title: Defining Ewing and Ewing-like small round cell tumors (SRCT): The need for molecular techniques in their categorization and differential diagnosis. A study of 200 cases.
abstract: BACKGROUND
  Differentiation of Ewing sarcoma family of tumors (ESFT) and Ewing-like tumors remains problematic. Certain ESFT with morphological and immunohistochemical (IHC) profiles lack the EWSR1-ETS transcript. To improve diagnostic accuracy we investigated the presence of several specific transcripts in 200 small round cell tumors (SRCT) displaying ESFT morphology and immunophenotype in which EWSR1 FISH analysis was non-informative or negative.
  
  
  METHODS
  200 tumors (formalin-fixed, paraffin-embedded) were analyzed by RT-PCR. All tumors were tested for EWSR1-ETS, EWSR1/WT1, PAX3/7-FOX01 or SYT/SSX transcripts, and the negative tumors were subsequently analyzed for CIC/DUX4, BCOR/CCNB3 and CIC/FOX04 transcripts.
  
  
  RESULTS
  133 (66.5%) ESFT displayed one of the above EWSR1-ETS translocations. Three cases (1.5%) revealed the SYT-SSX transcript for Synovial sarcoma, and one (0.5%) a EWSR1-WT1 transcript for Desmoplastic Small Round Cell tumor. The CIC-DUX4 translocation was found in six Ewing-like tumors (3%) with CD99 positivity. The BCOR-CCNB3 gene fusion was observed in 5 tumors (2.5%) displaying round or spindle cells with strong CCNB3 IHC expression in 3 tumors. Moreover, RT-PCR failed to detect any gene fusion transcripts in 19 tumors (9.5%) and were considered "undifferentiated small round cell sarcoma" (SRCS). Molecular biology results were non-informative in 33 SRCTs (16.5%) due to RNA degradation through inadequate fixation and/or decalcification.
  
  
  CONCLUSIONS
  Our analysis of 200 SRCTs confirms the molecular heterogeneity of neoplasms with ESFT morphology and highlight that molecular studies with RT-PCR including new emerging gene fusion transcripts are mandatory for the diagnosis when EWSR1 FISH is negative or non-informative. The incidence of CIC-DUX4, BCOR-CCNB3 and CIC-FOX04 transcripts was relatively low. A small group of Ewing-like sarcomas or undifferentiated SRCS remains unclassified. Adopting appropriate tissue fixation and processing protocols is important to avoid degradation of fixed/embedded tissue when no frozen tumor is available.
authors.affiliation: Instituto Valenciano de Oncología (FIVO), Pathology Dept, Valencia,Spain. Electronic address: isidro.machado@uv.es.
authors.forename: Isidro
authors.initials: I
authors.lastname: Machado
authors.affiliation: University of Valencia, Pathology Dept, Valencia, Valencia, Spain.
authors.forename: Lara
authors.initials: L
authors.lastname: Navarro
authors.affiliation: University of Valencia, Pathology Dept, Valencia, Valencia, Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: Pellin
authors.affiliation: University of Valencia, Pathology Dept, Valencia, Valencia, Spain.
authors.forename: Samuel
authors.initials: S
authors.lastname: Navarro
authors.affiliation: Friedrich Alexander University Erlangen-Nuremberg, Institute of Pathology, University Hospital, Erlangen, Germany.
authors.forename: Abbas
authors.initials: A
authors.lastname: Agaimy
authors.affiliation: Hospital Universitario de Fuenlabrada, Pathology Dept, Madrid, Spain.
authors.forename: Juan C
authors.initials: JC
authors.lastname: Tardío
authors.affiliation: All Cancer Center, Pathology Dept, Moscow, Russian Federation.
authors.forename: Apollon
authors.initials: A
authors.lastname: Karseladze
authors.affiliation: Cancer Center, Pathology Dept and Kazan State Medical University, Pathology Dept, Kazan, Russian Federation.
authors.forename: Semyon
authors.initials: S
authors.lastname: Petrov
authors.affiliation: Rizzoli Inst, Laboratory of Experimental Oncology, Bologna, Italy.
authors.forename: Katia
authors.initials: K
authors.lastname: Scotlandi
authors.affiliation: Rizzoli Inst, Laboratory of Experimental Oncology, Bologna, Italy.
authors.forename: Piero
authors.initials: P
authors.lastname: Picci
authors.affiliation: University of Valencia, Pathology Dept, Valencia, Valencia, Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: Llombart-Bosch
mesh_terms: D014408:Biomarkers, Tumor / Q000032:analysis*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism; D003937:Diagnosis, Differential; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D015514:Oncogene Proteins, Fusion / Q000235:genetics; D057089:Pathology, Molecular / Q000379:methods; D034802:RNA-Binding Protein EWS; D016601:RNA-Binding Proteins / Q000378:metabolism; D012512:Sarcoma, Ewing / Q000175:diagnosis* / Q000235:genetics*; D018228:Sarcoma, Small Cell / Q000175:diagnosis* / Q000235:genetics*; D014178:Translocation, Genetic / Q000235:genetics
chemical_list: D014408:Biomarkers, Tumor; D002148:Calmodulin-Binding Proteins; C500986:EWSR1 protein, human; D015514:Oncogene Proteins, Fusion; D034802:RNA-Binding Protein EWS; D016601:RNA-Binding Proteins
keywords: Ewing sarcoma family of tumors; Ewing-like tumors; Molecular biology; RT-PCR