Background:Limited information is available on the applicative value of liquid biopsy (LB) in rare tumors, including Ewing’s sarcoma (ES). The accepted precision diagnostics standards would greatly benefit from a non-invasive LB test monitoring pathognomonic gene rearrangements in the bloodstream.Methods:Tissue and blood samples were collected from six and four ES patients, respectively. Plasma was cleared by two successive rounds of centrifugation and stored frozen until RNA extraction by the QIAmp CNA kit. RNA was retro-transcribed and subjected to real-time quantitative polymerase chain reaction (RT-qPCR) and digital polymerase chain reaction (dPCR). Reactions were set up using two custom primer sets identifying types 1 and 2 EWS-FLI1 fusion transcripts.Results:The two prevalent types of EWS-FLI1 rearrangements could be identified using only two sets of polymerase chain reaction primers, regardless of patient-specific EWS-FLI1 DNA breakpoints. RT-qPCR and dPCR discriminated the two variants in five tumor tissue RNAs and in four circulating tumor RNAs (ctRNAs). Of note, EWS-FLI1 molecular diagnosis was possible using blood samples even when tumor tissue was not available. ctRNA levels correlated (p <0.05) with volume-based positron emission tomography (PET) parameters (metabolic tumor volume and total lesion glycolysis), and allowed the fine tracking of disease course after surgery, during adjuvant as well as neoadjuvant chemotherapy, and at follow up in one patient.Conclusions:To our knowledge, this is one of the few single-marker LB assays in solid tumors specifically designed to detect rearranged RNAs in blood, and the first study describing EWS circulating tumor RNAs in ES patients. Altogether, our results support the idea that LB may have a considerable impact on ES patient monitoring and management.