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

Abstract

BackgroundTreatment 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 methodsWe 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.ResultsWe 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).ConclusionctDNA 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.

Targeted locus amplification to develop robust patient-specific assays for liquid biopsies in pediatric solid tumors

“…The relation to tumor burden and monitoring of relapse in Ewing sarcoma was also demonstrated in the recent study by Shulman et al. ( 35 ) They also designed a patient-specific assay for the fusion genes in 6 patients with Ewing sarcoma, using data from WGS of the tumor material. In 2 patients that remained in complete remission, the fusion breakpoint disappeared after initiation of treatment.…”

“…In 2 patients that remained in complete remission, the fusion breakpoint disappeared after initiation of treatment. In 4 patients that suffered from relapse, cfDNA levels of the breakpoint reflected presence of relapse and response to therapy ( 35 ). These findings from other reports underline the potential of a patient-specific target as a treatment response marker and early relapse detection.…”

Sarcoma care in the era of precision medicine

“…There is a growing number of studies describing how analysis of cell-free DNA can be utilized for sarcoma diagnostics [157], treatment monitoring [158], identification of resistance mechanisms [159], and minimal residual disease detection [160]. This application is bound to grow rapidly.…”

“…Cell‐free DNA can be isolated from a blood sample, and when a malignant tumor is present in the body, some of the cell‐free DNA comes from the cancer cells [156]. There is a growing number of studies describing how analysis of cell‐free DNA can be utilized for sarcoma diagnostics [157], treatment monitoring [158], identification of resistance mechanisms [159], and minimal residual disease detection [160]. This application is bound to grow rapidly.…”