Mohammad Tanhaemami

PURPOSE: Identification of discrete risk groups remains a high priority for patients with Ewing sarcoma (EWS). We sought to prospectively validate circulating tumor DNA (ctDNA) as a prognostic factor and develop clinical-molecular risk groups. METHODS: We conducted a prospective investigator-initiated biology study for patients with localized EWS (LEOPARD) and embedded ctDNA analysis into the North American frontline metastatic study AEWS1221. Eligible patients were younger than 50 years with newly diagnosed EWS. All patients provided a baseline blood sample for analysis, which was subjected to ultralow-pass whole-genome sequencing and hybrid capture panel sequencing for ctDNA quantification, fusion detection, and characterization of STAG2 and TP53 alterations. Serial ctDNA sequencing was conducted on a subset of patients in each study. We tested for associations between ctDNA burden and secondary genomic events, and clinical features and outcomes. RESULTS: One hundred forty patients with localized disease and 255 with metastatic disease provided evaluable pretreatment samples for ctDNA analysis. Elevated baseline ctDNA was associated with stage, tumor size, primary site, indeterminate pulmonary nodules, and metastatic pattern. Elevated pretreatment ctDNA burden was associated with inferior outcomes in patients with localized (n = 140, hazard ratio [HR] = 2.36, P = .032) and metastatic disease (n = 255, HR = 2.15, P = .001). Patients with metastatic disease and TP53 variants and/or persistent on-therapy ctDNA had dismal outcomes. Patients with localized disease, low ctDNA, small tumors, and favorable genomics had no events and constitute a novel low-risk group. Among patients with metastatic disease, those with lung-only disease, low ctDNA, and favorable genomics represent an intermediate-risk group. CONCLUSION: This study prospectively validates pretreatment ctDNA burden as prognostic in EWS. Risk groups that integrate ctDNA burden with clinical-molecular features differentiate patients with low-, intermediate-, and high-risk disease.

Supplemental Tables 1, 2, and 3

Purpose: ALRN-6924 is a stapled peptide that disrupts MDM2/MDMX-mediated inhibition of p53. We evaluated safety, pharmacokinetics, pharmacodynamics, and preliminary efficacy of ALRN-6924 in children with advanced malignancies. Experimental Design: Patients with TP53-wild-type malignancies were enrolled to a monotherapy arm (solid/CNS tumors) or combination arm with cytarabine (acute leukemia). Monotherapy dosing used the TARGET-CRM design for dose escalation. Pharmacodynamic assessment included serum MIC-1 as a biomarker of p53 activation. Circulating tumor DNA was analyzed for emergent TP53 mutations. Results: Twenty-two patients enrolled; 20 received treatment (17 monotherapy, 3 combination). The most common diagnosis was Ewing sarcoma (n=5). One dose-limiting toxicity (DLT) occurred at monotherapy dose level 2 (2.7 mg/kg). Six patients were treated at dose level 3 (3.5 mg/kg) without DLT, and one patient was treated at dose level 4 (4.3 mg/kg) without DLT before study closure. No DLTs occurred on the combination arm. Common treatment-related adverse events included anemia (90%) and nausea (70%). MIC-1 levels increased 30-50-fold by 24 hours post-dose at dose levels 2-4, confirming on-target p53 activation. Among 19 response-evaluable patients, one partial remission occurred in a patient with relapsed ALL on the combination arm. Drug exposure was lower than in adults at equivalent doses. One patient with Ewing sarcoma had an emergent TP53 mutation detected in their baseline on-therapy ctDNA sample. Conclusions: ALRN-6924 was well tolerated in children with on-target activity. Future efforts to evaluate this agent should focus on biomarker-selected populations, combination strategies, and evaluation of higher dose levels.

PURPOSE: Identifying discrete subgroups associated with treatment response and resistance in localized Ewing sarcoma (EWS) remains a challenge. The primary objective of the Children's Oncology Group (COG) biology study AEWS18B1-Q was to molecularly characterize patients with localized EWS on prospective modern-day trials. PATIENTS AND METHODS: We analyzed clinical and molecular features from patients with localized EWS enrolled on frontline COG trials. All patients had available formalin-fixed paraffin-embedded (FFPE) tissue, frozen tissue, or whole-genome–amplified material. Sequencing was performed for identification of canonical fusions, recurrent copy number alterations (CNAs), and alterations in TP53 and STAG2 . Available tissue was analyzed for loss of STAG2 protein expression. Molecular features were evaluated for their association with cumulative incidence of relapse in univariate and multivariable analyses. RESULTS: Three hundred fifty-one patients had sufficient tissue, which in most cases was extracted from two FFPE slides. EWS canonical fusions were identified in 282 patients (80.3%). Pathogenic mutations in TP53 and STAG2 were identified in 5.1% and 7.6% of patients, respectively. A total of 63.1% of patients were found to have recurrent CNAs. In univariate analysis, there was an increased cumulative incidence of relapse in patients with TP53 mutation (5-year cumulative incidence of relapse 43%, 95% CI [17% to 67%] v 22%, 95% CI [17% to 27%]; Gray's test P = .039), STAG2 mutation (53%, 95% CI [29% to 73%] v 21%, 95% CI [16% to 26%]; P < .001), and recurrent CNAs (30%, 95% CI [22% to 37%] v 16%, 95% CI [9% to 24%]; P = .005). In a multivariable analysis, STAG2 mutation was the only molecular biomarker that remained prognostic. CONCLUSION: This is a prospective validation of the molecular prognostic features of patients with localized EWS receiving standard-of-care therapy on therapeutic clinical trials. Building on previous work, patients with STAG2 mutations were at high risk of relapse.

PURPOSE: Identification of discrete sub-groups associated with treatment response and resistance in localized Ewing sarcoma (EWS) remains a challenge. The primary objective of the Children’s Oncology Group biology study AEWS18B1-Q was to perform molecular characterization of a large cohort of patients with localized Ewing sarcoma treated on prospective trials with modern standard of care therapy. METHODS: We analyzed clinical and molecular features from patients with localized EWS enrolled on AEWS0031, AEWS1031, or INT-0154 frontline trials. All patients had available FFPE tissue, frozen tissue, or whole-genome amplified material. Sequencing was performed for identification of canonical fusions, recurrent copy number alterations (CNAs), and alterations in TP53 and STAG2 . Where available, tissue was analyzed for loss of STAG2 protein expression. Molecular features were evaluated for their association with cumulative incidence of relapse in univariate and multivariable analyses. RESULTS: Three hundred and fifty-one cases had sufficient tissue, which in most cases was extracted from two FFPE slides. EWS canonical fusions were identified in 282 cases (80.3%). Pathogenic mutations in TP53 and STAG2 were identified in 5.1% and 7.6% of cases, respectively and 63.1% of cases were found to have recurrent CNAs. In univariate analysis, there was an increased cumulative incidence of relapse in patients with TP53 mutation (5-year cumulative incidence of relapse 43%, CI [17%, 67%] vs. 22%, CI [17%, 27%]; Gray’s test P = 0.039), STAG2 mutation (53%, CI [29%, 73%] vs. 21%, CI [16%, 26%]; P < 0.001), and recurrent CNAs (30%, CI [22%, 37%] vs. 16%, CI [9%, 24%]; P = 0.005). In a multivariable analysis, STAG2 mutation was the only molecular biomarker that remained prognostic. CONCLUSION: This is a prospective validation of the molecular prognostic features of localized EWS receiving standard of care therapy on therapeutic clinical trials. Building on prior work, patients with STAG2 mutations were at high risk of relapse. CONTEXT: Key Objective: To determine if molecular features can identify clinically relevant disease sub-groups applicable to frontline clinical trial design among patients with localized Ewing sarcoma. Knowledge Generated: Among 351 patients with localized Ewing sarcoma treated on prospective trials, canonical fusions were identified in 80% of cases. In a multivariable analysis of patients with genomically defined Ewing sarcoma, the presence of STAG2 mutations identified a high-risk population. Relevance (added by Associate Editor)

PURPOSE: Novel biomarkers are needed to differentiate outcomes in intermediate-risk rhabdomyosarcoma (IR RMS). We sought to evaluate strategies for identifying circulating tumor DNA (ctDNA) in IR RMS and to determine whether ctDNA detection before therapy is associated with outcome. PATIENTS AND METHODS: Pretreatment serum and tumor samples were available from 124 patients with newly diagnosed IR RMS from the Children's Oncology Group biorepository, including 75 patients with fusion-negative rhabdomyosarcoma (FN-RMS) and 49 with fusion-positive rhabdomyosarcoma (FP-RMS) disease. We used ultralow passage whole-genome sequencing to detect copy number alterations and a new custom sequencing assay, Rhabdo-Seq, to detect rearrangements and single-nucleotide variants. RESULTS: We found that ultralow passage whole-genome sequencing was a method applicable to ctDNA detection in all patients with FN-RMS and that ctDNA was detectable in 13 of 75 serum samples (17%). However, the use of Rhabdo-Seq in FN-RMS samples also identified single-nucleotide variants, such as MYOD1 ^L122R, previously associated with prognosis. Identification of pathognomonic translocations between PAX3 or PAX7 and FOXO1 by Rhabdo-Seq was the best method for measuring ctDNA in FP-RMS and detected ctDNA in 27 of 49 cases (55%). Patients with FN-RMS with detectable ctDNA at diagnosis had significantly worse outcomes than patients without detectable ctDNA (event-free survival, 33.3% v 68.9%; P = .0028; overall survival, 33.3% v 83.2%; P < .0001) as did patients with FP-RMS (event-free survival, 37% v 70%; P = .045; overall survival, 39.2% v 75%; P = .023). In multivariable analysis, ctDNA was independently associated with worse prognosis in FN-RMS but not in the smaller FP-RMS cohort. CONCLUSION: Our study demonstrates that baseline ctDNA detection is feasible and is prognostic in IR RMS.