Quantitative and sensitive neuroblastoma minimal residual disease detection using extrachromosomal DNA (ecDNA) breakpoints
- 1Department of Pediatric Oncology and Hematology, Charite Universitaetsmedizin Berlin, Corporate Member of Freie Universitaet Berlin and Humboldt-Universitaet zu
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.
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BibTeX
@misc{szymansky2026quantitative,
title = {{Quantitative and sensitive neuroblastoma minimal residual disease detection using extrachromosomal DNA (ecDNA) breakpoints}},
author = {A. Szymansky and F. Hertwig and A. Winkler and R. Chamorro Gonzalez and K. Helmsauer and A.-M. Schroeer and M. J. Witthauer and E. Karaman and M. Lodrini and J. Wuenschel and N. Wittstruck and S. Fuchs and J. Theissen and B. Hero and R. P. Koche and M. J. Barz and L. Heukamp and K. Astrahantseff and K. Schmelz and P. Hundsdoerfer and J. Toedling and A. Eggert and C. Eckert and B. Spanjaard and H. E. Deubzer and M. Fischer and A. G. Henssen and J. H. Schulte},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.06.749680},
url = {https://www.biorxiv.org/content/10.64898/2026.09.06.749680v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Quantitative and sensitive neuroblastoma minimal residual disease detection using extrachromosomal DNA (ecDNA) breakpoints
AU - A. Szymansky
AU - F. Hertwig
AU - A. Winkler
AU - R. Chamorro Gonzalez
AU - K. Helmsauer
AU - A.-M. Schroeer
AU - M. J. Witthauer
AU - E. Karaman
AU - M. Lodrini
AU - J. Wuenschel
AU - N. Wittstruck
AU - S. Fuchs
AU - J. Theissen
AU - B. Hero
AU - R. P. Koche
AU - M. J. Barz
AU - L. Heukamp
AU - K. Astrahantseff
AU - K. Schmelz
AU - P. Hundsdoerfer
AU - J. Toedling
AU - A. Eggert
AU - C. Eckert
AU - B. Spanjaard
AU - H. E. Deubzer
AU - M. Fischer
AU - A. G. Henssen
AU - J. H. Schulte
PY - 2026
DA - 2026/09/06
PB - bioRxiv
DO - 10.64898/2026.09.06.749680
UR - https://www.biorxiv.org/content/10.64898/2026.09.06.749680v1
AB - 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.
ER -