Utilisation of fluorescent multiplex PCR and laser-induced capillary electrophoresis for the diagnosis of Ewing family of tumours in formalin-fixed paraffin-embedded tissues
- Barbara Patócs1,
- Krisztina Németh1,
- Miklós Garami1,
- Gabriella Arató2,
- Ilona Kovalszky3,
- Miklós Szendrői4,
- György Fekete1
- 12nd Department of Paediatrics, Semmelweis University, Budapest, Hungary
- 2Department of Pathology, Szent János Hospital, Budapest, Hungary
- 31st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary
- 4Department of Orthopaedics, Semmelweis University, Budapest, Hungary
- Correspondence to Dr Barbara Patócs, 2nd Department of Paediatrics, Semmelweis University, Tűzoltó u. 7-9, Budapest 1094, Hungary;
- Accepted 31 August 2012
- Published Online First 26 September 2012
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.