title: The spectrum of EWSR1-rearranged neoplasms at a tertiary sarcoma centre; assessing 772 tumour specimens and the value of current ancillary molecular diagnostic modalities.
abstract: BACKGROUND
  EWSR1 rearrangements were first identified in Ewing sarcoma, but the spectrum of EWSR1-rearranged neoplasms now includes many soft tissue tumour subtypes including desmoplastic small round cell tumour (DSRCT), myxoid liposarcoma (MLPS), extraskeletal myxoid chondrosarcoma (EMC), angiomatoid fibrous histiocytoma (AFH), clear cell sarcoma (CCS) and myoepithelial neoplasms. We analysed the spectrum of EWSR1-rearranged soft tissue neoplasms at our tertiary sarcoma centre, by assessing ancillary molecular diagnostic modalities identifying EWSR1-rearranged tumours and reviewing the results in light of our current knowledge of these and other Ewing sarcoma-like neoplasms.
  
  
  METHODS
  We retrospectively analysed all specimens tested for EWSR1 rearrangements by fluorescence in situ hybridisation (FISH) and/or reverse transcription-PCR (RT-PCR) over a 7-year period.
  
  
  RESULTS
  There was a total of 772 specimens. FISH was performed more often than RT-PCR (n=753, 97.5% vs n=445, 57.6%). In total, 210 (27.9%) specimens were FISH-positive for EWSR1 rearrangement compared to 111 (14.4%) that showed EWSR1 fusion transcripts with RT-PCR. Failure rates for FISH and RT-PCR were 2.5% and 18.0%. Of 109 round cell tumours with pathology consistent with Ewing sarcoma, 15 (13.8 %) cases were FISH-positive without an identifiable EWSR1 fusion transcript, 4 (3.7%) were FISH-negative but RT-PCR positive and 4 (3.7%) were negative for both. FISH positivity for DSRCT, MLPS, EMC, AFH and CCS was 86.3%, 4.3%, 58.5%, 60.0% and 87.9%, respectively. A positive FISH result led to diagnostic change in 40 (19.0%) EWSR1-rearranged cases. 13 FISH-positive cases remained unclassifiable.
  
  
  CONCLUSIONS
  FISH is more sensitive for identifying EWSR1 rearrangements than RT-PCR. However, there can be significant morphologic and immunohistochemical overlap between groups of EWSR1-rearranged neoplasms, with important prognostic and therapeutic implications. FISH and RT-PCR should be used as complementary modalities in diagnosing EWSR1-rearranged neoplasms, but as tumour groups harbouring EWSR1 rearrangements are increasingly characterised and because given translocations involving EWSR1 and its partner genes are not always specific for tumour types, it is critical that these are evaluated by specialist soft tissue surgical pathologists noting the morphologic and immunohistochemical context. As RT-PCR using commercial primers is limited to only the most prevalent EWSR1 fusion transcripts, the incorporation of high-throughput sequencing technologies into the standard diagnostic repertoire to assess for multiple molecular abnormalities of soft tissue tumours in parallel (including detection of newly characterised Ewing sarcoma-like tumours) might be the most effective and efficient means of ancillary diagnosis in future.
authors.affiliation: Sarcoma Unit, Royal Marsden Hospital, London SW3 6JJ, UK.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Noujaim
authors.affiliation: Sarcoma Unit, Royal Marsden Hospital, London SW3 6JJ, UK.
authors.forename: Robin L
authors.initials: RL
authors.lastname: Jones
authors.affiliation: Clinical Cytogenetics, Royal Marsden Hospital, Surrey SM2 5NG, UK.
authors.forename: John
authors.initials: J
authors.lastname: Swansbury
authors.affiliation: Molecular Diagnostics, Royal Marsden Hospital, Surrey SM2 5NG, UK.
authors.forename: David
authors.initials: D
authors.lastname: Gonzalez
authors.affiliation: Sarcoma Unit, Royal Marsden Hospital, London SW3 6JJ, UK.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Benson
authors.affiliation: Sarcoma Unit, Royal Marsden Hospital, London SW3 6JJ, UK.
authors.forename: Ian
authors.initials: I
authors.lastname: Judson
authors.affiliation: Sarcoma Unit, Royal Marsden Hospital, London SW3 6JJ, UK.
authors.forename: Cyril
authors.initials: C
authors.lastname: Fisher
authors.affiliation: Sarcoma Unit, Royal Marsden Hospital, London SW3 6JJ, UK.
authors.forename: Khin
authors.initials: K
authors.lastname: Thway
mesh_terms: D002148:Calmodulin-Binding Proteins / Q000235:genetics*; D055088:Early Detection of Cancer; D015321:Gene Rearrangement; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D034802:RNA-Binding Protein EWS; D016601:RNA-Binding Proteins / Q000235:genetics*; D012189:Retrospective Studies; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D012983:Soft Tissue Neoplasms / Q000235:genetics*; D062606:Tertiary Care Centers
chemical_list: D002148:Calmodulin-Binding Proteins; C500986:EWSR1 protein, human; D034802:RNA-Binding Protein EWS; D016601:RNA-Binding Proteins