Background: Genomic EWSR1-FLI1 fusion sequences have been used as non-invasive tumor marker to assess therapy response in EwS patients by quantification of cell-free circulating tumor DNA in plasma samples. Since fresh-frozen tumor biopsies are often not available, fusion sequences have to be identified in highly fragmented DNA from formalin-fixed, paraffin-embedded (FFPE) specimen. Methods: Patient-specific EWS fusion genes were pre-amplified in a multiplex droplet digital PCR (mddPCR) using DNA from FFPE tumor samples. This library was then sequenced with the Ion S5 System. Results: The combination of mddPCR and targeted enrichment NGS improves the success rate for identification of genomic fusion sequences and facilitates their use as a biomarker for ctDNA monitoring. Analyses of ctDNA levels and clinical parameters from EWING2008 patients revealed correlation of ctDNA copy numbers and patients' risk factors. Discussion: MddPCR based targeted enrichment NGS strategy enables the identification of tumor specific fusion sequences from highly fragmented DNA samples and therefore allows their application as molecular tumor marker in a wider range of sample sources.
