title: 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: OBJECTIVE
  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.
authors.affiliation: 2nd Department of Paediatrics, Semmelweis University, Budapest, Hungary. barbarapatocs@gmail.com
authors.forename: Barbara
authors.initials: B
authors.lastname: Patócs
authors.forename: Krisztina
authors.initials: K
authors.lastname: Németh
authors.forename: Miklós
authors.initials: M
authors.lastname: Garami
authors.forename: Gabriella
authors.initials: G
authors.lastname: Arató
authors.forename: Ilona
authors.initials: I
authors.lastname: Kovalszky
authors.forename: Miklós
authors.initials: M
authors.lastname: Szendroi
authors.forename: György
authors.initials: G
authors.lastname: Fekete
mesh_terms: D001859:Bone Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D018242:Neuroectodermal Tumors, Primitive / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology; D016612:Paraffin Embedding; D012189:Retrospective Studies; D012512:Sarcoma, Ewing / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology