Circulating plasma tumor DNA is superior to plasma tumor RNA detection in Ewing sarcoma patients.

Abstract The detection of tumor-specific nucleic acids from blood is being increasingly used as a method of liquid biopsy and minimal residual disease detection. However, achieving high sensitivity and high specificity remains a challenge. Here, we perform a direct comparison of two droplet digital PCR (ddPCR)-based detection methods, circulating plasma tumor RNA (ptRNA) and circulating plasma tumor DNA (ptDNA), in blood samples from newly diagnosed Ewing sarcoma patients. First, we developed three specific ddPCR-based assays to detect EWS-ETS fusion transcripts, which naturally demonstrated superior sensitivity to DNA detection on in vitro control samples. Next, we identified the patient-specific EWS-ETS breakpoint from five patient tumor samples and designed ddPCR-based patient-specific ptDNA assays for each patient. These patient-specific assays demonstrate that, while ptRNA can be detected in select newly diagnosed patients, positive results are low and statistically unreliable compared to ptDNA assays, which reproducibly detect robust positive results across most patients. Furthermore, the unique disease biology of Ewing sarcoma enabled us to demonstrate that most cell-free RNA is not tumor-derived, while cell-free-DNA burden is strongly affected by tumor-derived DNA burden. Here, we conclude that, even with optimized highly sensitive and specific assays, tumor DNA detection is superior to RNA detection in Ewing sarcoma patients.