Establishment of multiplex RT-PCR to detect fusion genes for the diagnosis of Ewing sarcoma

Hitomi Ueno-Yokohata, Hajime Okita, Keiko Nakasato, Chikako Kiyotani, Motohiro Kato, Kimikazu Matsumoto, Nobutaka Kiyokawa, Atsuko Nakazawa, Takako Yoshioka

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)


Background: Detection of the tumor-specific EWSR1/FUS-ETS fusion gene is essential to diagnose Ewing sarcoma. Reverse transcription–polymerase chain reaction (RT–PCR) and fluorescence in situ hybridization are commonly used to detect the fusion gene, and assays using next-generation sequencing have recently been reported. However, at least 28 fusion transcript variants have been reported, making rapid and accurate detection difficult. Methods: We constructed two sets of multiplex PCR assays and evaluated their utility using cell lines and clinical samples. Results: EWSR1/FUS-ETS was detected in five of six tumors by the first set, and in all six tumors by the second set. The fusion gene detected only by the latter was EWSR1-ERG, which completely lacked exon 7 of EWSR1. The fusion had a short N-terminal region of EWSR1 and showed pathologically atypical features. Conclusions: We developed multiplex RT–PCR assays to detect EWSR1-ETS and FUS-ETS simultaneously. These assays will aid the rapid and accurate diagnosis of Ewing sarcoma. In addition, variants of EWSR1/FUS-ETS with a short N-terminal region that may have been previously missed can be easily detected.

Original languageEnglish
Article number102
JournalDiagnostic Pathology
Issue number1
Publication statusPublished - 2021 Dec


  • Breakpoint
  • EWSR1
  • Ewing sarcoma
  • Fusion gene
  • Genetic diagnosis
  • Multiplex RT–PCR
  • Transcription factor

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Histology


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