Dystrophin Is a Tumor Suppressor in Human Cancers with Myogenic Programs

SUPPLEMENTARY INFORMATION Dystrophin Is a Tumor Suppressor in Human Cancers with Myogenic Programs Yuexiang Wang 1, Adrian Marino-Enriquez 1, Richard R. Bennett 2, Meijun Zhu 1, Yiping Shen 3,4, Grant Eilers 1, Jen-Chieh Lee 1,5, Joern Henze 1, Benjamin S. Fletcher 1, Zhizhan Gu 6, Edward A. Fox 7, Cristina R. Antonescu 8, Christopher D.M. Fletcher 1, Xiangqian Guo 9, Chandrajit P. Raut 10, George D. Demetri 11, Matt van de Rijn 9, Tamas Ordog 12-14, Louis M. Kunkel 2, Jonathan A. Fletcher 1 * 1 Department of Pathology, Brigham and Women s Hospital and Harvard Medical School, Boston, MA, USA. 2 Division of Genetics and Genomics, Manton Center for Orphan Disease Research, Children's Hospital and Department of Genetics, Harvard Medical School, Boston, MA, USA. 3 Genetic Diagnostic Laboratory, Department of Laboratory Medicine, Children's Hospital, and Department of Pathology, Harvard Medical School, Boston, MA, USA. 4 Department of Laboratory Medicine, Shanghai Children s Medical Center, Jiaotong University, Shanghai, China. 5 Department of Pathology, National Taiwan University Hospital, Taipei, Taiwan. 6 Division of Rheumatology, Immunology, and Allergy, Department of Medicine, Brigham and Women s Hospital and Harvard Medical School, Boston, MA, USA. 7 Molecular Diagnostics Laboratory, Dana-Farber Cancer Institute, Boston, MA, USA. 8 Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA. 9 Department of Pathology, Stanford University Medical Center, Stanford, CA, USA. 10 Department of Surgery, Brigham and Women s Hospital and Harvard Medical School, Boston, MA, USA. 11 Ludwig Center at Harvard, Harvard Medical School and Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA. 12 Center for Individualized Medicine, Mayo Clinic, Rochester, MN, USA. 13 Enteric Neuroscience Program, Mayo Clinic, Rochester, MN, USA. 14 Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA. *Correspondence to: Jonathan A. Fletcher, M.D. Department of Pathology Brigham and Women s Hospital 75 Francis Street Boston, MA 02115 Phone: 617-732-7883 Fax: 617-278-6921 E-mail: jfletcher@partners.org 1

SUPPLEMENTARY INFORMATION Table of contents 1. Supplementary Figures 1-10 Supplementary Figure 1. Identical DMD deletions in primary GIST and subsequent metastasis. Supplementary Figure 2. Identical DMD deletions in multiple GIST metastases from one patient. Supplementary Figure 3. DMD intragenic deletions in 1003 human cancers. Supplementary Figure 4. Detailed characterization of intragenic DMD deletions by MLPA. Supplementary Figure 5. Expression of Dp71 dystrophin in non-myogenic sarcomas. Supplementary Figure 6. Dp71 dystrophin is a positive regulator of cell viability in myogenic sarcoma. Supplementary Figure 7. minidmd expression in GIST, RMS, and LMS cells at levels physiologic for dystrophin in the corresponding cell lineages. Supplementary Figure 8. Effect of minidmd expression on cell viability in DMD-inactivated GIST, RMS and LMS cells. Supplementary Figure 9. Dystrophin is not expressed in Ewing s sarcoma (EWS502 and EWS894), fibrosarcoma (HT-1080) and HEK 293. Supplementary Figure 10. Dystrophin immunohistochemistry in clinical samples. 2. Supplementary Tables 1-6 Supplementary Table 1. SNP assay DMD deletion status in high grade myogenic cancers. Supplementary Table 2. SNP DMD deletion status for 58 primary or advanced high grade nonmyogenic sarcomas. Supplementary Table 3. MLPA ascertainment and localization of DMD deletions in myogenic sarcomas. Supplementary Table 4. Clinicopathologic classification and DMD status for all study specimens. Supplementary Table 5. Tissue microarray evaluation of dystrophin expression by immunohistochemistry in non-myogenic sarcomas. Supplementary Table 6. Dp71-specific sirna sequences. 2

a Patient 31 Xp Normal Primary Metastatic Normal Primary Metastatic DMD Xq -2.0 0 2.0 b DMD Dp71 Dp427 SNP_A-4228311, rs7061167 SNP_A-1998480, rs16998151 SNP_A-2281060, rs16989523 SNP_A-2194627, rs2692986 SNP_A-2033400, rs16989676 SNP_A-2264951, rs5971580 SNP_A-1900199, rs5971587 SNP_A-1835726, rs2047373 SNP_A-2024180, rs1350474 SNP_A-2134043, rs6527130 SNP_A-1861193, rs5927914 SNP_A-2293008, rs1699078 SNP_A-2209720, rs12556700 SNP_A-2191337, rs3788896 SNP_A-4218755, rs12387861 SNP_A-1826241, rs228392 SNP_A-2247703, rs228379 SNP_A-1903787, rs5928062 SNP_A-1998518, rs10522011 SNP_A-1854357, rs5972687 SNP_A-1998521, rs10522017 SNP_A-2108943, rs5972728 SNP_A-4233015, rs6527261 SNP_A-1916499, rs17341267 SNP_A-4195359, rs6631766 SNP_A-1836581, rs5972829 SNP_A-2163579, rs5928271 SNP_A-4240875, rs5928327 SNP_A-1998536, rs1518982 SNP_A-2194368, rs6628815 SNP_A-1998541, --- SNP_A-2237986, rs1604604 SNP_A-1898250, rs1948804 SNP_A-1961507, rs6628828 SNP_A-1803413, rs12391355-2.0 0 2.0 Supplementary Figure 1. Identical DMD deletions in a primary gastric GIST and a subsequent metastasis, diagnosed one year later, from patient 31. SNP profiles of nonneoplastic DNA from the patient, the primary gastric GIST, and the subsequent metastasis are shown. The top panel (a) shows the entire chromosome X; the bottom panel (b) focuses on the DMD locus. Data are shown as dchip log 2 ratio copy number. 3

a Patient 61 Wang et al., Supplementary Information Xp met 1 met 2 met 3 met 4 met 5 met 6 met 7 met 8 met 9 normal met 11 met 12 met 10 met 13 met 14 met 15 met 16 met 17 met 18 met 19 met 20 met 21 met 22 met 23 met 24 met 25 met 26 met 27 met 28 met 10 DMD Xq -2.0-1.7-1.5-1.2-0.9-0.7-0.4-0.1 0.1 0.4 0.7 0.9 1.2 1.5 1.7 2.0 b DMD Dp71 Dp427 SNP_A-4228311, rs7061167 SNP_A-1998480, rs16998151 SNP_A-2281060, rs16989523 SNP_A-2194627, rs2692986 SNP_A-2033400, rs16989676 SNP_A-2264951, rs5971580 SNP_A-1900199, rs5971587 SNP_A-1835726, rs2047373 SNP_A-2024180, rs1350474 SNP_A-2134043, rs6527130 SNP_A-1861193, rs5927914 SNP_A-2293008, rs1699078 SNP_A-2209720, rs12556700 SNP_A-2191337, rs3788896 SNP_A-4218755, rs12387861 SNP_A-1826241, rs228392 SNP_A-2247703, rs228379 SNP_A-1903787, rs5928062 SNP_A-1998518, rs10522011 SNP_A-1854357, rs5972687 SNP_A-1998521, rs10522017 SNP_A-2108943, rs5972728 SNP_A-4233015, rs6527261 SNP_A-1916499, rs17341267 SNP_A-4195359, rs6631766 SNP_A-1836581, rs5972829 SNP_A-2163579, rs5928271 SNP_A-4240875, rs5928327 SNP_A-1998536, rs1518982 SNP_A-2194368, rs6628815 SNP_A-1998541, --- SNP_A-2237986, rs1604604 SNP_A-1898250, rs1948804 SNP_A-1961507, rs6628828 SNP_A-1803413, rs12391355 SNP_A-4226087, rs5928493-2.0-1.7-1.5-1.2-0.9-0.7-0.4-0.1 0.1 0.4 0.7 0.9 1.2 1.5 1.7 2.0 Supplementary Figure 2. Identical DMD deletions in each of 28 GIST metastases from patient 61. The metastases have a DMD deletion not present in non-neoplastic DNA from the patient, demonstrating the somatic nature of DMD deletion. The top panel (a) shows the entire chromosome X; the bottom panel (b) focuses on the DMD locus. Data are shown as dchip SNP log 2 ratio copy number, with a representative SNP profile (metastasis 10) shown to the right. 4

Cancer type Myogenic sarcoma (GIST, RMS and LMS) Non-myogenic sarcoma Non-sarcoma cancers * DMD del No DMD del Total DMD del frequency 25 15 40 62.5% 0 58 58 0% 39 866 905 4.3% * DMD copy number data for non-sarcoma cancers were obtained from the Cancer Cell Line Encyclopedia. DMD del No DMD del 62.5% 0% 4.3% Myogenic sarcoma n=40 Non-myogenic sarcoma n=58 Non-sarcoma cancers n=905 Supplementary Figure 3. DMD intragenic deletions in 1,003 human cancers, including 40 myogenic sarcomas, 58 non-myogenic sarcomas and 905 non-sarcoma cancers. The 58 nonmyogenic sarcomas (Supplementary Table 2) include 4 malignant peripheral nerve sheath tumors, 10 liposarcomas, 15 Ewing s sarcomas, 1 fibrosarcoma, 1 clear cell sarcoma, 1 spindle cell sarcoma, 1 dermatofibrosarcoma protuberans, 1 giant cell tumor, 1 mesenchymal chondrosarcoma, and 23 osteosarcomas. The 905 non-sarcoma cancers (Cancer Cell Line Encyclopedia; Barretina et al, Nature, 2012) include 591 carcinomas, 175 hematologic neoplasms, 59 melanomas, 43 gliomas, 15 neuroblastomas, and 22 other tumors. DMD intragenic deletions are significantly more frequent in myogenic sarcomas than in non-myogenic sarcomas (P<0.0001, two-tailed Fisher s test) and non-sarcoma cancers (P<0.0001, two-tailed Fisher s test). 5

a Patient 17: Low-risk GIST (male) normal b Patient 7: Low-risk GIST (female) normal Wang et al., Supplementary Information c Patient 46: Metastatic GIST (male) ex1-44 del d Patient 52: Metastatic GIST (female) ex1-17, 37-45 hom del e Patient 40: M etastatic GIST (female) ex2-19, 40-79 het del Supplementary Figure 4. Detailed characterization of intragenic DMD deletions by MLPA. DMD MLPA capillary electropherograms are normal in low-risk GISTs from male ( a) and female (b) patients. MLPA shows intragenic hemizygous, homozygous and heterozygous deletions in metastatic GISTs from male ( c) and two female patients (d and e ), respectively. Each peak represents a single DMD exon, displayed non-consecutively based on the size of the ligated stuffer sequence. The reference trace is shown in red, the tumor sample is shown in blue. 6

RMS843 MPNST LPS DFSP SFT Dp71-76kDa - 52kDa GAPDH Supplementary Figure 5. Expression of Dp71 dystrophin in non-myogenic sarcomas. Protein blotting with dystrophin antibody 7A10 demonstrates Dp71 expression in malignant peripheral nerve sheath tumor (MPNST), liposarcoma (LPS), dermatofibrosarcoma protuberans (DFSP), and malignant solitary fibrous tumor (SFT). 7

a control Dp71 knockdown control Dp71 knockdown Dp71 Dp71 Dp427 Dp427 GAPDH GAPDH RMS176 RMS843 b 8 7 control Dp71 knockdown 8 7 control Dp71 knockdown Relative cell number 6 5 4 3 2 Relative cell number 6 5 4 3 2 1 1 0 0 1 2 3 4 Days RMS176 0 0 1 2 3 4 Days RMS843 Supplementary Figure 6. Dp71 dystrophin is a positive regulator of cell viability in myogenic sarcoma. (a) Protein blot validation of sirna-mediated inhibition of Dp71 in RMS176 (erms with DMD deletion) and RMS843 (arms without DMD deletion). (b) Dp71 inhibition reduces cell viability, as assessed by CellTiter-Glo assay. The control is a non-targeting sirna. Data shown as mean ± s.d. from three replicates. Dp71 and Dp427 were detected with dystrophin antibodies 7A10 and DYS1, respectively. 8

Skeletal muscle Lo w-risk GIST Parental EGFP MiniDMD Skelet al muscle Lo w-risk GIST Parental Wang et al., Supplementary Information EGFP MiniDMD Dystrophin 427kDa 240kDa GAPDH GIST-T1 GIST430 Skeletal Parental muscle EGFP MiniDMD Skelet al muscle Myo metrium Parent al EGFP MiniDMD Dystrophin 427kDa 240kDa GAPDH RMS176 LMS04 Skeletal Parental muscle EGFP MiniDMD Skeletal Parental muscle EGFP MiniDMD Dystrophin 427kDa 240kDa GAPDH HT-1080 EWS502 Skeletal Parental muscle EGFP MiniDMD Dystrophin 427kDa 240kDa GAPDH HEK293 Supplementary Figure 7. minidmd expression in GIST, RMS, and LMS cells at levels physiological for dystrophin in the corresponding cell lineages. Stable transfection with minidmd of GIST, RMS and LMS cells induces expression of 240-kDa minidmd dystrophin at levels comparable to those of endogeneous 427-kDa dystrophin in low-risk GIST, skeletal muscle and myometrium, respectively. minidmd was detected with DYS2 antibody. 9

RMS176 LMS04 GIST-T1 GIST430 HT-1080 EWS502 HEK 293 Supplementary Figure 8. Effect of minidmd expression on cell viability in DMD-inactivated GIST, RMS and LMS cells. Stable trnasfection with minidmd reduces cell viability in RMS176 and LMS04, as assessed by CellTiter-Glo viability assay, but does not alter viability of GIST (GIST-T1 and GIST430) or non-myogenic cells: fibrosarcoma (HT-1080), Ewing s sarcoma (EWS502) and HEK 293. Data shown as mean ± s.d. of three replicates. 10

RMS843 GIST882 EWS502 EWS894 RMS843 GIST882 HT-1080 RMS843 GIST882 HEK293 427kDa Dystrophin 225kDa 150kDa GAPDH Supplementary Figure 9. Dystrophin is not expressed in Ewing s sarcoma (EWS502 and EWS894), fibrosarcoma (HT-1080) and HEK 293. Dystrophin was detected with DYS1 antibody. 11

Wang et al., Supplementary Information a Skeletal muscle Cardiac muscle Myometrium Uterine leiomyoma Soft tissue leiomyoma Soft tissue leiomyoma Uterine LMS Soft tissue LMS Cardiac LMS b c Dystrophin IHC + - T otals Leiomyoma 20 0 20 Leiomyosarcoma 27 30 57 T otals 47 30 77 Supplementary Figure 10. Dystrophin immunohistochemistry in clinical samples. Immunohistochemical detection in formalin-fixed paraffin-embedded surgical samples shows robust dystrophin expression in skeletal, cardiac and smooth muscle (a) and in benign smooth muscle neoplasms (b, top panel), whereas dystrophin expression is inhibited in malignant smooth muscle tumors (b, bottom panel; LMS, leiomyosarcoma). (c) Contingency table summarizing dystrophin expression assessed by immunohistochemistry in 20 benign and 57 malignant smooth muscle tumors in a tissue microarray. Loss of dystrophin expression correlates with malignancy in smooth muscle tumors (P<0.0001, two-tailed Fisher s test). 12

Supplementary Table 1. SNP assay DMD deletion status in high grade myogenic cancers. Case # Gender Cancer type Primary or metastatic DMD Zygosity 15 F GIST Primary Nl 2 16 F GIST Primary Nl 2 28 F GIST Primary Nl 2 31-1 M GIST Primary Del 0 31-2 M GIST Metastatic Del 0 33 F GIST Primary Del 0 34 F GIST Primary Del 0 35 M GIST Primary Del 0 36 M GIST Primary Del 0 39 F GIST Metastatic Del 0 40 F GIST Metastatic Del 1 42 M GIST Metastatic Nl 1 43 M GIST Metastatic Nl 1 44 F GIST Metastatic Del 0 45 M GIST Metastatic Nl 1 47 F GIST Metastatic Nl 2 48 F GIST Metastatic Nl 2 50 M GIST Metastatic Nl 1 51 M GIST Metastatic Del 0 52 F GIST Metastatic Del 0 54 F GIST Metastatic Del 0 55 M GIST Metastatic Del 0 56 M GIST Metastatic Del 0 57 F GIST Metastatic Del 1 58 F GIST Metastatic Del 1 59 F GIST Metastatic Nl 2 61-10 M GIST Metastatic Del 0 62 M GIST Metastatic Del 0 64 F GIST Metastatic Del 0 72 F erms Primary Nl 2 80 M erms Metastatic Del 0 83 M erms Metastatic Del 0 84 M erms Metastatic Del 0 109 F LMS Primary Del 0 114 F LMS Metastatic Nl 2 116-4 F LMS Metastatic Del 1 116-5 F LMS Metastatic Del 0 118 F LMS Metastatic Nl 2 120 M LMS Metastatic Nl 1 121 M LMS Metastatic Nl 1 M denotes male; F denotes female; Del denotes deletion; Nl denotes normal. erms denotes embryonal rhabdomyosarcoma. 13

Supplementary Table 2. SNP DMD deletion status for 58 primary or advanced high grade non-myogenic sarcomas. Non-myogenic (NM) Case # Gender Tumor type Clinicopathologic Classification NM-01 M Malignant peripheral nerve sheath tumor Primary Nl NM-02 M Malignant peripheral nerve sheath tumor Primary Nl NM-03 F Malignant peripheral nerve sheath tumor Advanced Nl NM-04 F Malignant peripheral nerve sheath tumor Advanced Nl NM-05 M Liposarcoma Primary Nl NM-06 M Liposarcoma Advanced Nl NM-07 M Liposarcoma Advanced Nl NM-08 M Liposarcoma Advanced Nl NM-09 M Liposarcoma Advanced Nl NM-10 F Liposarcoma Advanced Nl NM-11 F Liposarcoma Advanced Nl NM-12 F Liposarcoma Advanced Nl NM-13 F Liposarcoma Advanced Nl NM-14 M Liposarcoma Advanced Nl NM-15 F Ewing s sarcoma Primary Nl NM-16 M Ewing s sarcoma Primary Nl NM-17 F Ewing s sarcoma Advanced Nl NM-18 M Ewing s sarcoma Advanced Nl NM-19 F Ewing s sarcoma Advanced Nl NM-20 M Ewing s sarcoma Advanced Nl NM-21 F Ewing s sarcoma Advanced Nl NM-22 M Ewing s sarcoma Advanced Nl NM-23 M Ewing s sarcoma Advanced Nl NM-24 M Ewing s sarcoma Advanced Nl NM-25 F Ewing s sarcoma Advanced Nl NM-26 F Ewing s sarcoma Advanced Nl NM-27 F Ewing s sarcoma Advanced Nl NM-28 F Ewing s sarcoma Advanced Nl NM-29 M Ewing s sarcoma Advanced Nl NM-30 M Fibrosarcoma Primary Nl NM-31 M Spindle-cell Sarcoma Advanced Nl NM-32 M Clear-cell sarcoma Advanced Nl NM-33 F Dermatofibrosarcoma protuberans Advanced Nl NM-34 M Giant-cell tumor Primary Nl NM-35 M Mesenchymal Chondrosarcoma Advanced Nl NM-36 M Osteosarcoma Primary Nl NM-37 M Osteosarcoma Primary Nl NM-38 F Osteosarcoma Primary Nl NM-39 M Osteosarcoma Primary Nl NM-40 F Osteosarcoma Primary Nl DMD 14

Supplementary Table 2. SNP DMD deletion status for 58 primary or advanced high grade non-myogenic sarcomas (continued). Non-myogenic (NM) Case # Gender Tumor type Clinicopathologic Classification NM-41 M Osteosarcoma Primary Nl NM-42 F Osteosarcoma Advanced Nl NM-43 F Osteosarcoma Advanced Nl NM-44 M Osteosarcoma Advanced Nl NM-45 M Osteosarcoma Advanced Nl NM-46 F Osteosarcoma Advanced Nl NM-47 M Osteosarcoma Advanced Nl NM-48 F Osteosarcoma Advanced Nl NM-49 F Osteosarcoma Advanced Nl NM-50 F Osteosarcoma Advanced Nl NM-51 M Osteosarcoma Advanced Nl NM-52 F Osteosarcoma Advanced Nl NM-53 M Osteosarcoma Advanced Nl NM-54 M Osteosarcoma Advanced Nl NM-55 M Osteosarcoma Advanced Nl NM-56 F Osteosarcoma Advanced Nl NM-57 M Osteosarcoma Advanced Nl NM-58 M Osteosarcoma Advanced Nl M denotes male; F denotes female; Del denotes deletion; Nl denotes normal. DMD 15

Supplementary Table 3. MLPA ascertainment and localization of DMD deletions in myogenic sarcomas. Case # Gender Tumor type Dystrophin Expression (IB)* DMD 1 M GIST 2 Nl 4 M GIST 2 Nl 5 M GIST 2 Nl 6 M GIST 2 Nl 7 F GIST 2 Nl 8 F GIST 2 Nl 9 M GIST 2 Nl 10 F GIST 2 Nl 11 F GIST 2 Nl 17 M GIST 2 Nl 20 F GIST 2 Nl 23 F GIST 2 Nl 31-1 M GIST 0 Del ex3-30 35 M GIST 0 Del ex1-47, 56-60 36 M GIST ND Del ex2-41 38 M GIST 0 Del ex2-43 39 F GIST 0 Del ex1 40 F GIST 0 Del ex2-19, 40-79 44 F GIST ND Del ex2-11, 30-61 45 M GIST 0 Nl 46 M GIST 0 Del ex1-44 47 F GIST 1 Nl 49 M GIST 0 Del ex1-4 51 M GIST 1 Del ex2-13 52 F GIST 0 Del ex1-17, 37-45 54 F GIST 0 Del ex1-7 55 M GIST 0 Del ex1-12, 44-55 56 M GIST 0 Del ex1-9 60 M GIST 1 Del ex1-47 63 M GIST 0 Del ex2-62 64 F GIST 1 Del ex1-6 65 M Rhabdomyoma 2 Nl 66 M Rhabdomyoma 2 Nl 68 F erms 0 Nl 69 M erms 2 Nl 70 F erms 0 Nl 71 M erms 2 Nl 72 F erms 0 Nl 73 M erms ND Nl 74 F erms 1 Nl 16

Supplementary Table 3. MLPA ascertainment and localization of DMD deletions in myogenic sarcomas (continued). Case # Gender Tumor type Dystrophin Expression (IB)* DMD 75 M erms 1 Nl 76 M erms 1 Nl 78 F erms 0 Nl 79 M erms 0 Nl 80 M erms 0 Del ex1-44 81 M erms 0 Nl 82-2 M erms 0 Nl 83 M erms 0 Del ex1-7, 21-44 84 M erms 0 Del ex44 85 M erms 1 Del ex11-44 86 M erms 0 Nl 87 M arms 2 Nl 88 M arms ND Nl 89 F arms 2 Nl 90 F arms 2 Nl 91 M arms 1 Nl 92 M arms ND Nl 93 F Leiomyoma 2 Nl 94 F Leiomyoma 2 Nl 95 F Leiomyoma 2 Nl 96 F Leiomyoma 2 Nl 97 F Leiomyoma 2 Nl 98 F Leiomyoma 2 Nl 101 F Leiomyoma 2 Nl 102 F LMS 2 Nl 106 M LMS 2 Nl 108 F LMS 1 Nl 109 F LMS 0 Del ex2-50 112 F LMS 2 Nl 113 F LMS 2 Nl 114 F LMS 0 Nl 115 F LMS 2 Nl 116-1 F LMS 1 Del ex3-12, 26-55 116-5 F LMS 0 Del ex3-12, 26-55 120 M LMS 0 Nl 121 M LMS 0 Nl 122 F LMS 2 Nl M denotes male; F denotes female; ND denotes not done. Del denotes deletion; Nl denotes normal; ex denotes exon. erms denotes embryonal rhabdomyosarcoma; arms denotes alveolar rhabdomyosarcoma. *0 denotes no expression; 1 denotes weak (reduced) expression; 2 denotes strong (normal for lineage) expression. 17

Supplementary Table 4. Clinicopathologic classification and DMD status for all study specimens. Case # Gender Tumor type Clinicopathologic Classification KIT/PDGFRA Genotype Dystrophin Expression (IB)* DMD (SNP and/or MLPA) 1 M GIST Low-risk KIT ex 11 2 Nl 2 M GIST Low-risk KIT ex 11 2 ND 3 M GIST Low-risk KIT ex 11 2 ND 4 M GIST Low-risk KIT ex 11 2 Nl 5 M GIST Low-risk KIT ex 11 2 Nl 6 M GIST Low-risk KIT ex 11 2 Nl 7 F GIST Low-risk KIT ex 11 2 Nl 8 F GIST Low-risk KIT ex 11 2 Nl 9 M GIST Low-risk KIT ex 11 2 Nl 10 F GIST Low-risk KIT ex 11 2 Nl 11 F GIST Low-risk KIT ex 11 2 Nl 12 F GIST Low-risk KIT ex 11 2 ND 13 F GIST Low-risk KIT ex 11 2 ND 14 F GIST Low-risk KIT ex 11 2 ND 15 F GIST Low-risk KIT ex 11 2 Nl 16 F GIST Low-risk KIT ex 11 2 Nl 17 M GIST Low-risk PDGFRA ex18 2 Nl 18 M GIST Low-risk PDGFRA ex18 2 ND 19 F GIST Low-risk PDGFRA ex18 2 ND 20 F GIST Low-risk WT 2 Nl 21 F GIST Low-risk WT 1 ND 22 M GIST Intermediate-risk KIT ex 11 2 ND 23 F GIST Intermediate-risk KIT ex 11 2 Nl 24 M GIST Intermediate-risk KIT ex 9 2 ND 25 F GIST Intermediate-risk KIT ex 11 2 ND 26 M GIST Intermediate-risk PDGFRA ex18 2 ND 27 F GIST Intermediate-risk PDGFRA ex18 2 ND 28 F GIST Intermediate-risk KIT ex 11 0 Nl 29 M GIST High-risk KIT ex 11 0 ND 30 M GIST High-risk KIT ex 9 1 ND 31-1 M GIST High-risk KIT ex 11 0 Del 31-2 M GIST Metastatic KIT ex 11 0 Del 32 M GIST High-risk KIT ex 11 1 ND 33 F GIST High-risk KIT ex 11 2 Del 34 F GIST High-risk KIT ex 11 2 Del 35 M GIST High-risk KIT ex 11 0 Del 36 M GIST High-risk KIT ex 11 ND Del 37 M GIST High-risk PDGFRA ex18 1 ND 38 M GIST Metastatic KIT ex 11 0 Del 39 F GIST Metastatic KIT ex 11 0 Del 40 F GIST Metastatic KIT ex 11 0 Del 41 F GIST Metastatic KIT ex 11 1 ND 42 M GIST Metastatic KIT ex 11 1 Nl 43 M GIST Metastatic KIT ex 11 0 Nl 18

Supplementary Table 4. Clinicopathologic classification and DMD status for all study specimens (continued). Case # Gender Tumor type Clinicopathologic Classification KIT/PDGFRA Genotype Dystrophin Expression (IB)* DMD (SNP and/or MLPA) 44 F GIST Metastatic KIT ex 9 ND Del 45 M GIST Metastatic KIT ex 13 0 Nl 46 M GIST Metastatic WT 0 Del 47 F GIST Metastatic KIT ex 9 1 Nl 48 F GIST Metastatic KIT ex 9 0 Nl 49 M GIST Metastatic KIT ex 11 0 Del 50 M GIST Metastatic KIT ex 11 1 Nl 51 M GIST Metastatic KIT ex 11 1 Del 52 F GIST Metastatic KIT ex 11 0 Del 53 M GIST Metastatic KIT ex 11 2 ND 54 F GIST Metastatic KIT ex 11 0 Del 55 M GIST Metastatic KIT ex 11 0 Del 56 M GIST Metastatic KIT ex 11 0 Del 57 F GIST Metastatic KIT ex 11 0 Del 58 F GIST Metastatic KIT ex 11 1 Del 59 F GIST Metastatic KIT ex 11 0 Nl 60 M GIST Metastatic KIT ex 9 1 Del 61-1 M GIST Metastatic KIT ex 9 ND Del 61-2 M GIST Metastatic KIT ex 9 ND Del 61-3 M GIST Metastatic KIT ex 9 ND Del 61-4 M GIST Metastatic KIT ex 9 ND Del 61-5 M GIST Metastatic KIT ex 9 ND Del 61-6 M GIST Metastatic KIT ex 9 ND Del 61-7 M GIST Metastatic KIT ex 9 ND Del 61-8 M GIST Metastatic KIT ex 9 ND Del 61-9 M GIST Metastatic KIT ex 9 ND Del 61-10 M GIST Metastatic KIT ex 9 0 Del 61-11 M GIST Metastatic KIT ex 9 ND Del 61-12 M GIST Metastatic KIT ex 9 ND Del 61-13 M GIST Metastatic KIT ex 9 ND Del 61-14 M GIST Metastatic KIT ex 9 ND Del 61-15 M GIST Metastatic KIT ex 9 ND Del 61-16 M GIST Metastatic KIT ex 9 ND Del 61-17 M GIST Metastatic KIT ex 9 ND Del 61-18 M GIST Metastatic KIT ex 9 ND Del 61-19 M GIST Metastatic KIT ex 9 ND Del 61-20 M GIST Metastatic KIT ex 9 ND Del 61-21 M GIST Metastatic KIT ex 9 ND Del 61-22 M GIST Metastatic KIT ex 9 ND Del 61-23 M GIST Metastatic KIT ex 9 ND Del 61-24 M GIST Metastatic KIT ex 9 ND Del 61-25 M GIST Metastatic KIT ex 9 ND Del 61-26 M GIST Metastatic KIT ex 9 ND Del 61-27 M GIST Metastatic KIT ex 9 ND Del 19

Supplementary Table 4. Clinicopathologic classification and DMD status for all study specimens (continued). Case # Gender Tumor type Clinicopathologic Classification KIT/PDGFRA Genotype Dystrophin Expression (IB)* DMD (SNP and/or MLPA) 61-28 M GIST Metastatic KIT ex 9 ND Del 62 M GIST Metastatic KIT ex 9 1 Del 63 M GIST Metastatic KIT ex 13 0 Del 64 F GIST Metastatic KIT ex 13 1 Del 65 M Rhabdomyoma Primary ND 2 Nl 66 M Rhabdomyoma Primary ND 2 Nl 67 F erms Primary ND 1 ND 68 F erms Primary ND 1 Nl 69 M erms Primary ND 2 Nl 70 F erms Primary ND 0 Nl 71 M erms Primary ND 2 Nl 72 F erms Primary ND 1 Nl 73 M erms Primary ND ND Nl 74 F erms Primary ND 1 Nl 75 M erms Primary ND 1 Nl 76 M erms Primary ND 1 Nl 77 M erms Primary ND 2 ND 78 F erms Metastatic ND 1 Nl 79 M erms Metastatic ND 1 Nl 80 M erms Metastatic ND 0 Del 81 M erms Metastatic ND 0 Nl 82-1 M erms Metastatic ND 0 ND 82-2 M erms Metastatic ND 0 Nl 83 M erms Metastatic ND 0 Del 84 M erms Metastatic ND 0 Del 85 M erms Metastatic ND 1 Del 86 M erms Metastatic ND 0 Nl 87 M arms Primary ND 2 Nl 88 M arms Metastatic ND ND Nl 89 F arms Metastatic ND 2 Nl 90 F arms Metastatic ND 2 Nl 91 M arms Metastatic ND 1 Nl 92 M arms Metastatic ND ND Nl 93 F Leiomyoma Primary ND 2 Nl 94 F Leiomyoma Primary ND 2 Nl 95 F Leiomyoma Primary ND 2 Nl 96 F Leiomyoma Primary ND 2 Nl 97 F Leiomyoma Primary ND 2 Nl 98 F Leiomyoma Primary ND 2 Nl 99 F Leiomyoma Primary ND 2 ND 100 F Leiomyoma Primary ND 2 ND 101 F Leiomyoma Primary ND 2 Nl 102 F LMS Primary ND 2 Nl 103 F LMS Primary ND 2 ND 20

Supplementary Table 4. Clinicopathologic classification and DMD status for all study specimens (continued). Case # Gender Tumor type Clinicopathologic Classification KIT/PDGFRA Genotype Dystrophin Expression (IB)* DMD (SNP and/or MLPA) 104 F LMS Primary ND 2 ND 105 F LMS Primary ND 1 ND 106 M LMS Primary ND 2 Nl 107 F LMS Primary ND 2 ND 108 F LMS Primary ND 1 Nl 109 F LMS Primary ND 0 Del 110-1 F LMS Primary ND 2 ND 110-2 F LMS Metastatic ND 2 ND 111 F LMS Metastatic ND 2 ND 112 F LMS Metastatic ND 1 Nl 113 F LMS Metastatic ND 0 Nl 114 F LMS Metastatic ND 0 Nl 115 F LMS Metastatic ND 2 Nl 116-1 F LMS Metastatic ND 1 Del 116-2 F LMS Metastatic ND 1 ND 116-3 F LMS Metastatic ND 0 ND 116-4 F LMS Metastatic ND 0 Del 116-5 F LMS Metastatic ND 0 Del 117 F LMS Metastatic ND 2 ND 118 F LMS Metastatic ND 0 Nl 119 M LMS Metastatic ND 0 ND 120 M LMS Metastatic ND 0 Nl 121 M LMS Metastatic ND 0 Nl 122 F LMS Metastatic ND 2 Nl M denotes male; F denotes female; ND denotes not done. Del denotes deletion; Nl denotes normal; ex denotes exon. erms denotes embryonal rhabdomyosarcoma; arms denotes alveolar rhabdomyosarcoma. *0 denotes no staining; 1 denotes weak (reduced) staining; 2 denotes strong (normal for lineage) staining. 21

Supplementary Table 5. Tissue microarray evaluation of dystrophin expression by immunohistochemistry in non-myogenic sarcomas. Sarcoma type No dystrophin expression Dystrophin expression Totals Myxofibrosarcoma 5 0 5 Dermatofibrosarcoma protuberans 10 0 10 Malignant Solitary Fibrous Tumor 18 0 18 Angiosarcoma 13 0 13 Totals 46 0 46 22

Supplementary Table 6. Dp71 -specific sirna sequences. Sense sequence (5 > 3 ) Antisense sequence (5 > 3 ) CAGCCAUGAGGGAACAGCUtt AGCUGUUCCCUCAUGGCUGca 23