Detection of sarcoma fusions by a next-generation sequencing based-ligation-dependent multiplex RT-PCR assay

Mod Pathol. 2022 May;35(5):649-663. doi: 10.1038/s41379-021-00980-x. Epub 2022 Jan 24.


Morphological, immunohistochemical, and molecular methods often need to be combined for accurate diagnosis and optimal clinical management of sarcomas. Here, we have developed, a new molecular diagnostic assay, for the detection of gene fusions in sarcomas. This targeted multiplexed next-generation sequencing (NGS)-based method utilizes ligation dependent reverse-transcriptase polymerase chain reaction (LD-RT-PCR-NGS) to detect oncogenic fusion transcripts involving 137 genes, leading to 139 gene fusions known to be recurrently rearranged in soft-tissue and bone tumors. 158 bone and soft-tissue tumors with previously identified fusion genes by fluorescent in situ hybridization (FISH) or RT-PCR were selected to test the specificity and the sensitivity of this assay. RNA were extracted from formalin-fixed paraffin-embedded (n = 143) or frozen (n = 15) material (specimen; n = 42 or core needle biopsies; n = 116). Tested tumors encompassed 23 major translocation-related sarcomas types, including Ewing and Ewing-like sarcomas, rhabdomyosarcomas, desmoplastic small round-cell tumors, clear-cell sarcomas, infantile fibrosarcomas, endometrial stromal sarcomas, epithelioid hemangioendotheliomas, alveolar soft-part sarcomas, biphenotypic sinonasal sarcomas, extraskeletal myxoid chondrosarcomas, myxoid/round-cell liposarcomas, dermatofibrosarcomas protuberans and solitary fibrous tumors. In-frame fusion transcripts were detected in 98.1% of cases (155/158). Gene fusion assay results correlated with conventional techniques (FISH and RT-PCR) in 155/158 tumors (98.1%). These data demonstrate that this assay is a rapid, robust, highly sensitive, and multiplexed targeted RNA sequencing assay for the detection of recurrent gene fusions on RNA extracted from routine clinical specimens of sarcomas (formalin-fixed paraffin-embedded or frozen). It facilitates the precise diagnosis and identification of tumors with potential targetable fusions. In addition, this assay can be easily customized to cover new fusions.

MeSH terms

  • Endometrial Neoplasms* / genetics
  • Female
  • Formaldehyde
  • High-Throughput Nucleotide Sequencing / methods
  • Humans
  • In Situ Hybridization, Fluorescence / methods
  • Oncogene Proteins, Fusion / genetics
  • RNA
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sarcoma* / genetics
  • Sarcoma* / pathology
  • Soft Tissue Neoplasms* / genetics
  • Soft Tissue Neoplasms* / pathology


  • Oncogene Proteins, Fusion
  • Formaldehyde
  • RNA