Highly personalized detection of minimal Ewing sarcoma disease burden from plasma tumor DNA

Abstract

Background Even though virtually all Ewing sarcoma patients achieve a radiographic complete response, up to 30% of patients who present with localized disease and up to 90% of those who present with metastases suffer a metastatic relapse, highlighting our inability to identify patients with residual disease at the end of therapy. Up to 95% of Ewing sarcomas carry a driving EWS-ETS translocation which has an intronic breakpoint that is specific to each tumor, and we developed a system to quantitatively detect the specific breakpoint DNA fragment in patient plasma. Methods We used a long range multiplex PCR technique to identify tumor-specific EWS-ETS breakpoints in Ewing sarcoma cell lines, patient derived xenografts, and patient tumors, and this sequence was used to design tumor specific primer sets to detect plasma tumor DNA (ptDNA) by droplet digital PCR (ddPCR) in xenograft bearing mice and patients. Results Tumor specific breakpoint DNA fragments were detected in the plasma of xenograft bearing mice, and signal correlated with tumor burden during primary tumor growth, following surgical resection, and upon metastatic relapse. Furthermore, we were able to detect the specific breakpoint in plasma DNA obtained from 3 patients with Ewing sarcoma, and in 2 patients, we were able to detect ptDNA when there was radiographically undetectable disease present. Conclusions Use of ddPCR to detect tumor specific EWS-ETS fusion gene breakpoint ptDNA fragments can be developed into a highly personalized biomarker of relapse that can be optimized in animal studies for ultimate use in patients.

Reduced-Intensity Haploidentical Bone Marrow Transplantation with Post-Transplant Cyclophosphamide for Solid Tumors in Pediatric and Young Adult Patients

“…These patients both developed CTC more than 6 months post-transplant, at a time when immune reconstitution is more robust. These findings are consistent with our recent findings [75] demonstrating that circulating tumor DNA strongly predicted imminent relapse in sarcoma patients without radiographic evidence of disease who were receiving standard chemotherapy. We speculate that a subclinical relapse occurring in a transplant patient after meaningful immune reconstitution can be controlled by the donor immune system, whereas an early relapse, before immune reconstitution, cannot.…”

Detection of Somatic Structural Variants Enables Quantification and Characterization of Circulating Tumor DNA in Children With Solid Tumors

“…36–38 To compare TranSS-Seq with this previously validated approach, patient-specific polymerase chain reaction primers were developed for a subset of Ewing sarcoma and alveolar rhabdomyosarcoma samples (Data Supplement). The percentage of ctDNA levels identified by TranSS-Seq correlated with the percentage of ctDNA detected by ddPCR (Fig 3E).…”

“…36–38 Although this approach has proven to be highly sensitive and quantitative, the development of each assay requires genomic profiling of large amounts of tumor biopsy material to establish the patient-specific oncogenic translocation. One recent study used a combination of ddPCR and hybrid capture sequencing to detect EWSR1 translocations in Ewing sarcoma and desmoplastic small round-cell tumors.…”

Genotyping of circulating cell‐free DNA enables noninvasive tumor detection in myxoid liposarcomas

“…As amplification of the target region is performed with primers binding to separate chromosomal regions and detection of the target amplicon occurs by a probe that is placed on the breakpoint region, these assays offer unprecedented specificity. The feasibility of this approach has also recently been confirmed in Ewing sarcomas . The assays to detect synovial sarcoma t(X;18) ctDNA from cell‐lines showed similar sensitivity and specificity than with myxoid liposarcomas (Fig.…”