Clinical implementation of plasma cell-free circulating tumor DNA quantification by digital droplet PCR for the monitoring of Ewing sarcoma in children and adolescents
Open accessAbstract
Background: Treatment stratification and response assessment in pediatric sarcomas has relied on imaging studies and surgical/histopathological evidence of vital tumor cells. Such studies and evidence collection processes often involve radiation and/or general anesthesia in children. Cell-free circulating tumor DNA (ctDNA) detection in blood plasma is one available method of so-called liquid biopsies that has been shown to correlate qualitatively and quantitatively with the existence of vital tumor cells in the body. Our clinical observational study focused on the utility and feasibility of ctDNA detection in pediatric Ewing sarcoma (EWS) as a marker of minimal residual disease (MRD). Patients and methods: We performed whole genome sequencing (WGS) to identify the exact breakpoints in tumors known to carry the EWS-FLI1 fusion gene. Patient-specific fusion breakpoints were tracked in peripheral blood plasma using digital droplet PCR (ddPCR) before, during, and after therapy in six children and young adults with EWS. Presence and levels of fusion breakpoints were correlated with clinical disease courses. Results: We show that the detection of ctDNA in the peripheral blood of EWS patients (i) is feasible in the clinical routine and (ii) allows for the longitudinal real-time monitoring of MRD activity in children and young adults. Although changing ctDNA levels correlated well with clinical outcome within patients, between patients, a high variability was observed (inter-individually). Conclusion: ctDNA detection by ddPCR is a highly sensitive, specific, feasible, and highly accurate method that can be applied in EWS for follow-up assessments as an additional surrogate parameter for clinical MRD monitoring and, potentially, also for treatment stratification in the near future.
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Fusion-driven tumors may be best suited to breakpoint-guided ddPCR or fusion-aware NGS.
“Fusion-driven tumors may be best suited to breakpoint-guided ddPCR or fusion-aware NGS [ 13 , 14 , 15 ].”
From Signals Alongside Scans: A Genomics-Guided Framework for Liquid Biopsy in Bone and Soft-Tissue Sarcomas (2026) · cites this paper as [13]
Tumor-informed ctDNA monitoring in osteosarcoma and breakpoint-guided ctDNA monitoring in translocation-associated sarcomas currently have the clearest longitudinal clinical support.
“Tumor-informed ctDNA monitoring in osteosarcoma and breakpoint-guided ctDNA monitoring in translocation-associated sarcomas currently have the clearest longitudinal clinical support, although both still require prospective multicenter validation before routine treatment decisions can be based on ctDNA alone [ 13 , 16 , 17 , 22 , 23 , 24 ].”
From Signals Alongside Scans: A Genomics-Guided Framework for Liquid Biopsy in Bone and Soft-Tissue Sarcomas (2026) · cites this paper as [13]
In Ewing sarcoma and other breakpoint-defined tumors, the disease-defining structural variant provides a stable and highly specific target for longitudinal monitoring.
“In Ewing sarcoma and other breakpoint-defined tumors, the disease-defining structural variant provides a stable and highly specific target for longitudinal monitoring [ 13 , 22 , 23 ].”
From Signals Alongside Scans: A Genomics-Guided Framework for Liquid Biopsy in Bone and Soft-Tissue Sarcomas (2026) · cites this paper as [13]
Ewing sarcoma and other translocation-associated sarcomas provide a different model because disease-defining structural variants can serve as highly specific molecular targets for longitudinal monitoring.
“Ewing sarcoma and other translocation-associated sarcomas provide a different model because disease-defining structural variants can serve as highly specific molecular targets for longitudinal monitoring [ 13 , 22 , 23 ].”
From Signals Alongside Scans: A Genomics-Guided Framework for Liquid Biopsy in Bone and Soft-Tissue Sarcomas (2026) · cites this paper as [13]
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“Selected miRNAs of interest (miR-21, miR-26a, miR-125b, and miR-199a) [31–33] were quantified using droplet digital PCR (ddPCR) on the QX200 Droplet Digital PCR System (Model: QX200; Bio-Rad Laboratories).”
Joch, S., Smolle, M. A., Kashofer, K., Thüringer, A., Szkandera, J., Benesch, M., El-Heliebi, A., Liegl-Atzwanger, B., Leithner, A., & Seidel, M. G. (2024) · Oncology Research and Treatment · doi:10.1159/000543223 · Cited by 2 · see citing works
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- Authors
- Seidel, M. G., Kashofer, K., Moser, T., Thueringer, A., Liegl-Atzwanger, B., Leithner, A., Szkandera, J., Benesch, M., El-Heliebi, A., & Heitzer, E.
- Affiliations
- Medical University of Graz (AT)
- Corresponding author
- Kashofer, Karl
- ORCID
- Venue
- Frontiers in Pediatrics
- Publisher
- Frontiers Media SA
- Published
- 2022-08-15
- Volume
- 10
- Pages
- 926405-926405
- ISSN
- 2296-2360 (ISSN-L)
- PMC
- PMC9420963
- OpenAlex
- W4291377988
- Cited by
- 24
- References
- 42
- Citations by year
- 2026: 7 · 2025: 8 · 2024: 5 · 2023: 3 · 2022: 1
- FWCI
- 2.74
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Frontiers in Pediatrics (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- Karl-Franzens-Universität Graz
- Medizinische Universität Graz
- Topics
- Health Sciences › Medicine › Pulmonary and Respiratory Medicine › Sarcoma Diagnosis and Treatment
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer Genomics and Diagnostics
- Health Sciences › Medicine › Public Health, Environmental and Occupational Health › Acute Lymphoblastic Leukemia research
- Keywords
- Ewing sarcoma, circulating tumor DNA, liquid biopsy, droplet digital PCR, minimal residual disease, pediatric sarcoma, treatment response monitoring, treatment stratification, children and adolescents
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