immune responses detectable in the CSF and peripheral blood, and patients receiving lymphodepletion had higher numbers of CAR+T cells detected in CSF over the course of therapy. Conclusions This study demonstrates the safety, tolerability, and biological activity of locoregionally-delivered IL13BBζ-CAR T cells for children and young adults with neuromalignancies. Moreover, we show anti-CAR immune responses in patients not receiving lymphodepletion, but not in patients receiving systemic lymphodepletion. ClinicalTrials.gov:NCT04510051.

Abstract Background Approximately one third of pediatric patients with embryonal tumors of the central nervous system will experience a recurrence, with poor prognosis. Patients with recurrent or progressive disease may be treated with multi-drug metronomic antiangiogenic approaches. Methods We performed a retrospective review of 13 patients treated at a single institution from 2010-2023 using a multimodal metronomic antiangiogenic regimen which includes oral thalidomide, celecoxib and fenofibrate, and alternating etoposide/cyclophosphamide, intravenous bevacizumab, and alternating intraventricular etoposide, cytarabine, and topotecan. Results Toxicities included myelosuppression with at least one grade 3 hematologic toxicity in 11/12 (92%) patients. Other toxicities included infection, neurologic (cerebritis, irritability), hepatic (transaminitis), and a secondary leukemia. Seven patients required one or more drugs to be held or modified due to toxicity. The overall response rate was 69.2% (95% confidence interval (CI): 38.6%-90.9%) with median time to best response for patients who achieved CR or PR of 5 months. The median event-free survival (EFS) and overall survival (OS) time was 6 months (range: 1-21 months) and 15 months (range: 1.1-42.6 months), respectively. The 12-months EFS was 46.1% (95% CI: 19.2%-69.6%) with 12-months OS 83.9% (95% CI: 49.4%-95.7%). Conclusion Metronomic multi-drug approaches combining oral, intravenous and intraventricular/intrathecal therapy were generally well tolerated and provided a survival benefit.

BACKGROUND:Intracranial mesenchymal tumors, FET::CREB fusion-positive (ICMT), show fusions involving FET RNA-binding protein family genes (EWSR1 or FUS) and CREB family of transcription factors (ATF1, CREB1, or CREM). The methylation signature(s), gene expression characteristics, and clinical behavior of this important tumor type require further characterization. METHODS:We study the methylation profiles of 81 ICMT cases (61 newly profiled cases and 20 cases from publicly available sources). Clinicopathologic and genomic data were recorded for each case when available. RESULTS:ICMT showed a relatively distinct methylation signature compared to related tumors. Among the 65 cases where fusion types were documented, the identified fusions included EWSR1::ATF1 (25 cases), EWSR1::CREB1 (12 cases), EWSR1::CREM (21 cases), FUS::CREM (3 cases), and SMARCA2::CREM (4 cases). We confirmed the prior description of 2 distinct subgroups of ICMT (subclasses A and B). The majority of the cases belonged to subclass A (n = 69; 85%), which showed a higher median age compared to subclass B patients (26 years vs. 15 years). Subclass B cases (n = 12; 15%) showed shorter progression-free survival (P < 0.01). Gene expression analysis of ICMT showed key overexpressed markers in ICMT, with significant CREM overexpression regardless of fusion type, when compared to either meningioma alone or a larger group of CNS tumors. CONCLUSIONS:This work provides further characterization of ICMT as an important CNS mesenchymal neoplasm that is prone to tumor recurrence, showing 2 prognostically relevant methylation subclasses and warranting diagnostic distinction from other epigenetically and histologically related tumors. ICMTs show substantial overexpression of the CREM gene, independent of fusion type.

e22005 Background: WNT-activated medulloblastoma (WNT-MB), the least common molecular subtype of medulloblastoma with a putative brainstem cell of origin, is associated with excellent survival outcomes. Recent clinical trials have focused on decreasing morbidity related to craniospinal irradiation and chemotherapy. Little is known about the role of surgery in contributing to long-term morbidity in this population. Objective: To describe the incidence and persistence of post-operative neurologic morbidity in patients with WNT-MB. Methods: We conducted a retrospective cohort study of pediatric patients with molecularly confirmed WNT-MB who underwent tumor resection and treatment at our institution between 2003–2024. Demographic, clinical, treatment, and neurologic outcome data were extracted. Neurologic findings were assessed pre-operatively, post-operatively prior to the initiation of radiation or chemotherapy, and at last follow-up. Changes in functional neurologic exam domains and posterior fossa syndrome symptoms were analyzed. Results: One hundred sixty-four patients with medulloblastoma who had available molecular subgroup data were identified; 16 (10%) had WNT-activated tumors of whom 13 met study inclusion criteria. The median age at diagnosis was 9 years (range 5-16). Median follow-up time was 8 years (range 0.6-16.1). One patient died early in their treatment course; the remaining 12 patients (92%) were alive at last follow-up. New post-operative neurologic deficits were seen in all 13 patients (100%), most commonly involving loss of independent ambulation (n=12, 92%), motor function (n = 11, 85%), language (n=10, 77%), and cranial nerve abnormalities (n= 8, 62%). Post-operative posterior fossa symptoms were frequent, including dysarthria (n=10, 77%) and ataxia (n=9, 69%). Ten patients (77%) exhibited persistent deficits at last follow-up. Conclusions: Despite excellent overall survival, patients with WNT-MB experience notable post-operative neurologic morbidity. More conservative resection strategies should be considered in future prospective studies for this population.

Abstract Background Embryonal tumors with multi-layered rosettes (ETMRs) are rare pediatric brain tumors with poorly defined prognostic features, standard of care treatments or outcome data. Recent data suggest that high-dose chemotherapy and radiotherapy is correlated with improved survival when compared to chemotherapy alone. This case series is updated with new long-term survivorship information. Case Descriptions Four patients with newly diagnosed ETMRs were treated with 2 cycles of induction chemotherapy as per PBTC-026 using isotretinoin, vorinostat, vincristine, cisplatin, etoposide, cyclophosphamide with added intrathecal topotecan. Second look surgery was performed if not in complete remission (CR). Consolidation given with three cycles of marrow-ablative chemotherapy (carboplatin and thiotepa) with autologous hematopoietic cell rescue followed by focal irradiation and 12 cycles of maintenance chemotherapy with intrathecal topotecan, vorinostat and isotretinoin. Patient 1 was a 3-year-old female with right parietal tumor, localized, and achieved a gross total resection (GTR). Patient 2 was an 11-month-old male with posterior fossa tumor, localized, and achieved subtotal resection. Patient 3 was a 9-month-old female with posterior fossa tumor with near GTR, and metastasis to T12/L1 and L3/L4. Patient 4 was a 34-month-old male with a right frontal lobe tumor, localized and achieved GTR. Patient 1 is now 66 months from diagnosis and in CR. Patient 2 had second look surgery both after induction and consolidation but suffered neurological injury to the brainstem which led family to decline further therapy and is currently 62 months from diagnosis. Patient 3 had local disease recurrence following radiation therapy at 10 months post diagnosis and died from disease. Patient 4 remains in CR at 61 months from diagnosis and received maintenance without intrathecal chemotherapy. Conclusion This case series adds a cohort of patients in long-term remission to the current knowledge of intensive multi-modal therapy for newly diagnosed ETMR. Further study to define standard optimal treatments in this high-risk group of patients is warranted, but long-term remission can be achieved.

Supplementary Figure 1. Mitochondrial gene variant hotspot analysis in the pediatric chordoma cohort based on VEP variant classifications.

Abstract Radiation-induced high-grade gliomas (RIG) are rare, aggressive secondary malignancies arising after cranial or spinal irradiation for pediatric CNS tumors, typically associated with poor prognosis and limited therapeutic response. Here we describe a patient who achieved prolonged progression-free survival (PFS) after nivolumab therapy for recurrent RIG. A female patient, initially treated for WNT-activated medulloblastoma at 6 years of age with gross-total resection, craniospinal irradiation (23.4 Gy with a 30.6 Gy posterior fossa boost), and adjuvant chemotherapy, developed a left brachium pontis IDH-wildtype, TP53-mutant, CDKN2A-homozygous deleted RIG with complex intrachromosomal copy number alterations, 66 months after completing radiation. Following resection, she received 60 Gy focal irradiation in 30 fractions with concurrent temozolomide. A small residual nodule was identified on her post-radiation scan. She continued chemotherapy with adjuvant temozolomide ± CCNU complicated by prolonged, recurrent thrombocytopenia. The decision was made for further tumor resection, resulting in a 1-2mm residual nodule. A single dose of neo-adjuvant nivolumab (3 mg/kg) was administered prior to surgery and then followed by adjuvant nivolumab (3mg/kg) continued every two weeks. Treatment was briefly paused for cutaneous toxicity and successfully resumed with antihistamine premedication. At 36 months after nivolumab initiation, the patient remains radiographically stable with no evidence of progression or new neurological deficits. Her baseline left-sided weakness and ataxia remain unchanged. No immune-related adverse events have recurred since premedication was introduced. This case demonstrates an exceptional, durable response to neo-adjuvant followed by adjuvant PD-1 blockade in radiation-induced HGG, a setting typically refractory to immunotherapy. Prolonged PFS in this molecularly characterized tumor suggests potential immunogenic vulnerability in radiation-induced gliomas, possibly due to an altered tumor microenvironment. Further studies are warranted to identify biomarkers predictive of durable response in this rare and challenging entity.

Abstract Liquid biopsy (LB) is emerging in the care of pediatric central nervous system (CNS) tumors. Low-pass whole genome sequencing of cerebrospinal fluid (CSF) was validated as a clinical test (LBSeq4Kids) at our institution in 2022. We report 123 CSF LBs from 48 patients (ages 7 months to 20 years) with CNS tumors at timepoints ranging from diagnosis through therapy to radiographic recurrence. Histologic diagnoses included medulloblastoma (n = 24), other embryonal tumor (n = 8), glioma (n = 9), others (n = 7). Collection utilized lumbar puncture (n = 73), Ommaya (n = 30), ventricular shunt (n = 10), ventriculostomy (n = 4), and other (n = 6). Twenty-four patients (50%) underwent serial sampling (mean: 4.2; range 2–10+ samples), enabling longitudinal molecular monitoring. Circulating tumor DNA (ctDNA) was detected in 72/123 samples (59%), variable by timepoint: 14/22 (64%) at diagnosis, 8/18 (44%) at end-of-therapy (EOT), and 9/13 (69%) at radiographic recurrence. Compared to primary tumor sequencing, 31/48 (65%) LB results were different from primary tumors, with 17/48 (35%) detecting additional variants. In cases with serial sampling 35/63 (56%) of LBs demonstrated clonal evolution as evidenced by accrual of additional alterations. In patients with positive CSF cytology, 13/15 (87%) had detectable ctDNA and in patients with negative cytology 54/98 (55%) had positive ctDNA. In patients with radiographic disease at the time of LB, 44/60 (73%) had detectable ctDNA and in patients without radiographic disease, 25/56 (45%) had ctDNA present. Outcomes for 7 patients with positive ctDNA at EOT varied: 2 has disease recurrence and died of disease, 3 received continuation therapy without recurrence, and 2 remain recurrence-free off therapy (8 and 14 months off therapy). LBSeq4Kids is feasible across various collection methods, demonstrates higher sensitivity than cytology, and provides insights into tumor evolution and sub-radiographic disease.

Abstract DNA methylation profiling for classification of central nervous system (CNS) tumors currently relies on Infinium (EPIC) arrays that interrogate a fraction of CpG loci, require high DNA input that precludes cfDNA applications, and are subject to manufacturer-driven platform changes. Methylation sequencing offers a scalable alternative to EPIC arrays that is not constrained by fixed feature sets or high input requirements and supports high-resolution copy number profiling. We assessed the clinical feasibility of whole-genome enzymatic methylation sequencing (WG-EMSeq) for CNS tumor classification. We constructed a neural network CNS classifier model from publicly available array data (N = 7372) to perform hierarchical (methylation family and class) prediction. For WG-EMSeq, DNA from 163 pediatric CNS tumors, including medulloblastomas, ependymomas, and gliomas, was extracted from fresh frozen (N = 136) and FFPE (N = 27) samples; enzymatic conversion was performed using the NEBNext Enzymatic Methyl-seq workflow. Libraries were sequenced to a median depth of 34X. Paired-end reads were aligned with DRAGEN and resulting methylation profiles were used to generate methylation family and class predictions. Predicted methylation families and classes were compared to reference labels based on pathologic analysis, methylation array profiling, and/or chromosomal microarray. WG-EMSeq classifications achieved a macro-averaged one-versus-rest AUROC=0.96. 144 of 163 (88%) WG-EMSeq samples matched a methylation family. The prediction accuracy of family-matched samples was 90% (sensitivity=0.90, specificity=0.99, F1=0.91). For class concordance, samples without a family match were excluded. 71% of samples matched to a methylation class (sensitivity=0.92, specificity=0.99, F1=0.98). High classification accuracy was observed for medulloblastomas, ependymomas, and high-grade gliomas, with reduced accuracy in low-grade gliomas. WG-EMSeq copy number profiles were highly concordant with patient-matched chromosomal microarrays. Classifier predictions were consistent down to 1ng DNA input, indicating WG-EMSeq may be viable for cfDNA applications. Clinical validation of WG-EMSeq is currently in progress as an alternative to arrays for CNS tumor classification.

Pineoblastoma is a clinically aggressive childhood brain tumor composed of distinct molecular subgroups with divergent driver genes, demographics, and clinical outcomes. To identify developmental origins and mechanisms governing disease pathogenesis, we derive single-cell transcriptomes from pineal parenchymal tumors, aligning malignant cells with developmental counterparts to retrace cellular origins. Integrative computational analyses map pineoblastoma origins to transient, cycling pinealocyte progenitors during development. Lineage-specific perturbation of suspected drivers in the early pineal gland yields preclinical models representative of consensus molecular subgroups. Multi-omic characterization of patient tumors and these models uncover a tumor-associated photoreceptor signature (TAPS) common to pineoblastoma, retinoblastoma, and Group 3 medulloblastoma. Transcriptional activity of this signature within respective cellular origins establishes a developmental basis for molecular similarities between entities. Photoreceptor signature constituents are selective dependencies across these anatomically distinct central nervous system malignancies, motivating future studies evaluating developmentally encoded programs of malignancy as potential therapeutic liabilities.