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Clinical and Molecular Characteristics of MEF2D Fusion-Positive B-Cell

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Clinical and Molecular Characteristics of MEF2D Fusion-Positive B-Cell Acute Lymphoblastic Leukemia SUPPLEMENTARY APPENDIX Clinical and molecular characteristics of MEF2D fusion-positive B-cell precursor acute lymphoblastic leukemia in childhood, including a novel translocation resulting in MEF2D-HNRNPH1 gene fusion Kentaro Ohki, 1 Nobutaka Kiyokawa, 1 Yuya Saito, 1,2 Shinsuke Hirabayashi, 1,3 Kazuhiko Nakabayashi, 4 Hitoshi Ichikawa, 5 Yukihide Momozawa, 6 Kohji Okamura, 7 Ai Yoshimi, 1,8 Hiroko Ogata-Kawata, 4 Hiromi Sakamoto, 5 Motohiro Kato, 1 Keitaro Fukushima, 9 Daisuke Hasegawa, 3 Hiroko Fukushima, 10 Masako Imai, 11 Ryosuke Kajiwara, 12 Takashi Koike, 13 Isao Komori, 14 Atsushi Matsui, 15 Makiko Mori, 16 Koichi Moriwaki, 17 Yasushi Noguchi, 18 Myoung-ja Park, 19 Takahiro Ueda, 20 Shohei Yamamoto, 21 Koichi Matsuda, 22 Teruhiko Yoshida, 5 Kenji Matsumoto, 23 Kenichiro Hata, 4 Michiaki Kubo, 6 Yoichi Matsubara, 24 Hiroyuki Takahashi, 25 Takashi Fukushima, 26 Yasuhide Hayashi, 27 Katsuyoshi Koh, 16 Atsushi Man - abe 3 and Akira Ohara 25 for the Tokyo Children’s Cancer Study Group (TCCSG) 1Department of Pediatric Hematology and Oncology Research, National Research Institute for Child Health and Development, Seta - gaya-ku, Tokyo; 2Department of Hematology/Oncology, Tokyo Metropolitan Children’s Medical Center, Fuchu-shi; 3Department of Pedi - atrics, St. Luke's International Hospital, Chuo-ku, Tokyo; 4Department of Maternal-Fetal Biology, National Research Institute for Child Health and Development, Setagaya-ku, Tokyo; 5Fundamental Innovative Oncology Core, National Cancer Center Research Institute, Chuo-ku, Tokyo; 6Laboratory for Genotyping Development, RIKEN Center for Integrative Medical Sciences (IMS), Yokohama-shi, Kana - gawa; 7Department of Systems BioMedicine, National Research Institute for Child Health and Development, Setagaya-ku, Tokyo; 8Divi - sion of Pediatric Hematology and Oncology, Ibaraki Children’s Hospital, Mito-shi; 9Department of Pediatrics, Dokkyo Medical University, Tochigi; 10 Department of Pediatrics, University of Tsukuba Hospital, Ibaraki; 11 Department of Pediatrics, Japanese Red Cross Musashino Hospital, Tokyo; 12 Department of Pediatrics, Yokohama City University Hospital, Kanagawa; 13 Department of Pediatrics, Tokai University School of Medicine, Kanagawa; 14 Department of Pediatrics, Matsudo City Hospital, Chiba; 15 Department of Pediatrics, Japanese Red Cross Maebashi Hospital, Gunma; 16 Department of Hematology/Oncology, Saitama Children’s Medical Center; 17 Depart - ment of Pediatrics, Saitama Medical Center, Saitama Medical University; 18 Department of Pediatrics, Japanese Red Cross Narita Hos - pital, Chiba; 19 Department of Hematology/Oncology, Gunma Children’s Medical Center, Shibukawa-shi; 20 Department of Pediatrics, Nippon Medical School, Bunkyo-ku, Tokyo; 21 Department of Pediatrics, Showa University Fujigaoka Hospital, Yokohama-shi, Kanagawa; 22 Laboratory of Clinical Genome Sequencing Department of Computational Biology and Medical Sciences Graduate School of Frontier Sciences, The University of Tokyo, Minato-ku; 23 Department of Allergy and Clinical Immunology, National Research Institute for Child Health and Development, Setagaya-ku, Tokyo; 24 Director, National Research Institute for Child Health and Development, Setagaya-ku, Tokyo; 25 Department of Pediatrics, Toho University Omori Medical Center, Tokyo; 26 Department of Child Health, Faculty of Medicine, University of Tsukuba, Ibaraki and 27 Institute of Physiology and Medicine, Jobu University, Takasaki-shi, Gunma, Japan ©2019 Ferrata Storti Foundation. This is an open-access paper. doi:10.3324/haematol. 2017.186320 Received: December 11, 2017. Accepted: August 29, 2018. Pre-published: August 31, 2018. Correspondence: [email protected] Identification of MEF2D fusion positive cases by microarray analysis Initially, 2 cases of MEF2D fusion (Table 1, Cases 9 and 16) were included in neither the discovery cohort nor validation cohort and we performed microarray analysis by including these 2 cases in “B-others”. Since these 2 were located in the same cluster as MEF2D fusion-positive patients (Figure S2), we performed RT-PCR for Cases 9 and 16 and found that they were positive for MEF2D-BCL9 and MEF2D-HNRNPUL1, respectively (Table 1). Therefore, we included them in the group of MEF2D and conducted all microarray analyses again, but mostly similar results were initially obtained. Supplementary methods Microarray and data analyses The cDNAs amplified and labeled were hybridized to Human Genome U133 Plus 2.0 Arrays (Affymetrix, Santa Clara, CA, USA), and the obtained data were normalized and filtered with the steps as described previously.S1 The gene expression signature for MEF2D fusion-positive B-ALL was investigated by comparing 14 MEF2D fusion-positive cases: 8 MEF2D-BCL9, 5 MEF2D-HNRNPUL1, and 1 MEF2D-HNRNPH1, and 359 control B-ALL samples: 21 BCR-ABL1+, 6 CRLF2 fusions+, 72 ETV6-RUNX1+, 24 TCF3-PBX1+, 22 MLL fusions+, 20 ZNF384 fusions+, and 196 B-others. For the clustering analysis, we selected the top 10 up- and top 10 down-regulated genes under all paired conditions of B-ALL with MEF2D fusion-positive ALL and 6 other genetic abnormalities (presented in Table S7). After the exclusion of those that overlapped, 146 subsequent gene probes, listed in Table S8, were used as the selected probe sets of differentially expressed genes. Fold change analysis was performed to select differentially expressed genes under all paired conditions of the 7 genetic abnormalities and B-others. In comparison with the B-others, the differential expression of 1,063 genes (up: 549, down: 514, fold change >2.0) was identified in MEF2D fusion-positive cases (Table S9 A). Similarly, the differential expression of 756 genes (up: 470, down: 286, fold change >2.0) was identified in TCF3-PBX1-positive cases in comparison with the B-others (Table S9 B). The functional analyses of genes characteristic of MEF2D fusion-positive ALLs in comparison with B-ALL with other types of genetic abnormalities were conducted using gene set enrichment analysis (GSEA), as described previously. S1 The analysis included 18 curated gene sets of B lymphocytes in various differentiation stagesS2-S4 selected from human immunologic signatures (C7) and curated gene sets (C2) from MsigDB (http://software.broadinstitute.org/gsea/index.jsp), as well as gene sets for 7 early hematopoietic stages including stem cells.S5 Supplementary references S1 Hirabayashi S, Ohki K, Nakabayashi K, et al. ZNF384-related fusion genes define a subgroup of childhood B-cell precursor acute lymphoblastic leukemia with a characteristic immunotype. Haematologica. 2017;102(1):118-129. S2 Zhan F, Tian E, Bumm K, Smith R, Barlogie B, Shaughnessy J Jr. Gene expression profiling of human plasma cell differentiation and classification of multiple myeloma based on similarities to distinct stages of late-stage B-cell development. Blood. 2003;101(3):1128-1140. S3 Haddad R, Guardiola P, Izac B, et al. Molecular characterization of early human T/NK and B-lymphoid progenitor cells in umbilical cord blood. Blood. 2004;104(13):3918-3926. S4 Hoffmann R, Lottaz C, Kühne T, Rolink A, Melchers F. Neutrality, Compensation, and Negative Selection during Evolution of B-Cell Development Transcriptomes. Mol Biol Evol. 2007;24(12):2610–2618. S5 Laurenti E, Doulatov S, Zandi S, et al. The transcriptional architecture of early human hematopoiesis identifies multilevel control of lymphoid commitment. Nat Immunol. 2013;14(7):756-763. Supplementary Tables Table S1. Summary of the cases and detected MEF2D fusions. Table S2. Primer sequences for RT-PCR used for detection of MEF2D fusions. Table S3. Summary of the results of detected MEF2D-related fusion genes by whole transcriptome sequencing. Table S4. Immunophenotypes of B-ALL cases with MEF2D fusions. Table S5. Genomic copy number alterations detected by MLPA of B-ALL cases with MEF2D fusions. Table S6. Additional genetic abnormalities of MEF2D fusion-positive cases detected by whole exome sequencing. Table S7. Differentially expressed genes under all paired conditions of genetic abnormalities. Table S8. Probe set ID used for hierarchical clustering and principal component analysis. Table S9. Differentially expressed genes between MEF2D fusion- or TCF3-PBX1-positive ALL and B-others (up and down fold change >2.0). Table S10. Results of the gene set enrichment analysis (GSEA). Table S11. Summary of the results of gene set enrichment analysis (GSEA) presented in Table S10. Table S1. Summary of the cases and detected MEF2D fusions TCCSG L04-16 study Others BCP*1-LBL*2 L04-16/L06-16 L07-1602 L09-1603 (outside the 2004.Nov-2007.Mar 2007 Apr-2009 May 2009 Jun-2013 Jun L04-16 cohort) ALL*3 328 *4 259 547 BCP-ALL 290 234 492 95 2 BCP-ALL without conventional genetic abnormalities Registered 126 117 238 Analyzed 124 ( 98.4% registered ) 74 55 75 MEF2D fusions 7 ( 2.4% in BCP-ALL ) 2 3 4 1 ( 5.6% in BCP-ALL w/o CGA ) MEF2D-BCL9 5 2 1 1 1 ( 1.7% in BCP-ALL ) ( 4.0% in BCP-ALL w/o CGA ) MEF2D-HNRNPUL1 2 2 2 MEF2D-HNRNPH1 1 CRLF2 fusions 3 4 7 1 Other Ph-like 3 *5 3 *6 4 *7 ZNF384 fusions 12 7 *8 5 7 TCF3-HLF 0 0 1 B-others 99 61 36 59 1 Undetermined 2 43 183 BCP-ALL with conventional genetic abnormalities Determined 164 117 254 20 Hypodiploid≦40 2 1 3 Hyperdiploid≧51 65 33 99 2 BCR-ABL1 16 13 21 1 ETV6-RUNX1 56 48 84 9 MLL-AF4 4 2 8 1 MLL-AF9 3 2 2 2 MLL-ENL 0 1 1 TCF3-PBX1 18 17 36 5 T-ALL 37 25 55 Unclassified 1 *1; BCP, B-cell precursor *2; LBL, lymphoblastic lymphoma *3; ALL, acute lymphoblastic leukemia *4; As described in our recent publication regarding TCCSG L04-16 Study (reference 18), of 1,225 patients registered for these studies, 192 were excluded due to various reasons and thus the number of patients is different from our previous publication (reference 10). *5; Ph-like, Ph-like ALL-related tyrosine kinase fusions, including 2 Igh-EPOR and 1 PAX5-JAK2 (reference 18) Table S3. Summary of the results of detected MEF2D -related fusion genes by whole transcriptome sequencing Chromosome, genomic Chromosome, genomic 5'-Partner gene 3'-Partner gene Gene_name1 Gene_name2 Case ID position of fusion position of fusion Flanking sequences Gene locations accession No.
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