Diagnostic Utility of Molecular Investigation in Extraskeletal Myxoid Chondrosarcoma  Stefania Benini, Stefania Cocchi, Gabriella Gamberi, Giovanna Magagnoli, Daniela Vogel, Cristina Ghinelli, Alberto Righi, Piero Picci, Marco Alberghini, Marco Gambarotti  The Journal of Molecular Diagnostics  Volume 16, Issue 3, Pages 314-323 (May 2014) DOI: 10.1016/j.jmoldx.2013.12.002 Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Algorithm shows the protocol used in our institution to evaluate the presence of gene rearrangements in tumors with a presumptive diagnosis of EMC. EMC, extraskeletal myxoid chondrosarcoma; FFPE, formalin-fixed, paraffin-embedded; FISH, fluorescence in situ hybridization. The Journal of Molecular Diagnostics 2014 16, 314-323DOI: (10.1016/j.jmoldx.2013.12.002) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Detection of EWSR1-NR4A3 fusion gene in EMC. A: EWSR1-NR4A3 chimeric transcripts detected by RT-PCR in various frozen tumor tissues. Cases 4 and 22 amplified type 1(198 bp), case 13 type 2 (146 bp), and case 18 type 6 (367 bp). Numbers of the cases are identical to the case numbers in Table 1. B: qPCR amplification plot observed for type 1 and type 2 transcripts in some cases of FFPE EMC tumor tissue. C: Partial nucleotide sequences of EWSR1-NR4A3 type 1, 2, and 6. D: Diagrams show variants of the EWSR1-NR4A3 chimeric transcripts identified in this study. The number of cases with different types of transcripts is given in parentheses. Arrowheads indicate the positions of the primers used for RT-PCR analysis. B, distilled water; EMC, extraskeletal myxoid chondrosarcoma; EWSR1, EWS RNA-binding protein 1; M, size marker (100 bp); N, tissue negative control; NR4A3, nuclear receptor subfamily 4, group A, member 3l qPCR, real-time quantitative PCR. The Journal of Molecular Diagnostics 2014 16, 314-323DOI: (10.1016/j.jmoldx.2013.12.002) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Detection of TAF15-NR4A3 fusion gene that results from t(9;17) in EMC. A: TAF15-NR4A3 chimeric transcripts (137 bp) detected by RT-PCR in frozen tumor tissue of EMC case 20. B: Partial nucleotide sequences of TAF15-NR4A3. C: Schematic of TAF15-NR4A3 chimeric transcript. The numbers refer to exons of the TAF15 and NR4A3 genes. Arrowheads indicate the positions of the primers used for RT-PCR analysis. B, distilled water; C, case; EMC, extraskeletal myxoid chondrosarcoma; M, size marker (100 bp); N, tissue negative control; NR4A3, nuclear receptor subfamily 4, group A, member 3; TAF15, TAF 15 RNA polymerase II, TATA box binding protein (TBP)-associated factor. The Journal of Molecular Diagnostics 2014 16, 314-323DOI: (10.1016/j.jmoldx.2013.12.002) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 A: Detection of TCF12-NR4A3 fusion gene results from t(9;15) in EMC. TCF12-NR4A3 chimeric transcripts (427 bp) detected by RT-PCR in the frozen tumor tissue of EMC (case 37). B: Partial nucleotide sequences of TCF12-NR4A3. C: Schematic of TCF12-NR4A3 chimeric transcript. The numbers refer to exons of the TCF12 and NR4A3 genes. Arrowheads indicate the positions of the primers used for RT-PCR analysis. B, distilled water; C, case; EMC, extraskeletal myxoid chondrosarcoma; M, size marker (100 bp); N, tissue negative control; NR4A3, nuclear receptor subfamily 4, group A, member 3; TCF12, transcription factor 12. The Journal of Molecular Diagnostics 2014 16, 314-323DOI: (10.1016/j.jmoldx.2013.12.002) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Case 31. A: EWSR1 rearrangement by FISH (left panel) shows signals detected in the form of split signals (centromere, orange; telomere, green), in contrast to normal fused signals. The right panel shows the break-apart FISH for NR4A3, showing split signals (centromere, green; telomere, orange) in contrast to normal fused signals. This was observed in all EMC cases successfully tested. B: H&E staining of the surgical specimen of the same case. Original magnification: DAPI ×1000 (A); H&E ×400 (B). EMC, extraskeletal myxoid chondrosarcoma; EWSR1, EWS RNA-binding protein 1; NR4A3, nuclear receptor subfamily 4, group A, member 3. The Journal of Molecular Diagnostics 2014 16, 314-323DOI: (10.1016/j.jmoldx.2013.12.002) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions