Molecular Analysis of Gene Fusions in Bone and Soft Tissue Tumors by Anchored Multiplex PCR–Based Targeted Next-Generation Sequencing Suk Wai Lam, Anne-Marie.

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Molecular Analysis of Gene Fusions in Bone and Soft Tissue Tumors by Anchored Multiplex PCR–Based Targeted Next-Generation Sequencing Suk Wai Lam, Anne-Marie Cleton-Jansen, Arjen H.G. Cleven, Dina Ruano, Tom van Wezel, Karoly Szuhai, Judith V.M.G. Bovée The Journal of Molecular Diagnostics Volume 20, Issue 5, Pages 653-663 (September 2018) DOI: 10.1016/j.jmoldx.2018.05.007 Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Overview of samples assessed by AMP-based targeted NGS, and compared to conventional molecular assays (FISH and reverse transcriptase-PCR), specific immunohistochemistry, or conventional morphology. Asterisk indicates initially discordant cases; however, after analysis with a third independent technique, they were found concordant. AMP, anchored multiplex PCR; FISH, fluorescence in situ hybridization; IHC, immunohistochemistry; NGS, next-generation sequencing. The Journal of Molecular Diagnostics 2018 20, 653-663DOI: (10.1016/j.jmoldx.2018.05.007) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Case 1, dermatofibrosarcoma protuberans. A: Hematoxylin and eosin staining shows classic morphology with storiform-arranged spindle tumor cells. B: Although FISH using commercial probes revealed no rearrangement, specifically designed FISH probes reveal colocalization of red (PDGFB) and green (COL1A1) signals, indicating a COL1A1-PDGFB fusion. C: Archer analysis software version 5.0 reveals both fusion partners (COL1A1, exon 10; and PDGFB, exon 2). D: Sanger sequencing of PCR product confirms a COL1A1-PDGFB fusion. Original magnification: ×20 (A); ×140 (B). The Journal of Molecular Diagnostics 2018 20, 653-663DOI: (10.1016/j.jmoldx.2018.05.007) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Case 2, Ewing sarcoma with EWSR1-ERG fusion. A: Hematoxylin and eosin staining shows typical appearance of small blue round cells. B: Fluorescence in situ hybridization with self-selected probes reveals colocalization of red (5′EWSR1) and blue (ERG) signals, indicating an EWSR1-ERG fusion. Note the loss of the other red (5′EWSR1) signal, leading to a difficult-to-interpret atypical break-apart pattern when using only split-apart probes for EWSR1. C: Archer analysis software version 5.0 reveals both fusion partners (EWSR1, exon 7; and ERG, exon 7). Original magnification: ×20 (A); ×160 (B). The Journal of Molecular Diagnostics 2018 20, 653-663DOI: (10.1016/j.jmoldx.2018.05.007) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Case 3, Ewing sarcoma with EWSR1-FLI1 fusion. A: Hematoxylin and eosin staining shows the typical appearance of small blue round cells. B: Although fluorescence in situ hybridization with commercial probes revealed no break in EWSR1, self-selected probes reveal colocalization of red (5′EWSR1) and blue (FLI1) signals, indicating an EWSR1-FLI1 fusion. C: Archer analysis software version 5.0 reveals both fusion partners (EWSR1, exon 7; and FLI1, exon 7). Original magnification: ×20 (A); ×160 (B). The Journal of Molecular Diagnostics 2018 20, 653-663DOI: (10.1016/j.jmoldx.2018.05.007) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Case 4, Ewing sarcoma with EWSR1-FLI1 fusion. A: Hematoxylin and eosin staining shows the typical appearance of small blue round cells. B: Fluorescence in situ hybridization with commercial probes reveals distantly located red (5′EWSR1) and green (3′EWSR1) signals, indicating a break in the EWSR1 locus. C: Archer analysis software version 5.0 reveals a less common fusion transcript involving EWSR1, exon 10; and FLI1, exon 6. D: Sanger sequencing of PCR product confirms an EWSR1-FLI1 fusion. Original magnification: ×20 (A); ×140 (B). The Journal of Molecular Diagnostics 2018 20, 653-663DOI: (10.1016/j.jmoldx.2018.05.007) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 6 Case 5, malignant plasmacytoid neoplasm with a broad differential diagnosis including extraskeletal myxoid chondrosarcoma and myoepithelial carcinoma. A: Hematoxylin and eosin staining shows atypical plasmacytoid tumor cells. B: Fluorescence in situ hybridization with commercial probes reveals a break within the EWSR1 region, with distantly located red (5′EWSR1) and green (3′EWSR1) signals, whereas anchored multiplex PCR–based targeted next-generation sequencing repeatedly fails to show an EWSR1 fusion. Original magnification: ×40 (A); ×140 (B). The Journal of Molecular Diagnostics 2018 20, 653-663DOI: (10.1016/j.jmoldx.2018.05.007) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Supplemental Figure S1 Proportion of split-apart signals generated by fluorescence in situ hybridization is plotted against the amount of fusion reads generated by anchored multiplex PCR–based targeted next-generation sequencing for nodular fasciitis and Ewing sarcoma cases. No correlation was found for either nodular fasciitis or Ewing sarcoma. The Journal of Molecular Diagnostics 2018 20, 653-663DOI: (10.1016/j.jmoldx.2018.05.007) Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions