Abstract

BackgroundDetection 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.MethodsWe constructed two sets of multiplex PCR assays and evaluated their utility using cell lines and clinical samples.ResultsEWSR1/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.ConclusionsWe 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.

Highlights

  • Detection of the tumor-specific EWSR1/FUS-ETS fusion gene is essential to diagnose Ewing sarcoma

  • Clinical samples The pathological diagnosis was confirmed by H.O., A.N., and/or T.Y. based on morphological observations and existing Reverse transcription–polymerase chain reaction (RT–PCR) and/or Fluorescence in situ hybridization (FISH) analysis

  • Generation of the multiplex RT–PCR method to detect EWSR1/FUS-ETS Design of the multiplex RT–PCR primers We aimed to set up a multiplex Reverse transcription–polymerase chain reaction (RT-PCR) system to detect all EWSR1/FUS-ETS fusion variants for rapid and practical genetic diagnosis

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Summary

Introduction

Detection of the tumor-specific EWSR1/FUS-ETS fusion gene is essential to diagnose Ewing sarcoma. Ewing sarcoma primarily occurs in the bones and soft tissues of children and young adults. It is characterized by fusion genes between a gene of the RNA-binding FET family (EWSR1 or FUS) with a gene of the ETStranscription factor family (FLI1, ERG, ETV1, ETV4 (E1AF), and FEV) [1,2,3,4,5], which are called EWSR1/FUSETS fusion genes. EWSR1 exon 7 and ERG exon 7 or EWSR1 exon 7 and ERG exon 9 are common exon combinations in EWSR1-ERG These fusion genes contain the ETS consensus sequence in-frame [9, 10].

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