Molecular Analysis of Gene Fusions in Bone and Soft Tissue Tumors by Anchored Multiplex PCR–Based Targeted Next-Generation Sequencing

• Published in: The Journal of molecular diagnostics : JMD Vol. 20; no. 5; pp. 653 - 663
• Main Authors: Lam, Suk Wai, Cleton-Jansen, Anne-Marie, Cleven, Arjen H.G., Ruano, Dina, van Wezel, Tom, Szuhai, Karoly, Bovée, Judith V.M.G.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-09-2018
• ISSN: 1525-1578; 1943-7811
• DOI: 10.1016/j.jmoldx.2018.05.007

Molecular assays for translocation detection in bone and soft tissue tumors have gradually been incorporated into routine diagnostics. However, conventional methods such as fluorescence in situ hybridization (FISH) and reverse transcriptase-PCR come with several drawbacks. In this study, the applicability of a novel technique termed anchored multiplex PCR (AMP) for next-generation sequencing (NGS), using the Archer FusionPlex Sarcoma kit, aimed at 26 genes, was evaluated and compared with FISH and reverse transcriptase-PCR. In case of discrepant results, further analysis occurred with a third independent technique. Eighty-one samples were subjected to AMP-based targeted NGS, and 86% (n = 70) were successfully conducted and were either fusion positive (n = 48) or fusion negative, but met all criteria for good quality (n = 22). A concordance of 90% was found between NGS and conventional techniques. AMP-based targeted NGS showed superior results, as in four cases reverse transcriptase-PCR and FISH were false negative. Moreover, because the assay targets one partner of a gene fusion, novel or rare fusion partners can be identified. Indeed, it revealed COL1A1 and SEC31A as novel fusion partners for USP6 in nodular fasciitis. Despite the fact that fusions involving genes outside the selectively captured region cannot be detected and false-negative results due to poor quality samples can be encountered, this method has demonstrated excellent diagnostic utility for translocation detection in sarcomas.