1. 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 (September 2018)
DOI: /j.jmoldx
Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

2. 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  , DOI: ( /j.jmoldx )
Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

3. 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  , DOI: ( /j.jmoldx )
Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

4. 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  , DOI: ( /j.jmoldx )
Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

5. 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  , DOI: ( /j.jmoldx )
Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

6. 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  , DOI: ( /j.jmoldx )
Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

7. 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  , DOI: ( /j.jmoldx )
Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

8. 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  , DOI: ( /j.jmoldx )
Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions