PURPOSE SJMB03 (ClinicalTrials.gov identifier: NCT00085202 ) was a phase III risk-adapted trial that aimed to determine the frequency and clinical significance of biological variants and genetic alterations in medulloblastoma. PATIENTS AND METHODS Patients 3-21 years old were stratified into average-risk and high-risk treatment groups based on metastatic status and extent of resection. Medulloblastomas were molecularly classified into subgroups (Wingless [WNT], Sonic Hedgehog [SHH], group 3, and group 4) and subtypes based on DNA methylation profiles and overlaid with gene mutations from next-generation sequencing. Coprimary study end points were (1) to assess the relationship between ERBB2 protein expression in tumors and progression-free survival (PFS), and (2) to estimate the frequency of mutations associated with WNT and SHH tumors. Clinical and molecular risk factors were evaluated, and the most robust were used to model new risk-classification categories. RESULTS Three hundred thirty eligible patients with medulloblastoma were enrolled. Five-year PFS was 83.2% (95% CI, 78.4 to 88.2) for average-risk patients (n = 227) and 58.7% (95% CI, 49.8 to 69.1) for high-risk patients (n = 103). No association was found between ERBB2 status and PFS in the overall cohort ( P = .74) or when patients were stratified by clinical risk ( P = .71). Mutations in CTNNB1 (96%), DDX3X (37%), and SMARCA4 (24%) were most common in WNT tumors and PTCH1 (38%), TP53 (21%), and DDX3X (19%) in SHH tumors. Methylome profiling classified 53 WNT (17.4%), 48 SHH (15.7%), 65 group 3 (21.3%), and 139 group 4 (45.6%) tumors. A comprehensive clinicomolecular risk factor analysis identified three low-risk groups (WNT, low-risk SHH, and low-risk combined groups 3 and 4) with excellent (5-year PFS > 90%) and two very high-risk groups (high-risk SHH and high-risk combined groups 3 and 4) with poor survival (5-year PFS < 60%). CONCLUSION These results establish a new risk stratification for future medulloblastoma trials.

PURPOSE We sought to investigate clinical outcomes of relapsed medulloblastoma and to compare molecular features between patient-matched diagnostic and relapsed tumors. METHODS Children and infants enrolled on either SJMB03 (NCT00085202) or SJYC07 (NCT00602667) trials who experienced medulloblastoma relapse were analyzed for clinical outcomes, including anatomic and temporal patterns of relapse and postrelapse survival. A largely independent, paired molecular cohort was analyzed by DNA methylation array and next-generation sequencing. RESULTS A total of 72 of 329 (22%) SJMB03 and 52 of 79 (66%) SJYC07 patients experienced relapse with significant representation of Group 3 and wingless tumors. Although most patients exhibited some distal disease (79%), 38% of patients with sonic hedgehog tumors experienced isolated local relapse. Time to relapse and postrelapse survival varied by molecular subgroup with longer latencies for patients with Group 4 tumors. Postrelapse radiation therapy among previously nonirradiated SJYC07 patients was associated with long-term survival. Reirradiation was only temporizing for SJMB03 patients. Among 127 patients with patient-matched tumor pairs, 9 (7%) experienced subsequent nonmedulloblastoma CNS malignancies. Subgroup (96%) and subtype (80%) stabilities were largely maintained among the remainder. Rare subgroup divergence was observed from Group 4 to Group 3 tumors, which is coincident with genetic alterations involving MYC, MYCN, and FBXW7. Subgroup-specific patterns of alteration were identified for driver genes and chromosome arms. CONCLUSION Clinical behavior of relapsed medulloblastoma must be contextualized in terms of up-front therapies and molecular classifications. Group 4 tumors exhibit slower biological progression. Utility of radiation at relapse is dependent on patient age and prior treatments. Degree and patterns of molecular conservation at relapse vary by subgroup. Relapse tissue enables verification of molecular targets and identification of occult secondary malignancies.

Biomarkers which better match anticancer drugs with cancer driver genes hold the promise of improved clinical responses and cure rates. We developed a precision medicine platform of rapid highthroughput drug screening (HTS) and patient-derived xenografting (PDX) of primary tumor tissue, and evaluated its potential for treatment identification among 56 consecutively enrolled high-risk pediatric cancer patients, compared with conventional molecular genomics and transcriptomics. Drug hits were seen in the majority of HTS and PDX screens, which identified therapeutic options for 10 patients for whom no targetable molecular lesions could be found. Screens also provided orthogonal proof of drug efficacy suggested by molecular analyses and negative results for some molecular findings. We identified treatment options across the whole testing platform for 70% of patients. Only molecular therapeutic recommendations were provided to treating oncologists and led to a change in therapy in 53% of patients, of whom 29% had clinical benefit. These data indicate that in vitro and in vivo drug screening of tumor cells could increase therapeutic options and improve clinical outcomes for high-risk pediatric cancer patients.

10525 Background: Absence of integrase interactor 1 (INI1) expression is a defining molecular feature of rhabdoid tumors (RT), epithelioid sarcoma (ES), and chordomas, inducing dependence on enhancer of zeste homolog-2 (EZH2). Tazemetostat (TAZ) is a selective EZH2 inhibitor approved by the FDA for treatment of patients (pts) ≥16 yrs with metastatic or locally advanced ES ineligible for complete resection. Data from a Phase 1 (Ph1) pediatric dose-escalation study (Ph1a) of TAZ were previously reported; herein we report interim efficacy and safety from the Ph1 pediatric dose-expansion study (Ph1b). Methods: NCT02601937 is a Ph1, multicenter study in pts 6 months – 18 yrs evaluating TAZ administered BID at 1200 mg/m2 in Ph1b, per Ph1a recommendation. Ph1b cohorts enrolled pts based on tumor type: Atypical teratoid RT (ATRT), RT, and other INI1-negative tumors (including ES and chordoma). The Ph1b primary endpoint was overall response rate (ORR). Secondary endpoints included safety/tolerability, duration of response (DOR), and survival. Results: Ph1b has enrolled 47 pts who received TAZ oral suspension. Across all tumor types, ORR was 17% (Table). Responses were observed in ATRT (4/21), chordoma (2/4), and ES (2/7); 1 pt dosed at 520mg/m2 and 7pts at 1200mg/m2. In the ATRT cohort, 19% of pts responded to TAZ with a median DOR of 6.5 months. The median DOR has not yet been reached in the other cohorts, with ongoing responses in 3 pts. TAZ was generally well tolerated with no drug-related deaths. Most common adverse events (AE) include vomiting, nausea, and cough. During Ph1b enrollment, 1 pt with chordoma (dosed at TAZ 900 mg/m2 for 15 months in Ph1a) developed a secondary malignancy (T-cell lymphoblastic lymphoma). In response, the pediatric recommended Ph2 dose was revised to limit exposure in pts without CNS involvement to 520 mg/m2 TAZ (maximum dosing of 1 yr after response, pts to go off-treatment until disease progression). Conclusions: Interim results indicate TAZ is generally well tolerated in children with an AE profile similar to adults. Pt enrollment in the non-ATRT, INI1-negative cohorts is ongoing. TAZ shows promising anti-tumor activity in a subset of pediatric tumors, including ATRT, chordoma, and ES. Clinical trial information: NCT02601937. [Table: see text]

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Background: MYCN amplification (MNA), segmental chromosomal aberrations (SCA) and ALK activating mutations are biomarkers for risk-group stratification and for targeted therapeutics for neuroblastoma, both of which are currently assessed on tissue biopsy. Increase in demand for tumor genetic testing for neuroblastoma diagnosis is posing a challenge to current practice, as the small size of the core needle biopsies obtained are required for multiple molecular tests. We evaluated the utility of detecting these biomarkers in the circulation. Methods: Various pre-analytical conditions tested to optimize circulating-tumor DNA (ctDNA) copy number changes evaluations. Plasma samples from 10 patients diagnosed with neuroblastoma assessed for SCA and MNA using single nucleotide polymorphism (SNP) array approach currently used for neuroblastoma diagnosis, with MNA status assessed independently using digital-droplet PCR (ddPCR). Three patients (one in common with the previous 10) tested for ALK activating mutations p.F1174L and p.F1245I using ddPCR. Results: Copy number detection is highly affected by physical perturbations of the blood sample (mimicking suboptimal sample shipment), which could be overcome using specialized preservative collection tubes. Pre-analytical DNA repair procedures on ctDNA before SNP chromosome microarray processing improved the lower limit of detection for SCA and MNA, defined as 20% and 10%, respectively. We detected SCA in 10/10 (100%) patients using SNP array, 7 of which also presented MNA. Circulating-free DNA (cfDNA) and matched tumor DNA profiles were generally identical. MNA was detected using ddPCR in 7/7 (100%) of MNA and 0/12 (0%) non-MNA cases. MNA and ALK mutation dynamic change was assessed in longitudinal samples from 4 and 3 patients (one patient with both), respectively, accurately reflected response to treatment in 6/6 (100%) and disease recurrence in 5/6 (83%) of cases. Samples taken prior to targeted treatment with the ALK inhibitor Lorlatinib and 6–8 weeks on treatment showed reduction/increase in ALK variants according to response to treatment. Conclusions: These results demonstrate the feasibility of ctDNA profiling for molecular risk-stratification, and treatment monitoring in a clinically relevant time frame and the potential to reduce fresh tissue requirements currently embedded in the management of neuroblastoma.

TPS10062 Background: RAF gene fusions ( BRAF and RAF1) and BRAF V600E mutations are oncogenic drivers found on a mutually exclusive basis in most pediatric low-grade gliomas (LGGs). In addition, RAF fusions ( BRAF and RAF1) have also been identified in other pediatric solid tumors. Tovorafenib (DAY101) is an investigational, oral, highly selective, CNS-penetrant, small molecule, type II pan-RAF inhibitor. In contrast to type I BRAF inhibitors, tovorafenib does not induce RAS-dependent paradoxical activation of the MAPK pathway. In the phase 1 PNOC014 study in pediatric patients with recurrent/progressive LGG, tovorafenib was well tolerated and 7/8 patients with tumor harboring RAF fusions had meaningful clinical benefit. Recently, a child with a novel SNX8-BRAF fusion spindle cell sarcoma demonstrated a rapid and deep response when treated with tovorafenib. Methods: FIREFLY-1 (NCT04775485) is an open-label, multicenter, phase 2 study evaluating the safety and efficacy of tovorafenib monotherapy in pediatric patients with RAF-altered recurrent or progressive LGG or advanced solid tumors. The initial design included only patients with LGG (arm 1). Two new arms have now been added; arm 2 will allow tovorafenib treatment for patients with LGG harboring an activating RAF alteration after completion of enrollment to arm 1 and prior to tovorafenib regulatory approval; arm 3 will enroll patients with advanced solid tumors harboring an activating RAF fusion. Eligible patients are 6 months to 25 years of age, who have received ≥1 prior line of systemic therapy with documented radiographic progression, have evaluable and/or measurable disease by appropriate criteria, a Karnofsky or Lansky performance score of at least 50, and adequate organ function. Patients are excluded if their tumor has other driver mutations, they have neurofibromatosis type 1, central serous retinopathy, retinal vein occlusion, clinically significant active cardiovascular disease, or are currently being treated with a strong CYP2C8 inhibitor or inducer other than those allowed per protocol. Approximately 140 patients in total will be enrolled including 60 in arm 1, 60 in arm 2 and 20 in arm 3. Tovorafenib will be administered at 420 mg/m2 (not to exceed 600 mg) weekly (days 1, 8, 15 and 22) for 26, 28-day cycles (in the absence of disease progression or unacceptable toxicity). They may then continue tovorafenib or enter a drug holiday period. The primary endpoint is overall response rate, as defined by the RANO criteria (arm 1) or RECIST v1.1 (arm 3) and as determined by an independent radiology review committee. Secondary endpoints (arms 1 and 3) include safety and tolerability, pharmacokinetics, duration of response, time to response and progression-free survival. Tovorafenib is available in tablet or liquid suspension formulations. Clinical trial information: NCT04775485.