Utilisation of fluorescent multiplex PCR and laser-induced capillary electrophoresis for the diagnosis of Ewing family of tumours in formalin-fixed paraffin-embedded tissues.

Abstract

AIMS: The localisation of the translocation breakpoint of the Ewing sarcoma family of tumours shows significant variability on relatively large regions of fusion partner genes. As a consequence, many alternative forms of EWSR1-ETS translocation exist which make the RNA-based molecular diagnostics of Ewing sarcoma family of tumours complicated. In addition to the heterogeneity of fusion transcripts, the degradation of RNA also presents a significant difficulty in the molecular analysis of formalin-fixed paraffin-embedded (FFPE) tissues. Our aim was to establish a sensitive method which is able to identify all combinatorially possible EWSR1-FLI1 and EWSR1-ERG translocation transcripts in FFPE tissue samples despite significant RNA-degradation. METHODS: The combination of fluorescent multiplex PCR with laser-induced capillary electrophoresis was used to detect and identify EWSR1-FLI1 and EWSR1-ERG chimeric transcripts on the basis of amplicon size, and forward primers labelled by distinct fluorophores. RESULTS: Using this method, we processed 60 FFPE samples of Ewing sarcoma family of tumours, and identified six types EWSR1-FLI1 and one type EWSR1-ERG chimeric transcripts acceptable for RT-PCR analysis in 27 out of 45 samples. This result shows 60% sensitivity for detecting the most frequent Ewing family of tumour (EFT)-related fusion transcripts. CONCLUSIONS: The utilisation of fluorescent multiplex PCR and laser-induced fluorescent capillary electrophoresis is effective for the diagnosis of EFT in FFPE tissue, and after the defined modifications it can offer a sensitive method to overcome the diagnostic difficulties connected with heterogeneity of the variant translocations in EFT.