Abstract A009-PR010: Implementation and Performance of a Neuroblastoma Liquid Biopsy Panel for Non-Invasive Molecular Monitoring

Abstract Liquid biopsy surveillance of patients with neuroblastoma can be used to monitor for relapse, detect therapeutic targets, and track drug resistance. As current commercially available liquid biopsy assays are tailored to adult malignancies and do not comprehensively capture variants relevant to neuroblastoma, especially at low variant allele frequency (VAF), we developed and clinically validated a Neuroblastoma Liquid Biopsy Panel (NBLBP) focused on genes recurrently mutated in neuroblastoma to maximize sequencing depth and VAF sensitivity. The NBLBP interrogates 23 genes enriched for somatic alterations in relapsed neuroblastoma identified from comprehensive literature review (e. g. , ALK, ATRX, MYCN, TP53, NF1) using anchored multiplex PCR with unique molecular identifiers, followed by next-generation sequencing, detecting both sequence and copy number variants (CNVs). The initial validation cohort contained 43 samples for single-nucleotide variant (SNV) and small insertion-deletion (indel) analysis and 21 samples for MYCN amplification. The limit of detection was established at 0. 5% VAF or lower for certain variants using 30 ng input of cell-free DNA (cfDNA). Validation for additional gene amplifications and ATRX deletion was performed using 80 samples following the initial phase. We conducted a retrospective review of 282 samples tested within the first 16 months of test launch from 137 individual neuroblastoma patients, most of whom had multiply relapsed high-risk disease. Tumor-associated ctDNA alterations were identified in 37. 6% (106/282). Subset analysis of assay input averaged at 52 ng, with only 3. 5% of samples having 30 ng due to low cfDNA at extraction. The most frequent Tier 1, 2 or pathogenic SNVs and indels included TP53 (n=39), ALK (n=37, including 6 germline variants), NF1 (n=28), and ATRX (n=22). Detected VAFs ranged from ≤0. 5% up to 50%LS1. Detected CNVs included MYCN gain or amplification (n=15), ATRX loss (n=5), and amplifications in CDK4 (n=2), MDM2 (n=2), MET (n=1), and ALK (n=1). Among patients with matched tumor testing (n=22), mutation profiles in ctDNA demonstrated high concordance with previously identified alterations in tumor tissue. Of patients tested (n=137), 58 had two or more serial samples, and some of these showed dynamic response of variants over time to treatment interventions and/or progressive disease. Individual patient analyses to assess clinical utility are ongoing and include ctDNA positivity preceding imaging confirmed relapse, adjustments in therapy based on ctDNA findings, and monitoring of ALK SNVs during targeted therapy. The NBLBP performs well at detecting disease-associated sequence and CNVs in patients with advanced neuroblastoma. Multiple variant types can be detected, including SNVs, small indels, and gene amplifications, allowing for sensitive disease tracking important for clinical decision making. NBLBP is a powerful non-invasive test that can supplement the currently available surveillance strategies for patients with advanced neuroblastoma. Citation Format: Jinhua Wu, Esther Berko, Feng Xu, Jeffrey Schubert, Jiani Chen, Virginia M Hamilton, Matthew Lueder, Minjie Luo, Grace Polkosnik, Yiming Zhong, John M Maris, Marilyn M Li, Yael P Mossé, Lea F Surrey. Implementation and Performance of a Neuroblastoma Liquid Biopsy Panel for Non-Invasive Molecular Monitoring abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85 (18Suppl₂): Abstract nr A009-PR010.