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Purpose Intraventricular compartmental radioimmunotherapy (cRIT) with 131-I-omburtamab is a potential therapy for recurrent primary brain tumors that can seed the thecal space. These patients often previously received external beam radiotherapy (EBRT) to a portion or full craniospinal axis (CSI) as part of upfront therapy. Little is known regarding outcomes after re-irradiation as part of multimodality therapy including cRIT. This study evaluates predictors of response, patterns of failure, and radiologic events after cRIT. Methods Patients with recurrent medulloblastoma or ependymoma who received 131-I-omburtamab on a prospective clinical trial were included. Extent of disease at cRIT initiation (no evidence of disease [NED] vs measurable disease [MD]) was assessed as associated with progression-free (PFS) and overall survival (OS) by Kaplan–Meier analysis. Results All 27 patients (20 medulloblastoma, 7 ependymoma) had EBRT preceding cRIT: most (22, 81%) included CSI (median dose 2340 cGy, boost to 5400 cGy). Twelve (44%) also received EBRT at relapse as bridging to cRIT. There were no cases of radionecrosis. At cRIT initiation, 11 (55%) medulloblastoma and 3 (43%) ependymoma patients were NED, associated with improved PFS (p = 0.002) and OS (p = 0.048) in medulloblastoma. Most relapses were multifocal. With medium follow-up of 3.0 years (95% confidence interval, 1.8–7.4), 6 patients remain alive with NED. Conclusion For patients with medulloblastoma, remission at time of cRIT was associated with significantly improved survival outcomes. Relapses are often multifocal, particularly in the setting of measurable disease at cRIT initiation. EBRT is a promising tool to achieve NED status at cRIT initiation, with no cases of radiation necrosis.

To elucidate the mechanisms underlying the divergent clinicopathologic spectrum of EWSR1/FUS::CREB translocation-associated tumors, we performed a comprehensive genomic analysis of fusion transcript variants, recurrent genetic alterations (mutations, copy number alterations), gene expression, and methylation profiles across a large cohort of tumor types. The distribution of the EWSR1/FUS fusion partners-ATF1, CREB1, and CREM-and exon involvement was significantly different across different tumor types. Our targeted sequencing showed that secondary genetic events are associated with tumor type rather than fusion type. Of the 39 cases that underwent targeted NGS testing, 18 (46%) had secondary OncoKB mutations or copy number alterations (29 secondary genetic events in total), of which 15 (52%) were recurrent. Secondary recurrent, but mutually exclusive, TERT promoter and CDKN2A mutations were identified only in clear cell sarcoma (CCS) and associated with worse overall survival. CDKN2A/B homozygous deletions were recurrent in angiomatoid fibrous histiocytoma (AFH) and restricted to metastatic cases. mRNA upregulation of MITF, CDH19, PARVB, and PFKP was found in CCS, compared to AFH, and correlated with a hypomethylated profile. In contrast, S100A4 and XAF1 were differentially upregulated and hypomethylated in AFH but not CCS. Unsupervised clustering of methylation profiles revealed that CREB family translocation-associated tumors form neighboring but tight, distinct clusters. A sarcoma methylation classifier was able to accurately match 100% of CCS cases to the correct methylation class; however, it was suboptimal when applied to other histologies. In conclusion, our comprehensive genomic profiling of EWSR1/FUS::CREB translocation-associated tumors uncovered mostly histotype, rather than fusion-type associated correlations in transcript variants, prognostically significant secondary genetic alterations, and gene expression and methylation patterns.

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.

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.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by the occurrence of pathogenic variants in BRCA1/2 in 5-6% of patients. Biallelic loss of BRCA1/2 enriches for response to platinum agents and poly (ADP-ribose) polymerase 1 inhibitors. There is a dearth of evidence on the mechanism of inactivation of the wild-type BRCA1 allele in PDAC tumors with a germline BRCA1 (gBRCA1) pathogenic or likely pathogenic variant (P/LPV). Herein, we examine promotor hypermethylation as a "second hit" mechanism in patients with gBRCA1-PDAC.We evaluated patients with PDAC who underwent Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) somatic and germline testing from an institutional database. DNA isolated from tumor tissue and matched normal peripheral blood were sequenced by MSK-IMPACT. In patients with gBRCA1-PDAC, we examined the somatic BRCA1 mutation status and promotor methylation status of the tumor BRCA1 allele via a methylation array analysis. In patients with sufficient remaining DNA, a second methylation analysis by pyrosequencing was performed.Of 1012 patients with PDAC, 19 (1.9%) were identified to harbor a gBRCA1 P/LPV. Fifteen patients underwent a methylation array and the mean percentage of BRCA1 promotor methylation was 3.62%. In seven patients in whom sufficient DNA was available, subsequent pyrosequencing confirmed an unmethylated BRCA1 promotor. Loss of heterozygosity was detected in 12 of 19 (63%, 95% confidence interval 38-84) patients, demonstrating loss of heterozygosity is the major molecular mechanism of BRCA1 inactivation in PDAC. Two (10.5%) cases had a somatic BRCA1 mutation.In patients with gBRCA1-P/LPV-PDAC, loss of heterozygosity is the main inactivating mechanism of the wild-type BRCA1 allele in the tumor, and methylation of the BRCA1 promoter is a distinctly uncommon occurrence.

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.

Pancreatic neoplasms are heterogenous and have traditionally been classified by assessing their lines of cellular differentiation using histopathologic methods, particularly morphologic and immunohistochemical evaluation. These methods frequently identify overlapping differentiation along ductal, acinar, and neuroendocrine lines, raising diagnostic challenges as well as questions regarding the relationship of these neoplasms. Neoplasms with acinar differentiation, in particular, frequently show more than one line of differentiation based on immunolabeling. Genome methylation signatures, in contrast, are better conserved within cellular lineages, and are increasingly used to support the classification of neoplasms. We characterized the epigenetic relationships between pancreatoblastomas, acinar cell carcinomas (including mixed variants), pancreatic neuroendocrine tumors, solid pseudopapillary neoplasms, and pancreatic ductal adenocarcinomas using a genome-wide array platform. Using unsupervised learning approaches, pancreatic neuroendocrine tumors, solid pseudopapillary neoplasms, ductal adenocarcinomas, and normal pancreatic tissue samples all localized to distinct clusters based on their methylation profiles, whereas all neoplasms with acinar differentiation occupied a broad overlapping region located between the predominantly acinar normal pancreatic tissue and ductal adenocarcinoma clusters. Our data provide evidence to suggest that acinar cell carcinomas and pancreatoblastomas are similar at the epigenetic level. These findings are consistent with genomic and clinical observations that mixed acinar neoplasms are closely related to pure acinar cell carcinomas rather than to neuroendocrine tumors or ductal adenocarcinomas.

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.

e19543 Background: LB-based CGP of ctDNA can facilitate the molecular evaluation of lymphoid and plasma cell neoplasms (PCNs), especially when traditional CGP is infeasible due to insufficient tumor in the blood or tissue or when tissue biopsy itself cannot be performed. Methods: Retrospective study of LB samples from patients reported to have lymphomas or PCNs tested using hybrid-capture next-generation sequencing using the FoundationOne Liquid CDx (targeting 324 genes), FoundationOne Liquid (70 genes), or FoundationACT (62 genes) assays during routine clinical care between 5/2016 and 9/2021. Results: Among 48,459 unique patient samples submitted for CGP, 133 were from lymphomas or PCNs: 69 non-Hodgkin lymphoma (NHL), 32 PCN, 16 diffuse large B-cell lymphoma (DLBCL), and 16 classic Hodgkin lymphoma (cHL) cases. The median cell-free DNA yield was 72.7 ng (interquartile range [IQR]: 46.0-157.0), well above the minimum quantity needed for analysis. Reportable pathogenic genomic alterations were detected in 67.7% (90/133) of cases. The median maximum somatic allele frequency (MSAF) was 3.8% (IQR: 1.1-19.6%), with no statistically significant differences noted between DLBCL (8.2%), NHL (5.6%), cHL (1.7%), and PCNs (1.4%) ( P= 0.13, Kruskal-Wallis test). In all, 322 pathogenic short variants and 3 pathogenic rearrangements were detected (median variant allele frequency [VAF]: 0.6%, IQR: 0.3-2.6%, range: 0.09-81.8%). One hundred ninety-seven had VAFs < 1.0%, indicating excellent sensitivity. NHLs exhibited pathogenic alterations in TP53 (53.2% of cases in which pathogenic alterations were identified), KMT2D (14.9%), NF1 (10.6%), MYD88 (8.5%), BCL2 (6.4%), BRAF (6.4%), EZH2 (6.4%), NRAS (6.4%), BTK (4.3%, all of which were C481X ibrutinib resistance mutations in cases of chronic lymphocytic leukemia detected at VAFs ranging from 0.18-5.5%), KRAS (4.3%), RB1 (4.3%), and TNFAIP3 (4.3%), among others. PCNs exhibited pathogenic alterations in TP53 (54.6%), KRAS (18.2%), NF1 (13.6%), PTPN11 (13.6%), BRAF (9.1%), NRAS (9.1%), MAP2K1 (4.6%), and RB1 (4.6%), among others. DLBCLs exhibited pathogenic alterations in TP53 (63.6%), MYD88 (27.3%), CCND2/3 (9.1%), EZH2 (9.1%), and KMT2D (9.1%), among others. CD274 (PD-L1) and PDCD1LG2 (PD-L2) amplification and IGH- PAX5 and IGH- MYC fusions were also detected in individual cases of DLBCL. cHLs exhibited pathogenic alterations in TP53 (40.0%), ATM (20.0%), BCOR (10.0%), KRAS (10.0%), MYD88 (10.0%), and PDCD1LG2 (PD-L2) (10.0%), among others. Conclusions: LB identified clinically actionable genomic alterations in lymphomas and PCNs and may be used when traditional CGP of tumor biopsy specimens cannot be performed. Given its low limit of detection, LB may also allow for minimally invasive dynamic monitoring of response to therapy.

e19064 Background: LB-based CGP of ctDNA can facilitate the diagnosis, molecular profiling, and monitoring of patients with myeloid neoplasia, particularly when standard CGP is not feasible due to a lack of circulating disease or insufficient tumor in the marrow or tissue. Methods: Retrospective study of LB samples from patients reported to have histiocytic neoplasms (HNs), myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), or acute myeloid leukemia (AML) tested using hybrid-capture next-generation sequencing using the FoundationOne Liquid CDx (targeting 324 genes), FoundationOne Liquid (70 genes), or FoundationACT (62 genes) assays during routine clinical care between 5/2016 and 9/2021. Results: Among 48,459 unique patient samples submitted for CGP, 83 were from myeloid neoplasia: 34 HN, 23 MDS, 15 MPN, and 11 AML. The median cell-free DNA yield was 74.9 ng (interquartile range [IQR]: 52.0-167.4), well above the minimum needed for analysis. Reportable pathogenic alterations were detected in 62.7% (52/83) of cases. The median maximum somatic allele frequency (MSAF) was 17.0% (IQR: 1.7-49.0%), with AML (45.7%), MDS (25.9%), and MPN (43.8%) having higher median MSAFs than HNs (3.2%), not unexpected given that the former naturally circulate. In all, 147 pathogenic short variants and 6 pathogenic rearrangements were detected (median variant allele frequency [VAF]: 2.1%, IQR: 0.3-20.5%, range: 0.11-99.8%). Sixty-one had VAFs < 1.0%, indicating excellent sensitivity. HNs exhibited activating RAS-RAF-MEK pathway alterations in BRAF (31.3% of cases in which pathogenic alterations were identified), NF1 (12.5%), MAP2K1 (6.3%), and NRAS (6.3%), detected at VAFs as low as 0.21%, 0.13%, 0.11%, and 4.0%, respectively. AML cases exhibited pathogenic alterations in TP53 (57.1%), FLT3 (28.6%), IDH2 (28.6%), NPM1 (28.6%), KRAS (14.3%), and NRAS (14.3%), among others. Subclonal heterogeneity was identified, including an AML case with KRAS G12A and G13R and NRAS G12A mutations detected at VAFs of 0.24%, 0.19%, and 0.2%, respectively. Altered genes in patients undergoing workup for MDS or MPN included TP53 (41.4%), JAK2 (37.9%), DNMT3A (13.8%), CHEK2 (10.3%), PTPN11 (10.3%), SF3B1 (10.3%), TET2 (10.3%), ASXL1 (6.9%), MPL (3.5%), and U2AF1 (3.5%), among others. Last, among 48,243 patients with a LB for a solid tumor diagnosis, 3,487 JAK2 V617F, 366 CALR C-terminal truncation, and 343 MPL W515X mutations were detected, potentially indicating a concurrent or occult MPN. Conclusions: LB identified clinically relevant genomic alterations in myeloid neoplasia, offering a powerful tool that may be used pre- or post-treatment when CGP of the buffy coat, marrow, or tissue is infeasible. Given its low limit of detection, LB can also identify low-level emerging or persistent subclones, which may facilitate monitoring and minimal residual disease testing.