Quantitative and sensitive neuroblastoma minimal residual disease detection using extrachromosomal DNA (ecDNA) breakpoints.

Authors

Annabell Szymansky, Falk Hertwig, Annika Winkler, Rocío Chamorro González, Konstantin Helmsauer, Anna-Marie Schroeer, Melanie J Witthauer, Eliz Karaman, Marco Lodrini, Jasmin Wünschel, Nadine Wittstruck, Steffen Fuchs, Jessica Theissen, Barbara Hero, Richard Koche, Malwine J Barz, Lukas C Heukamp, Kathy Astrahantseff, Karin Schmelz, Patrick Hundsdoerfer, Joern Toedling, Angelika Eggert, Cornelia Eckert, Bastiaan Spanjaard, Hedwig E Deubzer, Matthias Fischer, Anton G Henssen, Johannes H Schulte

Published in

bioRxiv : the preprint server for biology. Sep 12, 2026. Epub Sep 12, 2026.

Abstract

Sensitive detection of minimal residual disease (MRD) remains a major unmet need in high-risk neuroblastoma. MYCN amplification, a hallmark of high-risk disease, typically occurs on extrachromosomal DNA (ecDNA), but the potential of ecDNA-associated genomic rearrangements for individualized MRD monitoring has not been fully exploited. Here, we applied neuroblastoma-specific hybrid capture-based panel sequencing to identify patient-unique breakpoints within MYCN amplicons, and used Circle-seq and Nanopore sequencing to resolve the extrachromosomal amplicon structure in representative samples. Analysis of 8 neuroblastoma cell lines and 22 primary tumors identified 69 tumor-specific breakpoints. Those selected for assay development were validated by breakpoint-specific PCR and Sanger sequencing. Breakpoints detected in primary tumors remained detectable at relapse, supporting their stability as MRD markers. Breakpoint-specific real-time quantitative PCR and droplet digital PCR detected these junctions in bone marrow aspirates with high specificity and reached sensitivities down to a tumor DNA fraction of 10⁻⁶. We applied this approach to 53 serial bone marrow aspirates from 14 patients with high-risk neuroblastoma to monitor MRD dynamics, resolving treatment response and molecular persistence. In six samples, breakpoint-positive DNA was detected in bone marrow that was negative by conventional cytology and immunocytology, highlighting the added value of molecular monitoring. Together, these findings establish ecDNA breakpoint-based detection as a strategy for MRD assessment in neuroblastoma, that is, in principle, applicable to any ecDNA-amplified oncogene.
Breakpoints in extrachromosomal circular DNA, a substrate of oncogene amplification in cancer, can serve as diagnostic markers of minimal residual disease.

PMID:
42818635
Bibliographic data and abstract were imported from PubMed on 01 Oct 2026.

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