Cell-free DNA methylome and fragmentome analysis for relapse monitoring of Ewing sarcoma

Abstract Liquid biopsies and cell-free DNA (cfDNA) offer minimally invasive methods for the diagnosis and monitoring of Ewing Sarcoma (EwS). EwS have a low tumour mutational burden and their detection with plasma cfDNA is challenging. We hypothesised that analysing the cfDNA methylome and fragmentome could enhance sensitivity for detecting EwS and identifying disease recurrence. Using T7-MBD-seq, we conducted whole-genome and methylome sequencing of cfDNA from 87 serial samples of 23 patients with EwS and 3 patients with CIC-rearranged sarcoma (CIC). With EwingSign, a new machine learning model, we identified EwS or CIC in a test set for 11 out of 16 patients at diagnosis and 15 out of 18 clinically confirmed relapse events. 0 out of 24 non-cancer controls (NCC) were detected positive with EwingSign. When combined with global and regional fragmentome analysis, all 18 relapse cases were detected, with 15/18 detected by 2 or more modalities, and 1 out of 24 NCC was detected by one modality. These findings indicate that cfDNA methylome and fragmentome analysis, if validated in a larger cohort, could improve disease detection, monitoring and relapse identification in patients with EwS.