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. 2026 Mar 6.
doi: 10.1038/s44321-026-00396-7. Online ahead of print.

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

Sophie A Richardson #  1 , Aram Safrastyan #  1 , Mina Karimpour #  1 , Gayatri Gulati  1 , Patrick J B Harker  1 , Alan Redfern  1 , Simon P Pearce  1 , Vsevolod J Makeev  1 , Bernadette Brennan  2 , Alexander T J Lee  3 , Alexandra Clipson  1 , Steven M Hill  1 , Caroline Dive  1 , Dominic G Rothwell  1 , Martin G McCabe  4   5 , Florent Mouliere  6
Affiliations
Free article

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

Sophie A Richardson et al. EMBO Mol Med. .
Free article

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.

Keywords: Cell-Free DNA; Ewing Sarcoma; Fragmentomics; Liquid Biopsy; Methylome.

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Conflict of interest statement

Disclosure and competing interests statement. SMH, SPP, AC, CD, DGR and FM are co-inventors on patents related to cell-free DNA analysis. FM has consulted for Roche Dx. AC and FM have received support from Biomodal. CD has received research funding/educational research grants from Amgen, AstraZeneca, Astex Pharmaceuticals, Angle PLC, Boehringer Ingelheim, Carrick Therapeutics, Celgene, Clearbridge Biomedics, CV6 Therapeutics (NI) Ltd, Epigene Therapeutics Inc, GSK, Guardant, Menarini, Merck AG, Neomed Therapeutics, Novartis, RedX Pharma, Roche, Taiho Oncology, Thermo Fisher Scientific and UCB Pharma. CD has received honoraria for consultancy and/or advisory boards from Biocartis, Merck, AstraZeneca, GRAIL and Boehringer Ingelheim. ATJL has received honoraria and support with conference attendance from Alexion Pharmaceuticals. The remaining authors declare no competing interests.

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