Abstract Background Analysis of cerebrospinal fluid (CSF) circulating tumor DNA (ctDNA) has shown promise in multiple brain tumor types. The role of CSF-based liquid biopsy in early detection, surveillance, monitoring response and predicting relapse in patients with cancer predisposition syndromes has not been described. Methods We established a protocol for CSF-ctDNA collection from patients with hereditary replication repair deficiency (RRD) for surveillance, response assessment, and monitoring of gliomas post-therapy. ctDNA was assessed for single-nucleotide variants (SNVs), copy number variants(CNVs) and MMRD signatures (MMRDness score) using a combination of hybrid capture and low-pass whole-genome sequencing. Results A total of 84 CSF samples from 48 patients (CMMRD(32), Lynch (15), POLE(1)) were analyzed. Diagnostic sensitivity for high-grade glioma was 93% with 100% specificity. Three tumors were diagnosed >6 months prior to tumor evidence by diagnostic procedure. CSF-ctDNA status was significantly associated with survival - 58 samples were taken during RRD-glioma immunotherapy, overall survival was 91% and 15% for patients with negative versus positive CSF-ctDNA, respectively (p = 0.005). MMRDness scores were elevated at diagnosis and became negative in association with treatment response. These patients eventually had repeated negative CSF-ctDNA and are being monitored post therapy cessation. Ongoing monitoring of 12 patients post immunotherapy revealed 2 positive samples which were confirmed by biopsy upon positive imaging. One is alive upon changing immunotherapy. Comparative tumor and repeated CSF mutation analysis revealed unique patterns of spatial and temporal clonal evolution shaped by common drivers, new drivers and loss of antigens suggesting immune editing. Conclusions This is the first report of RRD patients benefiting from CSF-ctDNA for early detection, monitoring response and MRD with immunotherapy. In addition to CNV and SNV detection, MMRDness score is a unique quantitative tool to assess tumor burden during therapy. These data can be expanded to other cancer syndromes and malignant glioma management.

Background The safety and efficacy of stereotactic radiosurgery (SRS) in pediatric malignant central nervous system (CNS) tumors are not well established. This study evaluates disease control and toxicity outcomes following SRS in this population.Methods 53 SRS treatments in 35 pediatric patients (age <= 21) with primary (n = 27) or metastatic (n = 8) CNS malignancies treated between 2000 and 2023 were retrospectively reviewed. Kaplan-Meier and Cox proportional hazards analyses were performed.Results Median age at SRS was 12 years [interquartile range (IQR): 6.6-16.3]; 71% had prior external beam radiation to the brain. Median follow-up was 33 months. SRS was delivered in a single fraction in 91% (median dose 18 Gy). Median gross tumor volume (GTV) was 1.3cc (IQR: 0.36-4.1) for single-fraction and 23.2cc (IQR: 10.5-23.2) for fractionated SRS. The 1-year local control (LC) rate was 81.4%. 3-year intracranial progression-free survival (IC-PFS) and overall survival were 19.9% and 59.3%, respectively. Distant brain recurrence without local failure occurred in 54% at a median of 8 months. Patients treated for a single lesion had superior 3-year IC-PFS compared with those with 2-4 or >= 5 lesions (29.2% vs 6.7% and 0%, P = .01). Two patients (5%) developed grade >= 3 adverse radiation effects (AREs), both related to treatment effect within 1 year after SRS. No associations were found between ARE and dosimetric parameters or prior radiation.Conclusion SRS provides effective LC with low toxicity in pediatric malignant CNS tumors. Although there remains a substantial risk of distant CNS failures after SRS, all patients were able to receive additional salvage therapy including re-irradiation.

Childhood adamantinomatous craniopharyngiomas (ACP) are low-grade brain tumors associated with significant morbidity and treatment-related complications. Tocilizumab (TCZ), a monoclonal antibody targeting the IL-6 receptor, may mitigate tumor-associated inflammation, as IL-6 is elevated in both cystic and solid components of ACPs. Preliminary data suggests that TCZ may reduce or stabilize cystic volume within 3–6 months. This two-center retrospective series evaluated the impact of TCZ on tumor volume and clinical outcomes. Children with ACP received TCZ (10-12 mg/kg every 2-4 weeks) at different disease stages at UCSF (n=9) and the Princess Máxima Center (n=5). Tumor response was assessed by MRI at baseline and approximately every 3 months during treatment. Clinical effects were monitored through evaluations of vision, hypothalamic-pituitary function and adverse events. Fourteen children (median age 7.5 years (1–16y), 57% female) received TCZ with a median duration of 10.6 months (6months - 4 years). Thirteen had undergone surgery and/or cyst fenestration and five received radiation therapy prior TCZ initiation. A reduction in the cystic component was observed in 9 patients (64%), with 5 demonstrating stable solid components. Among these, 2 underwent cyst drainage during TCZ treatment, and 1 additionally received bevacizumab. The median time to best response was 6 months (3–12m). Cystic progression occurred in 5 patients (36%), 3 of these exhibited growth in the solid component after a median time of six months. One patient initially received tocilizumab monotherapy; bevacizumab was added after 1.5-3 years in response to cystic growth, resulting in sustained cyst stabilization. All patients had regular ophthalmologic evaluations. Two showed mild visual field improvement. Hypothalamic-pituitary function remained stable. Tocilizumab was well tolerated and associated with cystic volume reduction in a subset of patients. The addition of bevacizumab led to ongoing responses after single agent TCZ failed. Volumetric MRI assessment is ongoing.

PURPOSE:Pediatric recurrent medulloblastoma and atypical teratoid/rhabdoid tumor (ATRT) are largely incurable and warrant novel therapies. PNOC005 is a phase I clinical trial investigating the safety and tolerability of intratumoral or intrathecal administration of oncolytic measles virus (MV-NIS) in children and young adults with recurrent medulloblastoma or ATRT. PATIENTS AND METHODS:We investigated (i) the safety of a measles virus variant, MV-NIS, in a pediatric phase I study and (ii) the mechanisms of MV-NIS and the potential benefit of combination with immune checkpoint inhibition (ICI). Pediatric patients with recurrent medulloblastoma or ATRT were treated with intratumoral injections for local recurrence or via lumbar puncture for disseminated recurrence. We evaluated local immune responses to MV-NIS with and without ICI via single-cell and bulk RNA sequencing in an intracranial, immunocompetent, syngeneic murine model. RESULTS:MV-NIS given intratumorally or via repeat intrathecal dosing was safe. MV-NIS prolonged survival in murine models but did not demonstrate an additive benefit with ICI. No changes in tumor-infiltrating immune cell composition or activation were observed in response to MV-NIS treatment; however, MV-NIS induced local expression of neutralizing antibodies, complement cascade, and phagocytosis-related genes. CONCLUSIONS:This is the first trial investigating intratumoral as well as repeated intrathecal delivery of MV-NIS in children with medulloblastoma and ATRT. We show that the therapy is safe and well tolerated, with minimal adverse effects. Immune markers and biological correlates preliminarily indicate antiviral effects in tumors.

Supplementary Figure S5. Phenotypic states and cell type composition of MV-NIS-infected cells.

Supplementary Table S1. PNOC005 clinical trial sites.

Abstract BACKGROUND Children and young adults diagnosed with high-grade glioma (HGG) face extremely poor prognoses. Despite multiple clinical trials testing new treatments in this population, a survival advantage has yet to be achieved. Herein we assessed, in a single-arm, multi-center pilot trial, the feasibility of molecular profiling of primary HGG tumor tissue to create an individualized treatment plan with up to four FDA approved medications. METHODS Patients aged <21 years with newly diagnosed, localized, hemispheric HGG (Stratum A) or midline HGG (non-DIPG; Stratum B) were eligible. Tumor tissue underwent comprehensive molecular profiling (targeted gene panel, whole exome, and whole transcriptome sequencing). Based on detailed review of the molecular data by a dedicated tumor board, an individualized treatment plan that combined up to four FDA approved drugs was recommended. Circulating tumor DNA (ctDNA), imaging, and quality of life (QOL) measures were collected at multiple timepoints. RESULTS Fifty-five patients enrolled between 2018 and 2023 (median age 11 years [range 2-20], n=31 female, n=29 Stratum A). The most common integrated diagnoses included H3K27-altered diffuse midline glioma (n=17), H3/IDH-wildtype diffuse pediatric-type HGG (n=16), and H3G34-mutant diffuse hemispheric glioma (n=12). Median overall survival (OS) from the time of study enrollment was 26.5 months in Stratum A (lower 95% CI: 18.7) and 21.7 months in Stratum B (lower 95% CI: 16.8), with a median follow-up of 35.4 months for all patients (lower 95% CI: 32.5). The most common grade 3 or 4 treatment-related adverse events were decreased neutrophils (n=28), decreased platelets (n=22), and decreased white blood cells (n=16). As of December 2023, seven patients remain on therapy. Central imaging, ctDNA, and QOL analyses are underway. CONCLUSIONS A personalized treatment recommendation for children and young adults with HGG based on comprehensive transcriptomic and genomic analysis is feasible. Current survival data are encouraging, and molecular subgroup analyses are ongoing.

Abstract BACKGROUND H3K27-altered diffuse midline gliomas (DMGs) have poor prognosis with no standard of care therapy beyond radiation (RT). While RT prolongs survival, not all patients respond. Molecular biomarkers can be a strategy to predict RT response and thus patient outcomes. METHODS We performed a retrospective analysis of patients with biopsy-proven H3K27M-altered DMG between 2013-2023 from six US medical centers and the DMG Center in Zurich. Patients with tumor genomic sequencing and completion of RT were eligible. Genomic alterations were evaluated for somatic mutations, fusions, and chromosomal instability. Cox proportional hazard models were used to evaluate associations between genomic alterations and survival. Multivariate analysis included all significant variables at P <0.1 in initial analysis, including age at diagnosis, tumor location, TP53 and PIK3R1 alterations. RESULTS 297 patients (135 female; median age 8.3 years; range 0.2-71.4 years) were included. Median progression-free survival (PFS) and overall survival (OS) was 7.6 months (95%CI: 6.9-8.6) and 14.0 months (95%CI: 12.9-15.7). In univariate analysis, TP53 or PIK3R1 status correlated with shorter OS (TP53-mutant 12.5 vs. TP53-wildtype 17.5 months; HR=1.5; 95%CI: (1.1-2.0), P= 0.005); (PIK3R1-mutant 12.6 vs. 14.5 months; HR=1.9, 95%CI: (1.1-3.4), P = 0.03). Multivariate analysis corroborated TP53-mutant status with poorer OS (HR=1.5; 95%CI: (1.2-2.0), P= 0.003). Subgroup univariate analysis of pontine DMG patients showed poorer OS with TP53, NF1, or TERT mutations (TP53-mutant 11.9 vs. 15.2 months; HR= 1.6; 95%CI: (1.1-2.3), P= 0.02); (NF1-mutant 8.4 vs. 13.6 months; HR= 2.3; 95%CI: (1.1-5.0), P=0.03); (TERT 8.5 vs. 13.6 months; HR= 2.5; 95%CI: (1.1-5.7), P= 0.03). Subsequent multivariate analysis corroborated the association of TERT mutations with poorer OS in pontine DMG (HR=2.5; 95%CI:1.05, 5.9, P= 0.03). CONCLUSIONS This is the largest cohort of H3K27M-altered DMG patients with volumetric imaging analysis ongoing. Taken together, our data suggests these genetic alterations can risk-stratify patients for RT and future investigations of radiosensitizers targeting these alterations may improve survival.

Abstract BACKGROUND H3K27-altered diffuse midline gliomas (DMGs) have poor prognosis with no standard of care therapy beyond radiation (RT). While RT prologues survival, not all patients respond. Prior work has shown somatic TP53 mutations predict radiation resistance, and poorer outcomes. We report on a multi-center retrospective cohort study to identify molecular biomarkers of RT response in DMG patients. METHODS We performed retrospective chart reviews of patients with biopsy-proven H3K27M-altered DMG between 2013-2023 from six US medical centers and the DMG Center in Zurich. Patients with tumor genomic sequencing and completion of RT were eligible. Genomic alterations were evaluated for somatic mutations, fusions, and chromosomal instability. Cox proportional hazard models were used to evaluate associations between genomic alterations and survival. Multivariate analysis included all significant variables at P <0.1 in initial analysis, including age at diagnosis, tumor location, TP53 and PIK3R1 alterations. RESULTS 297 patients (135 female; median age 8.3years; range 0.2-71.4 years) were included. Median progression-free survival (PFS) and overall survival (OS) was 7.6months (95%-CI 6.9-8.6) and 14.0months (95%-CI 12.9-15.7). Univariate analysis identified TP53 or PIK3R1 status to be associated with shorter OS (TP53-mutant 12.5months vs. TP53-wildtype 17.5months; HR=1.5; 95%-CI (1.1-2.0), P=0.005); (PIK3R1-mutant 12.6months vs. PIK3R1-wildtype 14.5months; HR=1.9, 95%-CI (1.1-3.4), P=0.03). Multivariate analysis corroborated TP53 status association with shorter OS (HR=1.5; 95%-CI (1.2-2.0), P=0.003). Subgroup univariate analysis of pontine DMG patients showed reduced OS with TP53, NF1, or TERT mutations (TP53-mutant 11.9months vs. TP53-wildtype 15.2months; HR=1.6; 95%-CI (1.1-2.3), P=0.02); (NF1-mutant 8.4months vs. NF1-wildtype 13.6months; HR=2.3; 95%-CI (1.1-5.0), P=0.03); (TERT-mutant 8.5months vs. TERT-wildtype 13.6months; HR=2.5; 95%-CI (1.1-5.7), P=0.03). Subsequent multivariate analysis corroborated the association of TERT mutations with shorter OS in pontine DMG (HR=2.5; 95%-CI (1.05, 5.9), P=0.03). CONCLUSIONS This is one of the largest molecular characterized cohorts of H3K27M-altered DMG patients. DMGs with somatic TP53 or PIK3R1 mutations demonstrate inferior OS. Pontine DMG patients with somatic TP53, NF1 or TERT mutations demonstrate inferior OS.

Abstract BACKGROUND Despite multiple clinical trials in young patients with newly diagnosed high grade gliomas (HGG), survival remains poor. PNOC008 is a single-arm, multi-center pilot trial, investigating the feasibility, toxicity, and efficacy of a molecularly guided individualized treatment approach following radiotherapy. METHODS Patients aged ≤21 years with newly diagnosed, localized, hemispheric HGG (Stratum A) or non DIPG, diffuse midline glioma (DMG) (Stratum B) were eligible. Comprehensive molecular profiling (targeted gene panel, whole exome, and whole transcriptome sequencing) was performed on primary tumor tissue. The molecular data was reviewed by a dedicated tumor board that recommended an individualized treatment plan combining up to four FDA approved drugs. Patients were followed for toxicity and efficacy. Circulating tumor DNA (ctDNA), imaging, and quality of life (QOL) measures were collected at multiple timepoints. RESULTS Fifty-five HGG patients enrolled between 2018 and 2023 (median age 11 years [range 2-20], n=31 female, n=29 Stratum A), including H3K27-altered (n=17), H3/IDH-wildtype diffuse pediatric-type (n=16), and H3G34-mutant diffuse hemispheric glioma (n=12). In 44 patients that followed the recommended treatment, median overall survival (OS) from time of study enrollment was 26.5 months in Stratum A (lower 95% CI: 18.5) and 23.6 months in Stratum B (lower 95% CI: 16.8), and 30 months in H3G34-mutant patients (n=10, lower 95% CI:24.6) with median follow-up of 34.5 months for all patients (lower 95% CI: 32.2). The treatment recommendations most commonly included alkylator with targeted therapy combinations. The often novel drug combinations were generally well tolerated with grade 3 or 4 treatment-related adverse events being mostly hematologic. Central imaging, ctDNA, and QOL analyses are underway. CONCLUSIONS A personalized treatment approach using comprehensive transcriptomic and genomic analysis is feasible and well tolerated with encouraging survival data in children and young adults with HGG. Further analyses of molecular subgroups and correlatives are ongoing.

Background The objective of this study was to determine the safety, tolerability, and distribution of MTX110 (aqueous panobinostat) delivered by convection-enhanced delivery (CED) in patients with newly diagnosed diffuse intrinsic pontine glioma (DIPG) who completed focal radiation therapy (RT). Methods Patients with DIPG (2-21 years) were enrolled after RT. CED of MTX110 combined with gadoteridol was completed across 7 dose levels (DL) (30-90 & mu;M; volumes ranging from 3 mL to 2 consecutive doses of 6 mL). An accelerated dose escalation design was used. Distribution of infusate was monitored with real-time MR imaging. Repeat CED was performed every 4-8 weeks. Quality-of-life (QoL) assessments were obtained at baseline, every 3 months on therapy, and end of therapy. Results Between May 2018 and March 2020, 7 patients who received a total of 48 CED infusions, were enrolled (median age 8 years, range 5-21). Three patients experienced dose-limited toxicities. Four grade 3 treatment-related adverse events were observed. Most toxicities were transient new or worsening neurologic function. Median overall survival (OS) was 26.1 months (95% confidence interval: 14.8-not reached). Progression-free survival was 4-14 months (median, 7). Cumulative percentage of tumor coverage for combined CED infusions per patient ranged from 35.6% to 81.0%. Increased CED infusions were negatively associated with self-reported QoL assessments. Conclusion Repeat CED of MTX110 with real-time imaging with gadoteridol is tolerable for patients with DIPG. Median OS of 26.1 months compares favorably with historical data for children with DIPG. The results support further investigation of this strategy in a larger cohort.

Abstract BACKGROUND Subjects diagnosed with diffuse midline glioma (DMG) face extremely poor prognoses. PNOC DMG-ACT (DMG-Adaptive Combination Trial, PNOC022) is an open-label, multi-institutional, international trial that aims to investigate combination therapies for patients with DMG. Herein, we report on the combination study arm with ONC201, an orally available DRD2 and ClpP agonist, and paxalisib, a dual PI3K-mTOR inhibitor for patients who completed standard-of-care radiation (Cohort 2). METHODS Children and young adults aged 2-39 years were enrolled 4-14 weeks post-radiation and received maintenance therapy with weekly ONC201 (weight-based adult equivalent of 625 mg) and daily paxalisib (27mg/m2). Plasma and CSF samples for pharmacokinetics (PK) and circulating tumor DNA (ctDNA) were collected at multiple timepoints. RESULTS Sixty-eight patients with biopsy-proven DMG enrolled between November 2021 and June 2023 (median age 9 years [range 3-37], n=41 female [60%]). Site-determined tumor location includes 47 pontine (69%), 16 thalamic (24%), 3 spinal cord (4%) and 2 cerebellar (2%) DMGs. Median overall survival (OS) from time of diagnosis is 16.5 months (lower 95% CI 11.6 months) with a median follow-up time of 9.9 months (95% CI: 8.5, 11.4). The H3K27M mutation was found in 91% (n=50) and 9% were classified as H3K27-altered (n=5). TP53 was mutant in 50% (22/44 available). Most common grade 3 and above treatment-related adverse events were neutrophil count decreased (n=4); mucositis (n=3); and, colitis, DRESS, lymphocyte count decreased, hyperglycemia, hypokalemia (n=2). PK and ctDNA analyses are being finalized. CONCLUSIONS The current median OS is encouraging compared to historical controls. At the meeting, we will present updated molecular characterization and early biological correlates in association with clinical outcomes including toxicity, OS, PK, and central imaging confirmed progression-free survival.

Supplementary Figure from Upfront Biology-Guided Therapy in Diffuse Intrinsic Pontine Glioma: Therapeutic, Molecular, and Biomarker Outcomes from PNOC003

BACKGROUND: Diffuse intrinsic pontine glioma (DIPG) is a fatal brain tumor. Herein, we report on novel prognostic and predictive genomic biomarkers identified in PNOC003, a multi-center precision medicine trial for children and young adults diagnosed with DIPG. METHODS: Patients aged 3-25 years were enrolled on PNOC003 based on radiographic diagnosis of DIPG. Pre-treatment tumor biopsies were analyzed using tumor-normal whole-exome sequencing and mRNA-tumor sequencing to determine biology-informed, multi-agent therapy following radiation therapy (RT). Whole-genome sequencing was performed as an exploratory study aim. Genomic biomarkers were investigated to identify predictors of RT response and overall survival (OS) in patients with confirmed H3K27M-altered DIPG. Prognostic biomarkers were verified in a retrospective, H3K27M-altered diffuse midline glioma cohort (n=22) from the Children’s Brain Tumor Network (CBTN). RESULTS: Thirty patients enrolled on PNOC003 met molecular criteria for H3K27M-altered DIPG. TP53 was the most frequently altered driver gene (73%). Somatic alterations in PTEN>TP53>PDGFRA were independently associated with OS (P<0.05, in order of negative impact on survival). TP53 mutations associated with worse OS (TP53mut 11.1 mo [95% CI 8.7, 14]; TP53wt 13.3 mo [95% CI 11.8, NA]; P=3e-2), chromosomal instability (P=3e-3), and resistance to RT (P=6e-4). Moreover, loss of chromosome 10q, encoding tumor suppressor PTEN, was associated with worse OS, co-occurred with PTEN alterations, biallelic PTEN inactivation and loss of gene expression. The combination of TP53 alterations and loss of 10q/PTEN in H3K27M-altered DIPG was associated with the worst OS in a combined PNOC003 and CBTN cohort (TP53mut/10qdel, n=14, OS 8.4 mo [95% CI 7.4, 15.8]; TP53mut/10qwt, n=20, OS 13.1 mo [95% CI 10.1, 17.2]; TP53wt/10qwt, n=14, OS 15.5 mo [11.8, 29.4]; P=2e-3). CONCLUSION: PNOC003, a tissue-driven clinical trial, provided insights into prognostic and predictive genomic biomarkers and informed a novel molecular tumor classification system for H3K27M-altered DIPGs.