Abstract 4025: Sequential cell free DNA analysis reveals genetic heterogeneity in patients with relapsed neuroblastoma enrolled in the SIOPEN-ITCC BEACON study

Abstract Background: Outcome for high-risk neuroblastoma following relapse remains poor, with an urgent need to increase understanding of the biology to develop effective (combination) therapies. Aims: To characterize tumor-specific molecular alterations for patients enrolled in the SIOPEN-ITCC BEACON trial (EudraCT 2012-000072-42, evaluating different backbone chemotherapy regimens +/-bevacizumab in high risk refractory/relapsed NB) and to analyze resistance mechanisms based on sequential cfDNA. Methods: Samples were collected from 191 BEACON patients. Tumor samples were available for 73 patients (2:diagnosis, 24:refractory; 47:relapse), and liquid biopsies for 182 patients (1-6 samples per patient, mean: 3). cfDNA lcWGS (low coverage Whole-genome sequencing), cfDNA WES (Whole-exome sequencing or targeted panel-sequencing was conducted on all plasma samples, with paired germline analyses. Results: Molecular analysis of 63 tumor samples (41 large NGS-panel and 22 WES), , identified MYCN amplification in 17 cases, and mutations in RAS/MAPK/P53 pathway genes in 24% of patients, with 17.5% harboring an ALK mutation. cfDNA quantity at enrollment in BEACON did not correlate with disease burden (SIOPEN score), but showed correlation with outcome (HR p=0.02). From plasma samples obtained at inclusion and analyzed by WES, a mean of 35[5-96] SNVs were shared with tumor samples, while 31[7-73] and 11[2-29] SNVs were unique to tumor and plasma, respectively. cfDNA specific SNVs frequently targeted genes of pathways in cell-to-cell and cell-to-extracellular matrix connections. Sequential cfDNA quantities and ctDNA content evaluated by WES were measured for 139 patients, and showed distinct dynamics correlating with disease status. Notably, cfDNA quantity increases were associated with the emergence of new mutations in all analyzed cases, including alterations in genes of the RAS/MAPK pathway such as HRAS. Evidence of clonal hematopoiesis during treatment was observed in 40% of cases. Fragment size and nucleosome footprint analyses for inference of transcription factor and overall expression profiles is ongoing. Conclusions: In refractory/relapsed high-risk neuroblastoma patients, 1/4 had at least one mutation in RAS/MAPK/p53 pathway genes, which might correlate with poorer survival. 53% of SNVs observed in tumor were detectable in plasma, and plasma-specific SNVs were observed in all cases. This finding underscores the value of plasma sample collection during follow-up to study tumor heterogeneity and clonal evolution. Citation Format: Yasmine Iddir, Charles Bobin, Alexandra Saint-Charles, Julien Masliah-Planchon, Marion Gambart, Elnaz Saberi-Ansari, Antonio Colaprico, Angela Bellini, Charlotte Butterworth, Valery Attignon, Deborah A. Tweddle, Jennifer R. Tall, Debbie Hughes, Rebekah Weston, Andrea Vilaplana, Louis Chesler, Lucas Moreno, Sally George, Gudrun Schleiermacher. Sequential cell free DNA analysis reveals genetic heterogeneity in patients with relapsed neuroblastoma enrolled in the SIOPEN-ITCC BEACON study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4025.