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.
