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

Abstract

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.

• Authors: Marie-Delphine Lanic, François Le Loarer, Vinciane Rainville, Vincent Sater, Mathieu Viennot, Ludivine Beaussire, Pierre-Julien Viailly, Emilie Angot, Isabelle Hostein, Fabrice Jardin, Philippe Ruminy, Marick Laé
• Journal: Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc (Mod Pathol) Vol. 35 Issue 5 Pg. 649-663 (05 2022) ISSN: 1530-0285 [Electronic] United States
• PMID: 35075283 (Publication Type: Journal Article)
• Copyright: © 2022. The Author(s), under exclusive licence to United States & Canadian Academy of Pathology.

Chemical References

• Oncogene Proteins, Fusion
• Formaldehyde
• RNA

Topics

• 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, pathology)
• Soft Tissue Neoplasms (genetics, pathology)