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Liquid biopsy has emerged as a novel noninvasive tool in cancer diagnostics. While significant strides have been made in other malignancies using liquid biopsy for diagnosis, disease monitoring, and treatment selection, development of these assays has been more challenging for brain tumors. Recently, research in primary and metastatic brain tumors has begun to harness the potential utility of liquid biopsy—particularly using circulating tumor DNA (ctDNA). Initial studies to identify ctDNA in plasma of brain tumor patients have shown feasibility, but the yield of ctDNA is far below that for other malignancies. Attention has therefore turned to the cerebrospinal fluid (CSF) as a more robust source of ctDNA. This review discusses the unique considerations in liquid biopsy for glioma and places them in the context of the work to date. We address the utility of CSF liquid biopsy for diagnosis, longitudinal monitoring, tracking tumor evolution, clinical trial eligibility, and prognostication. We discuss the differences in assay requirements for each clinical application to best optimize factors such as efficacy, cost, and speed. Ultimately, CSF liquid biopsy has the potential to transform how we manage primary brain tumor patients.

Background The 2016 WHO classification described a subtype of midline gliomas harboring histone 3 (H3) K27M alterations, and the 2021 edition added a new subtype of hemispheric diffuse gliomas with H3 G34R/V mutations. The incidence and clinical behavior of leptomeningeal disease (LMD) in these patients is not well defined. Methods Retrospective study of patients with H3-altered gliomas diagnosed from 01/2012 to 08/2021; histone mutations were identified through next-generation sequencing (NGS) of tumor biopsy and/or cerebrospinal fluid (CSF). Results We identified 42 patients harboring H3 mutations (K27M mutations in 33 patients, G34R/V in 8, and both in one). Median age was 21 (4–70); 27 were male. LMD was diagnosed in 21/42 (50%) patients, corresponding to a three-year cumulative incidence of 44.7% (95% confidence interval (CI): 26.1%-63.4%) for the K27-mutant group and a one-year cumulative incidence of 37.5% in the G34-mutant group (95% CI: 0.01%-74.4%; no events after one year). Median time from tumor diagnosis to LMD was 12.9 months for H3-K27 patients and 5.6 months for H3-G34 patients. H3 mutation was detected in CSF in all patients with LMD who had NGS (8 H3-K27-mutant patients). In the H3-K27-mutant group, modeled risk of death was increased in patients who developed LMD (hazard ratio: 7.37, 95% CI: 2.98-18.23, p Conclusions In our cohort, 50% of patients developed LMD. Although further studies are needed, CSF ctDNA characterization may aid in identifying molecular tumor profiles in glioma patients with LMD, and neuroaxis imaging and CSF NGS should be considered for early LMD detection.

Background The prognosis for patients with pediatric high-grade glioma (pHGG) is poor despite aggressive multimodal therapy. Objective responses to targeted therapy with BRAF inhibitors have been reported in some patients with recurrent BRAF-mutant pHGG but are rarely sustained. Methods We performed a retrospective, multi-institutional review of patients with BRAF-mutant pHGG treated with off-label BRAF +/– MEK inhibitors as part of their initial therapy. Results Nineteen patients were identified, with a median age of 11.7 years (range, 2.3–21.4). Histologic diagnoses included HGG (n = 6), glioblastoma (n = 3), anaplastic ganglioglioma (n = 4), diffuse midline glioma (n = 3), high-grade neuroepithelial tumor (n = 1), anaplastic astrocytoma (n = 1), and anaplastic astroblastoma (n = 1). Recurrent concomitant oncogenic alterations included CDKN2A/B loss, H3 K27M, as well as mutations in ATRX, EGFR, and TERT. Eight patients received BRAF inhibitor monotherapy. Eleven patients received combination therapy with BRAF and MEK inhibitors. Most patients tolerated long-term treatment well with no grade 4–5 toxicities. Objective and durable imaging responses were seen in the majority of patients with measurable disease. At a median follow-up of 2.3 years (range, 0.3–6.5), three-year progression-free and overall survival for the cohort were 65% and 82%, respectively, and superior to a historical control cohort of BRAF-mutant pHGG patients treated with conventional therapies. Conclusions Upfront targeted therapy for patients with BRAF-mutant pHGG is feasible and effective, with superior clinical outcomes compared to historical data. This promising treatment paradigm is currently being evaluated prospectively in the Children’s Oncology Group ACNS1723 clinical trial.

Supplementary Table S1

Supplementary Figures and Legends

Figure S1 related to Figure 1 shows that MLN4924 reduces oligo-ubiquitylation of Lats2 and that Merlin-mutant schwannoma cells have a lower on-target sensitivity to MLN4924 than a genetically matched Merlin-positive Schwann line. Also shows that canonical YAP target genes are not expressed highly in FC-1801 cells.

Infant high-grade gliomas appear clinically distinct from their counterparts in older children, indicating that histopathologic grading may not accurately reflect the biology of these tumors. We have collected 241 cases under 4 years of age, and carried out histologic review, methylation profiling, and custom panel, genome, or exome sequencing. After excluding tumors representing other established entities or subgroups, we identified 130 cases to be part of an “intrinsic” spectrum of disease specific to the infant population. These included those with targetable MAPK alterations, and a large proportion of remaining cases harboring gene fusions targeting ALK (n = 31), NTRK1/2/3 (n = 21), ROS1 (n = 9), and MET (n = 4) as their driving alterations, with evidence of efficacy of targeted agents in the clinic. These data strongly support the concept that infant gliomas require a change in diagnostic practice and management.Significance:Infant high-grade gliomas in the cerebral hemispheres comprise novel subgroups, with a prevalence of ALK, NTRK1/2/3, ROS1, or MET gene fusions. Kinase fusion–positive tumors have better outcome and respond to targeted therapy clinically. Other subgroups have poor outcome, with fusion-negative cases possibly representing an epigenetically driven pluripotent stem cell phenotype.See related video: https://vimeo.com/438254885See related commentary by Szulzewsky and Cimino, p. 904.This article is highlighted in the In This Issue feature, p. 890

Supplementary Materials and Methods, Supplementary Figure Legends, Supplementary Table 1, Supplementary References

Inactivation of NF2/Merlin causes the autosomal-dominant cancer predisposition syndrome familial neurofibromatosis type 2 (NF2) and contributes to the development of malignant pleural mesothelioma (MPM). To develop a targeted therapy for NF2-mutant tumors, we have exploited the recent realization that Merlin loss drives tumorigenesis by activating the E3 ubiquitin ligase CRL4DCAF1, thereby inhibiting the Hippo pathway component Lats. Here, we show that MLN4924, a NEDD8-activating enzyme (NAE) inhibitor, suppresses CRL4DCAF1 and attenuates activation of YAP in NF2-mutant tumor cells. In addition, MLN4924 sensitizes MPM to traditional chemotherapy, presumably as a result of collateral inhibition of cullin-RING ubiquitin ligases (CRL) involved in DNA repair. However, even in combination with chemotherapy, MLN4924 does not exhibit significant preclinical activity. Further analysis revealed that depletion of DCAF1 or treatment with MLN4924 does not affect mTOR hyperactivation in NF2-mutant tumor cells, suggesting that loss of Merlin activates mTOR independently of CRL4DCAF1. Intriguingly, combining MLN4924 with the mTOR/PI3K inhibitor GDC-0980 suppresses the growth of NF2-mutant tumor cells in vitro as well as in mouse and patient-derived xenografts. These results provide preclinical rationale for the use of NAE inhibitors in combination with mTOR/PI3K inhibitors in NF2-mutant tumors.

Background Prospective data on maintenance therapy with bevacizumab for persons with NF2-related schwannomatosis (NF2-SWN) is lacking. In this prospective multicenter phase II study, we evaluated the efficacy, safety, and tolerability of bevacizumab for maintenance therapy in children and adults with NF2-SWN and hearing loss due to vestibular schwannomas (VS). Methods Following induction therapy, participants received bevacizumab 5 mg/kg every 3 weeks for 18 months. Participants were monitored for changes in hearing, tumor size, and quality of life (QOL), and for adverse events. Hearing loss was defined as a statistically significant decline in word recognition score (WRS) or pure-tone average compared to the study baseline; tumor growth was defined as >20% increase in volume compared to baseline. Results Twenty participants with NF2-SWN (median age 23.5 years; range, 12.5–62.5 years) with hearing loss in the target ear (median WRS 70%, range 2%–94%) received maintenance bevacizumab. Freedom from hearing loss in the target ear was 95% after 48 weeks, 89% after 72 weeks, and 70% after 98 weeks. Freedom from tumor growth in the target VS was 94% after 48 weeks, 89% after 72 weeks, and 89% after 98 weeks. NF2-related QOL remained stable for 98 weeks whereas tinnitus-related distress decreased. Maintenance bevacizumab was well tolerated, with 3 participants (15%) discontinuing treatment due to adverse events. Conclusions Maintenance bevacizumab (5 mg/kg every 3 weeks) is associated with high rates of hearing and tumor stability during 18 months of follow-up. No new unexpected adverse events related to bevacizumab were identified in this population.

Background The incidence and biology of IDH1/2 mutations in pediatric gliomas are unclear. Notably, current treatment approaches by pediatric and adult providers vary significantly. We describe the frequency and clinical outcomes of IDH1/2-mutant gliomas in pediatrics. Methods We performed a multi-institutional analysis of the frequency of pediatric IDH1/2-mutant gliomas, identified by next-generation sequencing (NGS). In parallel, we retrospectively reviewed pediatric IDH1/2-mutant gliomas, analyzing clinico-genomic features, treatment approaches, and outcomes. Results Incidence: Among 851 patients with pediatric glioma who underwent NGS, we identified 78 with IDH1/2 mutations. Among patients 0–9 and 10–21 years old, 2/378 (0.5%) and 76/473 (16.1%) had IDH1/2-mutant tumors, respectively. Frequency of IDH mutations was similar between low-grade glioma (52/570, 9.1%) and high-grade glioma (25/277, 9.0%). Four tumors were graded as intermediate histologically, with one IDH1 mutation. Outcome: Seventy-six patients with IDH1/2-mutant glioma had outcome data available. Eighty-four percent of patients with low-grade glioma (LGG) were managed observantly without additional therapy. For low-grade astrocytoma, 5-year progression-free survival (PFS) was 42.9% (95%CI:20.3–63.8) and, despite excellent short-term overall survival (OS), numerous disease-related deaths after year 10 were reported. Patients with high-grade astrocytoma had a 5-year PFS/OS of 36.8% (95%CI:8.8–66.4) and 84% (95%CI:50.1–95.6), respectively. Patients with oligodendroglioma had excellent OS. Conclusions A subset of pediatric gliomas is driven by IDH1/2 mutations, with a higher rate among adolescents. The majority of patients underwent upfront observant management without adjuvant therapy. Findings suggest that the natural history of pediatric IDH1/2-mutant glioma may be similar to that of adults, though additional studies are needed.

Purpose:Genomic aberrations in cell cycle and PI3K pathways are commonly observed in pediatric brain tumors. This study determined the MTD/recommended phase II dose (RP2D) of ribociclib and everolimus and characterized single-agent ribociclib concentrations in plasma and tumor in children undergoing resection.Patients and Methods:Patients were enrolled in the phase I study according to a rolling 6 design and received ribociclib and everolimus daily for 21 and 28 days, respectively. Surgical patients received ribociclib at the pediatric RP2D (350 mg/m2) for 7–10 days preoperatively followed by enrollment on the phase I study. Pharmacokinetics were analyzed for both cohorts.Results:Sixteen patients were enrolled on the phase I study (median age, 10.3 years; range, 3.9–20.4) and 6 patients in the surgical cohort (median age, 11.4 years; range: 7.2–17.1). Thirteen patients were enrolled at dose level 1 without dose-limiting toxicities (DLT). Two of the 3 patients at dose level 2 experienced DLTs (grade 3 hypertension and grade 4 alanine aminotransferase). The most common grade 3/4 toxicities were lymphopenia, neutropenia, and leukopenia. The RP2D of ribociclib and everolimus was 120 and 1.2 mg/m2 for 21 and 28 days, respectively. Steady-state everolimus exposures with ribociclib were 2.5-fold higher than everolimus administered alone. Ribociclib plasma, tumor concentrations, and cerebrospinal fluid (CSF) samples were collected. The mean tumor-to-plasma ratio of ribociclib was 19.8 (range, 2.22–53.4).Conclusions:Ribociclib and everolimus were well-tolerated and demonstrated pharmacokinetic properties similar to those in adults. Potential therapeutic ribociclib concentrations could be achieved in CSF and tumor tissue, although interpatient variability was observed.

Pharmacokinetic Data Analysis

BackgroundPreclinical studies have suggested that mTOR pathway signaling may be a potential therapeutic target for childhood ependymoma.MethodsA phase II clinical trial (ClinicalTrials.gov identifier: NCT02155920) of single-agent everolimus was performed to test the hypothesis that mTOR pathway inhibition would result in tumor responses for children with recurrent and/or progressive ependymomas.ResultsEleven subjects [sex: 4 females (36.4%); median age: 8 years (range: 2-15 years); race: 9 white; prior therapies: median 6 (range: 3-9)] were enrolled on the study. Ten primary tumors were located in the posterior fossa and one primary tumor was located in the spinal cord. Eight of 9 tumors were PF-A subtype epenydmomas. All subjects were treated with oral everolimus 4.5 mg/m2/day (each cycle = 28 days) that was titrated to achieve serum trough levels of 5-15 ng/ml. Overall, everolimus was well tolerated; except for a single event of grade 3 pneumonia, all adverse events were grade 1-2. No objective tumor responses were observed. Participating subjects experienced tumor progression and discontinued therapy after a median of 2 cycles of therapy (1 cycle = 2; 2 cycles = 6; 3, 4, and 8 cycles = 1 each).ConclusionsEverolimus does not appear to have activity for children with recurrent or progressive PF-A ependymoma.

Most pediatric central nervous system (CNS) tumors are located in eloquent anatomic areas, making surgical resection and, in some cases, even biopsy risky or impossible. This diagnostic predicament coupled with the move toward molecular classification for diagnosis has exposed an urgent need to develop a minimally invasive means to obtain diagnostic information. In non-CNS solid tumors, the detection of circulating tumor DNA (ctDNA) in plasma and other bodily fluids has been incorporated into routine practice and clinical trial design for selection of molecular targeted therapy and longitudinal monitoring. For primary CNS tumors, however, detection of ctDNA in plasma has been challenging. This is likely related at least in part to anatomic factors such as the blood-brain barrier. Due to the proximity of primary CNS tumors to the cerebrospinal fluid (CSF) space, our group and others have turned to CSF as a rich alternative source of ctDNA. Although multiple studies at this time have demonstrated the feasibility of CSF ctDNA detection across multiple types of pediatric CNS tumors, the optimal role and utility of CSF ctDNA in the clinical setting has not been established. This review discusses the work-to-date on CSF ctDNA liquid biopsy in pediatric CNS tumors and the associated technical challenges, and reviews the promising opportunities that lie ahead for integration of CSF ctDNA liquid biopsy into clinical care and clinical trial design.

Stage 4a metastatic retinoblastoma (RB) is curable with intensive multimodality therapy including myeloablative chemotherapy with autologous stem cell transplant (HDC-ASCT) and involved field radiation therapy (IFRT). To our knowledge, no data exist on the impact of (a) pre-ASCT disease status, and (b) IFRT to sites of metastatic disease post ASCT on survival.We retrospectively reviewed patients with stage 4a metastatic RB who underwent induction chemotherapy followed by HDC-ASCT, with or without IFRT, to residual tumor sites at Memorial Sloan Kettering Cancer Center (MSKCC) (n = 24).The degree of postinduction response prior to ASCT did not affect outcome, with 5-year overall survival (OS) of 68% and 86% in patients who achieved complete response (CR) and very good partial response (VGPR)/partial response (PR) prior to ASCT, respectively. IFRT administered post ASCT in patients with possible residual bony metastatic disease increases the likelihood of developing osteosarcoma in the radiation field.OS for patients with stage 4a metastatic RB treated with ASCT with VGPR or PR to pretransplant chemotherapy was not significantly different from patients with CR. In addition, IFRT does not seem to be required for bony disease control and increased the likelihood of developing osteosarcoma.

PURPOSE: The prognosis for SHH-medulloblastoma (MB) patients with Li-Fraumeni syndrome (LFS) is poor. Due to lack of comprehensive data for these patients, it is challenging to establish effective therapeutic recommendations. We here describe the largest retrospective cohort of pediatric LFS SHH-MB patients to date and their clinical outcomes. PATIENTS AND METHODS: N=31 patients with LFS SHH-MB were included in this retrospective multicenter study. TP53 variant type, clinical parameters including treatment modalities, event-free survival (EFS) and overall survival (OS), as well as recurrence patterns and incidence of secondary neoplasms, were evaluated. RESULTS: All LFS-MBs were classified as SHH subgroup, in 30/31 cases based on DNA methylation analysis. The majority of constitutional TP53 variants (72%) represented missense variants, and all except two truncating variants were located within the DNA-binding domain. 54% were large cell anaplastic, 69% gross totally resected and 81% had M0 status. The 2-(y)ear and 5-(y)ear EFS were 26% and 8,8%, respectively, and 2y- and 5y-OS 40% and 12%. Patients who received post-operative radiotherapy (RT) followed by chemotherapy (CT) showed significantly better outcomes (2y-EFS:43%) compared to patients who received CT before RT (30%) (p

BackgroundRecent genomic studies have shed light on the biology and inter-tumoral heterogeneity underlying pineal parenchymal tumors, in particular pineoblastomas (PBs) and pineal parenchymal tumors of intermediate differentiation (PPTIDs). Previous reports, however, had modest sample sizes and lacked power to integrate molecular and clinical findings. The different proposed subgroup structures also highlighted a need to reach consensus on a robust and relevant classification system.MethodsWe performed a meta-analysis on 221 patients with molecularly characterized PBs and PPTIDs. DNA methylation profiles were analyzed through complementary bioinformatic approaches and molecular subgrouping was harmonized. Demographic, clinical and genomic features of patients and samples from these pineal tumor subgroups were annotated.FindingsFour clinically and biologically relevant consensus PB subgroups were defined: PB-miRNA1 (n=96), PB-miRNA2 (n=23), PB-MYC/FOXR2 (n=34) and PB-RB1 (n=25); with PPTID (n=43) remaining as a molecularly distinct entity. Genomic and transcriptomic profiling allowed the characterization of oncogenic drivers for individual subgroups, specifically, alterations in the microRNA processing pathway in PB-miRNA1/2, MYC amplification and FOXR2 overexpression in PB-MYC/FOXR2, RB1 alteration in PB-RB1, and KBTBD4 insertion in PPTID. Age at diagnosis, sex predilection and metastatic status varied significantly among tumor subgroups. While patients with PB-miRNA2 and PPTID had superior outcome, survival was intermediate for patients with PB-miRNA1, and dismal for those with PB-MYC/FOXR2 and PB-RB1.InterpretationWe systematically interrogated the clinical and molecular heterogeneity within pineal parenchymal tumors and proposed a consensus nomenclature for disease subgroups, laying the groundwork for future studies as well as routine use in tumor classification.

Purpose: Genomic aberrations in cell cycle and PI3K pathways are commonly observed in pediatric brain tumors. This study determined the MTD/recommended phase II dose (RP2D) of ribociclib and everolimus and characterized single-agent ribociclib concentrations in plasma and tumor in children undergoing resection. Patients and Methods: Patients were enrolled in the phase I study according to a rolling 6 design and received ribociclib and everolimus daily for 21 and 28 days, respectively. Surgical patients received ribociclib at the pediatric RP2D (350 mg/m2) for 7–10 days preoperatively followed by enrollment on the phase I study. Pharmacokinetics were analyzed for both cohorts. Results: Sixteen patients were enrolled on the phase I study (median age, 10.3 years; range, 3.9–20.4) and 6 patients in the surgical cohort (median age, 11.4 years; range: 7.2–17.1). Thirteen patients were enrolled at dose level 1 without dose-limiting toxicities (DLT). Two of the 3 patients at dose level 2 experienced DLTs (grade 3 hypertension and grade 4 alanine aminotransferase). The most common grade 3/4 toxicities were lymphopenia, neutropenia, and leukopenia. The RP2D of ribociclib and everolimus was 120 and 1.2 mg/m2 for 21 and 28 days, respectively. Steady-state everolimus exposures with ribociclib were 2.5-fold higher than everolimus administered alone. Ribociclib plasma, tumor concentrations, and cerebrospinal fluid (CSF) samples were collected. The mean tumor-to-plasma ratio of ribociclib was 19.8 (range, 2.22–53.4). Conclusions: Ribociclib and everolimus were well-tolerated and demonstrated pharmacokinetic properties similar to those in adults. Potential therapeutic ribociclib concentrations could be achieved in CSF and tumor tissue, although interpatient variability was observed.

Background Safe sampling of central nervous system tumor tissue for diagnostic purposes may be difficult if not impossible, especially in pediatric patients, and an unmet need exists to develop less invasive diagnostic tests. Methods We report our clinical experience with minimally invasive molecular diagnostics using a clinically validated assay for sequencing of cerebrospinal fluid (CSF) cell-free DNA (cfDNA). All CSF samples were collected as part of clinical care, and results reported to both clinicians and patients/families. Results We analyzed 64 CSF samples from 45 pediatric, adolescent and young adult (AYA) patients (pediatric = 25; AYA = 20) with primary and recurrent brain tumors across 12 histopathological subtypes including high-grade glioma (n = 10), medulloblastoma (n = 10), pineoblastoma (n = 5), low-grade glioma (n = 4), diffuse leptomeningeal glioneuronal tumor (DLGNT) (n = 4), retinoblastoma (n = 4), ependymoma (n = 3), and other (n = 5). Somatic alterations were detected in 30/64 samples (46.9%) and in at least one sample per unique patient in 21/45 patients (46.6%). CSF cfDNA positivity was strongly associated with the presence of disseminated disease at the time of collection (81.5% of samples from patients with disseminated disease were positive). No association was seen between CSF cfDNA positivity and the timing of CSF collection during the patient’s disease course. Conclusions We identified three general categories where CSF cfDNA testing provided additional relevant diagnostic, prognostic, and/or therapeutic information, impacting clinical assessment and decision making: (1) diagnosis and/or identification of actionable alterations; (2) monitor response to therapy; and (3) tracking tumor evolution. Our findings support broader implementation of clinical CSF cfDNA testing in this population to improve care.

Infant high-grade gliomas appear clinically distinct from their counterparts in older children, indicating that histopathologic grading may not accurately reflect the biology of these tumors. We have collected 241 cases under 4 years of age, and carried out histologic review, methylation profiling, and custom panel, genome, or exome sequencing. After excluding tumors representing other established entities or subgroups, we identified 130 cases to be part of an “intrinsic” spectrum of disease specific to the infant population. These included those with targetable MAPK alterations, and a large proportion of remaining cases harboring gene fusions targeting ALK (n = 31), NTRK1/2/3 (n = 21), ROS1 (n = 9), and MET (n = 4) as their driving alterations, with evidence of efficacy of targeted agents in the clinic. These data strongly support the concept that infant gliomas require a change in diagnostic practice and management. Significance: Infant high-grade gliomas in the cerebral hemispheres comprise novel subgroups, with a prevalence of ALK, NTRK1/2/3, ROS1, or MET gene fusions. Kinase fusion–positive tumors have better outcome and respond to targeted therapy clinically. Other subgroups have poor outcome, with fusion-negative cases possibly representing an epigenetically driven pluripotent stem cell phenotype. See related video: https://vimeo.com/438254885 See related commentary by Szulzewsky and Cimino, p. 904. This article is highlighted in the In This Issue feature, p. 890

Tumors of the pineal region comprise several different entities with distinct clinical and histopathological features. Whereas some entities predominantly affect adults, pineoblastoma (PB) constitutes a highly aggressive malignancy of childhood with a poor outcome. PBs mainly arise sporadically, but may also occur in the context of cancer predisposition syndromes including DICER1 and RB1 germline mutation. With this study, we investigate clinico-pathological subgroups of pineal tumors and further characterize their biological features. We performed genome-wide DNA methylation analysis in 195 tumors of the pineal region and 20 normal pineal gland controls. Copy-number profiles were obtained from DNA methylation data; gene panel sequencing was added for 93 tumors and analysis was further complemented by miRNA sequencing for 22 tumor samples. Unsupervised clustering based on DNA methylation profiling separated known subgroups, like pineocytoma, pineal parenchymal tumor of intermediate differentiation, papillary tumor of the pineal region and PB, and further distinct subtypes within these groups, including three subtypes within the core PB subgroup. The novel molecular subgroup Pin-RB includes cases of trilateral retinoblastoma as well as sporadic pineal tumors with RB1 alterations, and displays similarities with retinoblastoma. Distinct clinical associations discriminate the second novel molecular subgroup PB-MYC from other PB cases. Alterations within the miRNA processing pathway (affecting DROSHA, DGCR8 or DICER1) are found in about two thirds of cases in the three core PB subtypes. Methylation profiling revealed biologically distinct groups of pineal tumors with specific clinical and molecular features. Our findings provide a foundation for further clinical as well as molecular and functional characterization of PB and other pineal tumors, including the role of miRNA processing defects in oncogenesis.

Large-scale molecular profiling studies in recent years have shown that central nervous system (CNS) tumors display a much greater heterogeneity in terms of molecularly distinct entities, cellular origins and genetic drivers than anticipated from histological assessment. DNA methylation profiling has emerged as a useful tool for robust tumor classification, providing new insights into these heterogeneous molecular classes. This is particularly true for rare CNS tumors with a broad morphological spectrum, which are not possible to assign as separate entities based on histological similarity alone. Here, we describe a molecularly distinct subset of predominantly pediatric CNS neoplasms (n = 60) that harbor PATZ1 fusions. The original histological diagnoses of these tumors covered a wide spectrum of tumor types and malignancy grades. While the single most common diagnosis was glioblastoma (GBM), clinical data of the PATZ1-fused tumors showed a better prognosis than typical GBM, despite frequent relapses. RNA sequencing revealed recurrent MN1:PATZ1 or EWSR1:PATZ1 fusions related to (often extensive) copy number variations on chromosome 22, where PATZ1 and the two fusion partners are located. These fusions have individually been reported in a number of glial/glioneuronal tumors, as well as extracranial sarcomas. We show here that they are more common than previously acknowledged, and together define a biologically distinct CNS tumor type with high expression of neural development markers such as PAX2, GATA2 and IGF2. Drug screening performed on the MN1:PATZ1 fusion-bearing KS-1 brain tumor cell line revealed preliminary candidates for further study. In summary, PATZ1 fusions define a molecular class of histologically polyphenotypic neuroepithelial tumors, which show an intermediate prognosis under current treatment regimens.

In contrast to adults, meningiomas are uncommon tumors in childhood and adolescence. Whether adult and pediatric meningiomas differ on a molecular level is unclear. Here we report detailed genomic analyses of 37 pediatric meningiomas by sequencing and DNA methylation profiling. Histologically, the series was dominated by meningioma subtypes with aggressive behavior, with 70% of patients suffering from WHO grade II or III meningiomas. The most frequent cytogenetic aberrations were loss of chromosomes 22 (23/37 [62%]), 1 (9/37 [24%]), 18 (7/37 [19%]), and 14 (5/37 [14%]). Tumors with NF2 alterations exhibited overall increased chromosomal instability. Unsupervised clustering of DNA methylation profiles revealed separation into three groups: designated group 1 composed of clear cell and papillary meningiomas, whereas group 2A comprised predominantly atypical meningiomas and group 2B enriched for rare high-grade subtypes (rhabdoid, chordoid). Meningiomas from NF2 patients clustered exclusively within groups 1 and 2A. When compared with a dataset of 105 adult meningiomas, the pediatric meningiomas largely grouped separately. Targeted panel DNA sequencing of 34 tumors revealed frequent NF2 alterations, while other typical alterations found in adult non-NF2 tumors were absent. These data demonstrate that pediatric meningiomas are characterized by molecular features distinct from adult tumors.

PURPOSE: By incorporating major developments in genetics, ophthalmology, dermatology, and neuroimaging, to revise the diagnostic criteria for neurofibromatosis type 1 (NF1) and to establish diagnostic criteria for Legius syndrome (LGSS). METHODS: We used a multistep process, beginning with a Delphi method involving global experts and subsequently involving non-NF experts, patients, and foundations/patient advocacy groups. RESULTS: We reached consensus on the minimal clinical and genetic criteria for diagnosing and differentiating NF1 and LGSS, which have phenotypic overlap in young patients with pigmentary findings. Criteria for the mosaic forms of these conditions are also recommended. CONCLUSION: The revised criteria for NF1 incorporate new clinical features and genetic testing, whereas the criteria for LGSS were created to differentiate the two conditions. It is likely that continued refinement of these new criteria will be necessary as investigators (1) study the diagnostic properties of the revised criteria, (2) reconsider criteria not included in this process, and (3) identify new clinical and other features of these conditions. For this reason, we propose an initiative to update periodically the diagnostic criteria for NF1 and LGSS. ispartof: GENETICS IN MEDICINE vol:23 issue:8 pages:1506-1513 ispartof: location:United States status: published

The spectrum of germline predisposition in pediatric cancer continues to be realized. Here we report 751 solid tumor patients who underwent prospective matched tumor-normal DNA sequencing and downstream clinical use (clinicaltrials.gov NCT01775072). Germline pathogenic and likely pathogenic (P/LP) variants were reported. One or more P/LP variants were found in 18% (138/751) of individuals when including variants in low, moderate, and high penetrance dominant or recessive genes, or 13% (99/751) in moderate and high penetrance dominant genes. 34% of high or moderate penetrance variants were unexpected based on the patient's diagnosis and previous history. 76% of patients with positive results completed a clinical genetics visit, and 21% had at least one relative undergo cascade testing as a result of this testing. Clinical actionability additionally included screening, risk reduction in relatives, reproductive use, and use of targeted therapies. Germline testing should be considered for all children with cancer.

The application of high-throughput sequencing approaches including paired tumor/normal sampling with therapeutic intent has demonstrated that 8%-19% of pediatric CNS tumor patients harbor a germline alteration in a classical tumor predisposition gene (NF1, P53). In addition, large-scale germline sequencing studies in unselected cohorts of pediatric neuro-oncology patients have demonstrated novel candidate tumor predisposition genes (ELP1 alterations in sonic hedgehog medulloblastoma). Therefore, the possibility of an underlying tumor predisposition syndrome (TPS) should be considered in all pediatric patients diagnosed with a CNS tumor which carries critical implications including accurate prognostication, selection of optimal therapy, screening, risk reduction, and family planning. The Pediatric Cancer Working Group of the American Association for Cancer Research (AACR) recently published consensus screening recommendations for children with the most common TPS. In this review, we provide an overview of the most relevant as well as recently identified TPS associated with the most frequently encountered pediatric CNS tumors with an emphasis on pathogenesis, genetic testing, clinical features, and treatment implications.

Outcomes in children and adolescents with recurrent or progressive high-grade glioma are poor, with a historical median overall survival of 5.6 months. Pediatric high-grade gliomas are largely immunologically silent or "cold," with few tumor-infiltrating lymphocytes. Preclinically, pediatric brain tumors are highly sensitive to oncolytic virotherapy with genetically engineered herpes simplex virus type 1 (HSV-1) G207, which lacks genes essential for replication in normal brain tissue.We conducted a phase 1 trial of G207, which used a 3+3 design with four dose cohorts of children and adolescents with biopsy-confirmed recurrent or progressive supratentorial brain tumors. Patients underwent stereotactic placement of up to four intratumoral catheters. The following day, they received G207 (10Twelve patients 7 to 18 years of age with high-grade glioma received G207. No dose-limiting toxic effects or serious adverse events were attributed to G207 by the investigators. Twenty grade 1 adverse events were possibly related to G207. No virus shedding was detected. Radiographic, neuropathological, or clinical responses were seen in 11 patients. The median overall survival was 12.2 months (95% confidence interval, 8.0 to 16.4); as of June 5, 2020, a total of 4 of 11 patients were still alive 18 months after G207 treatment. G207 markedly increased the number of tumor-infiltrating lymphocytes.Intratumoral G207 alone and with radiation had an acceptable adverse-event profile with evidence of responses in patients with recurrent or progressive pediatric high-grade glioma. G207 converted immunologically "cold" tumors to "hot." (Supported by the Food and Drug Administration and others; ClinicalTrials.gov number, NCT02457845.).

BACKGROUND: H3 K27M-mutant diffuse midline glioma is a fatal malignancy with no proven medical therapies. The entity predominantly occurs in children and young adults. ONC201 is a small molecule selective antagonist of dopamine receptor D2/3 (DRD2/3) with an exceptional safety profile. Following up on a durable response in the first H3 K27M-mutant diffuse midline glioma patient who received ONC201 (NCT02525692), an expanded access program was initiated. METHODS: Patients with H3 K27M-mutant gliomas who received at least prior radiation were eligible. Patients with leptomeningeal spread were excluded. All patients received open-label ONC201 orally once every week. Safety, radiographic assessments, and overall survival were regularly assessed at least every 8 weeks by investigators. As of August 2018, a total of 18 patients with H3 K27M-mutant diffuse midline glioma or DIPG were enrolled to single patient expanded access ONC201 protocols. Among the 18 patients: seven adult (>20 years old) and seven pediatric (

Neurofibromatosis type 2 (NF2) is an autosomal-dominant disorder characterized by the development of bilateral vestibular schwannomas. The NF2 gene encodes the tumor suppressor merlin, and loss of merlin activity promotes tumorigenesis and causes NF2. Cellular redox signaling has been implicated in different stages of tumor development. Among reactive nitrogen species, peroxynitrite is the most powerful oxidant produced by cells. We recently showed that peroxynitrite-mediated tyrosine nitration down-regulates mitochondrial metabolism in tumor cells. However, whether peroxynitrite supports a metabolic shift that could be exploited for therapeutic development is unknown. Here, we show that vestibular schwannomas from NF2 patients and human, merlin-deficient (MD) Schwann cells have high levels of endogenous tyrosine nitration, indicating production of peroxynitrite. Furthermore, scavenging or inhibiting peroxynitrite formation significantly and selectively decreased survival of human and mouse MD-Schwann cells. Using multiple complementary methods, we also found that merlin deficiency leads to a reprogramming of energy metabolism characterized by a peroxynitrite-dependent decrease of oxidative phosphorylation and increased glycolysis and glutaminolysis. In MD-Schwann cells, scavenging of peroxynitrite increased mitochondrial oxygen consumption and membrane potential, mediated by the up-regulation of the levels and activity of mitochondrial complex IV. This increase in mitochondrial activity correlated with a decrease in the glycolytic rate and glutamine dependence. This is the first demonstration of a peroxynitrite-dependent reprogramming of energy metabolism in tumor cells. Oxidized proteins constitute a novel target for therapeutic development not only for the treatment of NF2 schwannomas but also other tumors in which peroxynitrite plays a regulatory role.

BACKGROUND: The prognosis for pediatric high-grade glioma (pHGG) is poor despite aggressive multi-modal therapy. Objective responses to targeted therapy with BRAF inhibitors have been reported in some patients with recurrent BRAF-mutant pHGG but are rarely sustained. METHODS: We performed a retrospective, multi-institutional review of patients with BRAF-mutant pHGG treated with off-label BRAF +/- MEK inhibitors as part of their initial therapy. RESULTS: Nineteen patients were identified, with a median age of 10.7 years (range: 1.8–20.3). Histologic diagnoses included HGG (n=6), glioblastoma (n=3), anaplastic ganglioglioma (n=4), diffuse midline glioma (n=3), high-grade neuroepithelial tumor (n=1), anaplastic astrocytoma (n=1), and anaplastic astroblastoma (n=1). Recurrent concomitant oncogenic alterations included CDKN2A/B loss, H3 K27M, as well as mutations in ATRX, EGFR and TERT. Eight patients received BRAF inhibitor monotherapy. Eleven patients received combination therapy with BRAF and MEK inhibitors. Most patients tolerated long-term treatment well with no grade 4–5 toxicities. Objective and durable imaging responses were seen in the majority of patients with measurable disease. At a median follow-up of 2.3 years (range,0.3–6.5), three-year progression-free (PFS) and overall survival (OS) for the cohort were 65% and 82%, respectively, and superior to a historical control cohort treated with conventional therapies. CONCLUSIONS: Upfront targeted therapy for patients with BRAF-mutant pHGG is feasible and effective, with superior clinical outcomes observed compared to historical data. This promising treatment paradigm is currently being evaluated prospectively in the Children’s Oncology Group ACNS1723 clinical trial.

Knowledge on management of pediatric spinal cord low-grade glioma (LGG) is scarce.We analyzed clinical datasets of 128 pediatric patients with spinal LGG followed within the prospective multicenter trials HIT-LGG 1996 (n = 36), SIOP-LGG 2004 (n = 56), and the subsequent LGG-Interim registry (n = 36).Spinal LGG, predominantly pilocytic astrocytomas (76%), harbored KIAA1549-BRAF fusion in 14/35 patients (40%) and FGFR1-TACC1 fusion in 3/26 patients (12%), as well as BRAFV600E mutation in 2/66 patients (3%). 10-year overall survival (OS) and event-free survival (EFS) was 93% ± 2% and 38% ± 5%, respectively. Disseminated disease (n = 16) was associated with inferior OS and EFS, while age ≥11 years and total resection were favorable factors for EFS. We observed 117 patients following total (n = 24) or subtotal/partial resection (n = 74), biopsy (n = 16), or radiologic diagnosis only (n = 3). Eleven patients were treated first with chemotherapy (n = 9) or irradiation (n = 2). Up to 20.8 years after diagnosis/initial intervention, 73/128 patients experienced one (n = 43) or up to six (n = 30) radiological/clinical disease progressions. Tumor resections were repeated in 36 patients (range, 2-6) and 47 patients required nonsurgical treatment (chemotherapy, n = 20; radiotherapy, n = 10; multiple treatment lines, n = 17). Long-term disease control for a median of 6.5 (range, 0.02-20) years was achieved in 73/77 patients following one (n = 57) or repeated (n = 16) resections, and in 35/47 patients after nonsurgical treatment.The majority of patients experienced disease progression, even after years. Multiple interventions were required for more than a third, yet multimodal treatment enabled long-term disease control. Molecular testing may reveal therapeutic targets.

Inhibition of mTORC1 signaling has been shown to diminish growth of meningiomas and schwannomas in preclinical studies, and clinical data suggest that everolimus, an orally administered mTORC1 inhibitor, may slow tumor progression in a subset of patients with neurofibromatosis type 2 (NF2) with vestibular schwannoma. To assess the pharmacokinetics, pharmacodynamics, and potential mechanisms of treatment resistance, we performed a presurgical (phase 0) clinical trial of everolimus in patients undergoing elective surgery for vestibular schwannoma or meningiomas. Eligible patients with meningioma or vestibular schwannoma requiring tumor resection enrolled on study received everolimus 10 mg daily for 10 days immediately prior to surgery. Everolimus blood levels were determined immediately before and after surgery. Tumor samples were collected intraoperatively. Ten patients completed protocol therapy. Median pre- and postoperative blood levels of everolimus were found to be in a high therapeutic range (17.4 ng/mL and 9.4 ng/mL, respectively). Median tumor tissue drug concentration determined by mass spectrometry was 24.3 pg/mg (range, 9.2–169.2). We observed only partial inhibition of phospho-S6 in the treated tumors, indicating incomplete target inhibition compared with control tissues from untreated patients (P = 0.025). Everolimus led to incomplete inhibition of mTORC1 and downstream signaling. These data may explain the limited antitumor effect of everolimus observed in clinical studies for patients with NF2 and will inform the design of future preclinical and clinical studies targeting mTORC1 in meningiomas and schwannomas.

BACKGROUND Oncogenic driver alterations in fibroblast growth factor receptors (FGFRs) are present in a subset of pediatric gliomas. Debio1347 is an orally available, highly selective FGFR 1–3 inhibitor with a favorable safety profile and encouraging preliminary clinical activity in an adult phase 1 study. METHODS We treated five children with progressive/refractory CNS tumors harboring an FGFR gene alteration with Debio1347 on single patient use protocols. Patients were treated using the 20 mg tablet formulation at the adult recommended phase 2 dose (80 mg/1.73 m2 ′ BSA once daily). RESULTS All adverse events (AEs) were grade 1–2. Most common treatment-related AEs were hyperphosphatemia, ALT elevation and hypoalbuminemia. Two patients met criteria for partial response and two patients had stable disease. A 13-month-old patient with a spinal cord high-grade glioma harboring two FGFR1 mutations had tumor reduction of 96.3% maintained for 11 months. A 26 month-old patient with a pilomyxoid astrocytoma harboring an FGFR1-TACC1 fusion had a tumor reduction of 74.5% maintained for 9 months. Prolonged disease stabilization and clinically significant improvement in visual function was noted in an eight year-old patient with metastatic suprasellar pilomyxoid astrocytoma harboring two FGFR1 mutations (14 months overall and sustained for 6 months off therapy) and in a 14 year-old patient with posterior fossa glioneuronal tumor harboring an FGFR3-TACC3 fusion (26 months and ongoing). CONCLUSIONS FGFR targeted therapy with Debio1347 demonstrated tolerable toxicity and promising anti-tumor efficacy in pediatric patients with recurrent/refractory FGFR altered gliomas. Further studies in this population are warranted.

Preclinical studies suggested that the mTOR signaling pathway could be a potential therapeutic target in childhood ependymomas. As a result, a phase II clinical trial (ClinicalTrials.gov identifier: NCT02155920) of single-agent everolimus was performed to test the hypothesis that inhibition of the mTOR pathway would result in tumor responses for children with recurrent and/or progressive ependymoma. Eleven patients [sex: 4 females (36.4%); median age: 8 years (range: 2 – 15 years); race: 9 white and 2 other; prior therapies: median 6 (range: 3 – 9)] were enrolled on the study. The primary tumor location for 10 participants was the posterior fossa; the primary location of the remaining tumor was the cervical spine. All patients were treated with oral everolimus 4.5 mg/m2/day (each cycle = 28 days) that was titrated to achieve serum trough levels of 5 – 15 ng/mL. Overall, everolimus was well tolerated; adverse events were grade 1 – 2 and consistent with its previously established safety profile in children. No objective tumor responses were observed. All patients discontinued therapy due to tumor progression after a median of 2 cycles of therapy (1 cycle = 2; 2 cycles = 6; 3, 4, and 8 cycles = 1 each). This study does not support the use of everolimus for children with recurrent or progressive ependymoma.

Background To evaluate efficacy, pharmacokinetics (PK) and pharmacodynamics of single-agent everolimus in pediatric patients with radiographically progressive low-grade glioma (LGG). Methods Everolimus was administered at 5 mg/m2 once daily as a tablet or liquid for a planned 48-week duration or until unacceptable toxicity or disease progression. Patients with neurofibromatosis type 1 were excluded. PK and pharmacodynamic endpoints were assessed in consenting patients. Results Twenty-three eligible patients (median age 9.2 years) were enrolled. All patients received prior chemotherapy (median number of prior regimens two) and/or radiotherapy (two patients). By week 48, two patients had a partial response, 10 stable disease, and 11 clinical or radiographic progression; two discontinued study prior to 1 year (toxicity: 1, physician determination: 1). With a median follow up of 1.8 years (range 0.2-6.7 years), the 2-, 3-, and 5-year progression-free survivals (PFS) were 39 ± 11%, 26 ± 11%, and 26 ± 11%, respectively; two patients died of disease. The 2-, 3-, and 5-year overall survival (OS) were all 93 ± 6%. Grade 1 and 2 toxicities predominated; two definitively related grade 3 toxicities (mucositis and neutropenia) occurred. Grade 4 elevation of liver enzymes was possibly related in one patient. Predose blood levels showed substantial variability between patients with 45.5% below and 18.2% above the target range of 5-15 ng/mL. Pharmacodynamic analysis demonstrated significant inhibition in phospho-S6, 4E-BP1, and modulation of c-Myc expression. Conclusion Daily oral everolimus provides a well-tolerated, alternative treatment for multiple recurrent, radiographically progressive pediatric LGG. Based on these results, everolimus is being investigated further for this patient population.

Background Oncogenic driver alterations in FGFR are present in a subset of pediatric gliomas. Debio1347 is an orally available, highly selective FGFR 1–3 inhibitor that had a favorable safety profile and encouraging preliminary clinical activity in an adult phase 1 study. Methods Five children with progressive/refractory CNS tumors harboring an FGFR gene alteration following prior therapy were treated with Debio1347 at Memorial Sloan Kettering Cancer Center on single patient use protocols. Patients were treated using the 20 mg tablet formulation at the adult recommended phase 2 dose (80 mg/1.73 m2 * BSA once daily). Toxicities were graded using CTCAEv5.0 and imaging response assessments were performed every 8–12 weeks. RESULTS All AEs were grade 1–2. Most common treatment-related adverse events were hyperphosphatemia, ALT increased and hypoalbuminemia (4 patients). Two patients met criteria for partial response and two patients had stable disease. A 13 month-old patient with a spinal cord high-grade glioma harboring two FGFR1 mutations (V592M, K687E) had tumor reduction of 91.7% maintained for 12 months. A 26-month-old patient with a pilomyxoid astrocytoma harboring an FGFR1-TACC1 fusion had a tumor reduction of 74.5% maintained for 9 months. Molecular characterization of recurrent tumor from this patient demonstrated an NF1 deletion as a novel molecular mechanism of acquired resistance to FGFR inhibition. Prolonged disease stabilization was noted in an eight year-old patient with metastatic suprasellar pilomyxoid astrocytoma harboring an FGFR1 mutation (9 months) and in a 14 year-old patient with posterior fossa glioneuronal tumor harboring an FGFR3-TACC3 fusion (24 months and ongoing). Conclusions Debio1347 demonstrated tolerable toxicity and promising anti-tumor efficacy in pediatric patients with refractory FGFR altered gliomas. Specific attention to growth velocity and clinical symptoms with incorporation of imaging assessment of bone growth is warranted. Candidate biomarkers (FGFR1 V592M and K687E SNVs, FGFR-TACC fusions) may guide patient selection. Further studies in this population are warranted.

Cell-free DNA (cfDNA) from cerebrospinal fluid (CSF) offers unique opportunities for genomic profiling of tumors involving the central nervous system but remains uncommonly used in clinical practice. We describe our clinical experience using cfDNA from CSF for routine molecular testing using Memorial Sloan Kettering Integrated Mutation Profiling of Actionable Cancer Targets (targeting 468 cancer-related genes). In all, 148 cfDNA samples were assessed, comparing results of cfDNA versus genomic DNA (gDNA; gDNA from cell pellets) derived from the same CSF sample and the primary tumor. Of these, 71.6% (106/148) were successfully sequenced. Somatic alterations (mutations and fusions) were observed in 70.8% (75/106) of the samples; 97.3% (73/75) comprised variants confirming central nervous system involvement by a previously diagnosed tumor, 14.7% (11/75) had additional variants consistent with a therapy-related resistance mechanism, and 2.7% (2/75) had variants that independently diagnosed a new primary. Among samples with paired cfDNA and gDNA sequencing results, cfDNA was more frequently positive for at least one mutation [43.6% (55/126) versus 19.8% (25/126)] and harbored 1.6× more mutations (6.94 versus 4.65; P = 0.005), with higher mean variant allele fractions (41.1% versus 13.0%; P