Clinical implementation of plasma cell-free circulating tumor DNA quantification by digital droplet PCR for the monitoring of Ewing sarcoma in children and adolescents

Markus G Seidel; Karl Kashofer; Tina Moser; Andrea Thueringer; Bernadette Liegl-Atzwanger; Andreas Leithner; Joanna Szkandera; Martin Benesch; Amin El-Heliebi; Ellen Heitzer

doi:10.3389/fped.2022.926405

Clinical implementation of plasma cell-free circulating tumor DNA quantification by digital droplet PCR for the monitoring of Ewing sarcoma in children and adolescents

Front Pediatr. 2022 Aug 15:10:926405. doi: 10.3389/fped.2022.926405. eCollection 2022.

Authors

Markus G Seidel¹, Karl Kashofer^{2

3}, Tina Moser^{4

5}, Andrea Thueringer², Bernadette Liegl-Atzwanger², Andreas Leithner⁶, Joanna Szkandera⁷, Martin Benesch¹, Amin El-Heliebi^{3

8

9}, Ellen Heitzer^{4

5}

Affiliations

¹ Division for Pediatric Hematology-Oncology, Department of Pediatrics and Adolescent Medicine, Medical University of Graz, Graz, Austria.
² Diagnostic and Research Center for Molecular BioMedicine, Diagnostic and Research Institute of Pathology, Medical University of Graz, Graz, Austria.
³ BioTechMed-Graz, Graz, Austria.
⁴ Diagnostic and Research Center for Molecular BioMedicine, Diagnostic and Research Institute of Human Genetics, Medical University of Graz, Graz, Austria.
⁵ Christian Doppler Laboratory for Liquid Biopsies for Early Detection of Cancer, Medical University of Graz, Graz, Austria.
⁶ Department of Orthopedics and Trauma, Medical University of Graz, Graz, Austria.
⁷ Division of Oncology, Department of Internal Medicine, Medical University of Graz, Graz, Austria.
⁸ Division of Cell Biology, Histology and Embryology, Gottfried Schatz Research Center, Medical University of Graz, Graz, Austria.
⁹ Center for Biomarker Research in Medicine (CBmed), Graz, Austria.

Abstract

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.

Keywords: Ewing sarcoma (EWS); breaktracer; cell-free DNA (cfDNA); circulating cell-free tumor DNA (ctDNA); ddPCR assay; minimal residual disease (MRD); pediatric oncology.