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Frequency of Mutations in Spliceosome Genes and Poor Prognosis Isolated From www.bloodjournal.org by guest on November 4, 2014. For personal use only. 2014 124: 1304-1311 doi:10.1182/blood-2013-12-540716 originally published online June 12, 2014 Isolated trisomy 13 defines a homogeneous AML subgroup with high frequency of mutations in spliceosome genes and poor prognosis Tobias Herold, Klaus H. Metzeler, Sebastian Vosberg, Luise Hartmann, Christoph Röllig, Friedrich Stölzel, Stephanie Schneider, Max Hubmann, Evelyn Zellmeier, Bianka Ksienzyk, Vindi Jurinovic, Zlatana Pasalic, Purvi M. Kakadia, Annika Dufour, Alexander Graf, Stefan Krebs, Helmut Blum, Maria Cristina Sauerland, Thomas Büchner, Wolfgang E. Berdel, Bernhard J. Woermann, Martin Bornhäuser, Gerhard Ehninger, Ulrich Mansmann, Wolfgang Hiddemann, Stefan K. Bohlander, Karsten Spiekermann and Philipp A. Greif Updated information and services can be found at: http://www.bloodjournal.org/content/124/8/1304.full.html Articles on similar topics can be found in the following Blood collections Myeloid Neoplasia (1270 articles) Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved. From www.bloodjournal.org by guest on November 4, 2014. For personal use only. Regular Article MYELOID NEOPLASIA Isolated trisomy 13 defines a homogeneous AML subgroup with high frequency of mutations in spliceosome genes and poor prognosis Tobias Herold,1-4 Klaus H. Metzeler,1-4 Sebastian Vosberg,1-4 Luise Hartmann,1-4 Christoph R¨ollig,3-5 Friedrich St¨olzel,5 Stephanie Schneider,1 Max Hubmann,1,2 Evelyn Zellmeier,1 Bianka Ksienzyk,1 Vindi Jurinovic,6 Zlatana Pasalic,1 Purvi M. Kakadia,7 Annika Dufour,1 Alexander Graf,8 Stefan Krebs,8 Helmut Blum,8 Maria Cristina Sauerland,9 Thomas B¨uchner,10 Wolfgang E. Berdel,10 Bernhard J. Woermann,11 Martin Bornh¨auser,3-5 Gerhard Ehninger,3-5 Ulrich Mansmann,3,4,6 Wolfgang Hiddemann,1-4 Stefan K. Bohlander,12 Karsten Spiekermann,1-4 and Philipp A. Greif1-4 1Department of Internal Medicine 3, University Hospital Grosshadern, Ludwig-Maximilians-Universit¨at,Munich, Germany; 2Clinical Cooperative Group Leukemia, Helmholtz Zentrum M¨unchen,German Research Center for Environmental Health, Munich, Germany; 3German Cancer Consortium (DKTK), Heidelberg, Germany; 4German Cancer Research Center (DKFZ), Heidelberg, Germany; 5Medizinische Klinik und Poliklinik I, Universit¨atsklinikumDresden, Dresden, Germany; 6Institute for Medical Informatics, Biometry and Epidemiology, Ludwig-Maximilians-Universit¨at,Munich, Germany; 7Center for Human Genetics, Philipps University, Marburg, Germany; 8Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, Ludwig-Maximilians-Universit¨at, Munich, Germany; 9Institute of Biostatistics and Clinical Research, and 10Department of Medicine A - Hematology, Oncology and Pneumology, University of M¨unster, M¨unster, Germany; 11German Society of Hematology and Oncology, Berlin, Germany; and 12Department of Molecular Medicine and Pathology, The University of Auckland, Auckland, New Zealand Key Points In acute myeloid leukemia (AML), isolated trisomy 13 (AML113) is a rare chromosomal abnormality whose prognostic relevance is poorly characterized. We analyzed the clinical • AML patients with isolated course of 34 AML113 patients enrolled in the German AMLCG-1999 and SAL trials and trisomy 13 have a very poor performed exome sequencing, targeted candidate gene sequencing and gene expression clinical outcome profiling. Relapse-free (RFS) and overall survival (OS) of AML113 patients were inferior • Isolated trisomy 13 in compared to other ELN Intermediate-II patients (n5855) (median RFS, 7.8 vs 14.1 months, P 5 P 5 AMLisassociatedwith .006; median OS 9.3 vs. 14.8 months, .004). Besides the known high frequency of RUNX1 a high frequency of mutations (75%), we identified mutations in spliceosome components in 88%, including SRSF2 codon 95 mutations in 81%. Recurring mutations were detected in mutations in SRSF2 (81%) ASXL1 (44%) and BCOR (25%). Two patients carried mutations in CEBPZ, suggesting that and RUNX1 (75%) CEBPZ is a novel recurrently mutated gene in AML. Gene expression analysis revealed a homogeneous expression profile including upregulation of FOXO1 and FLT3 and downregulation of SPRY2. This is the most comprehensive clinical and biological characterization of AML113 to date, and reveals a striking clustering of lesions in a few genes, defining AML113 as a genetically homogeneous subgroup with alterations in a few critical cellular pathways. Clinicaltrials.gov identifiers: AMLCG-1999: NCT00266136; AML96: NCT00180115; AML2003: NCT00180102; and AML601: NCT00893373 (Blood. 2014;124(8):1304-1311) Introduction Acquired isolated trisomy 13 (113) is a rare cytogenetic alteration in a potential mechanism of leukemogenesis in AML113.6,7 The acute myeloid leukemia (AML). In a retrospective study of 22 856 AML possibility that AML113 might be a marker for treatment response patients from the Mayo Clinic, its incidence was 0.7%.1 So far, the to lenalidomide has recently been raised.8 prognostic relevance of AML113 has not been extensively studied, but Constitutional aneuploidy is linked to increased cancer risk.9 For assumed to be unfavorable based on small or heterogeneous patient example, Down syndrome (trisomy 21) predisposes to megakaryoblastic cohorts.2-4 However, according to the European LeukemiaNet (ELN) leukemia with a high frequency of acquired GATA1 mutations.10 classification, AML113 is currently classified in the Intermediate-II Trisomy 13 (Patau syndrome) is a severe congenital disorder genetic group.5 AML113 is frequently associated with FAB M0 with cerebral, cardiac, and renal malformations.11 An association of morphology and shows a high frequency (80% to 100%) of RUNX1 Patau syndrome and solid neoplasms including neuroblastoma and mutations.6,7 Overexpression of FLT3 (located in band q12 on nephroblastoma was reported.12 In the literature, we found a single chromosome 13) due to a gene dosage effect was proposed as case report of Patau syndrome with congenital myeloid leukemia.13 Submitted December 1, 2013; accepted May 28, 2014. Prepublished online as The publication costs of this article were defrayed in part by page charge Blood First Edition paper, June 12, 2014; DOI 10.1182/blood-2013-12- payment. Therefore, and solely to indicate this fact, this article is hereby 540716. marked “advertisement” in accordance with 18 USC section 1734. Presented in abstract form at the 55th annual meeting of the American Society of Hematology, New Orleans, LA, December 7-10, 2013. The online version of this article contains a data supplement. © 2014 by The American Society of Hematology 1304 BLOOD, 21 AUGUST 2014 x VOLUME 124, NUMBER 8 From www.bloodjournal.org by guest on November 4, 2014. For personal use only. BLOOD, 21 AUGUST 2014 x VOLUME 124, NUMBER 8 SRSF2 MUTATIONS IN AML 113 1305 Table 1. Patient characteristics Variable AML113* Control Group* P No. of patients 34 850 Median age, years (range) 64 (43-80) 59 (17-84) .004 Male sex, no. (%) 24 (70) 465 (55) .08 WBC count, G/l, median (range) 10 (1-318) 11 (0.1-365) .64 Hemoglobin, g/dl, median (range) 8.9 (4.6-12.8) 9.2 (2.9-17.2) .2 Platelet count, G/l, median (range) 77 (1-399) 54 (1-1760) .23 LDH (U/l), median (range) 269 (155-1011) 414 (115-11140) .009 BM blasts, %, median (range) 80 (11-100) 68 (11-100) .02 BM blasts at day 16, %, median (range) 5 (0-85) 9 (0-100) .78 Performance status (ECOG) $ 2 (%) 8 (26) 263 (34) .44 de novo AML (%) 26 (76) 646 (76) 1.0 Allogeneic transplantation, no. (%) 6 (18) 180 (21) .83 CR, no. (%) 21 (62) 471 (55) .49 Relapse, no. (%) 18 (86) 327 (69) .14 Deceased, no. (%) 31 (91) 644 (76) .04 Significant P values are indicated in bold. *All patients were enrolled in the AMLCG-99 or SAL trials and received intensive induction treatment. All patients are classified as ELN Intermediate-II; AML113: patients with isolated tri- or tetrasomy 13, additional aberrations of the sex chromosomes are allowed. Considering that the vast majority of infants with Patau syndrome die Targeted amplicon sequencing before 1 year of age,11 it remains unclear whether constitutional A selection of genes identified by exome sequencing (n 5 9) and a panel of genes trisomy 13 predisposes to myeloid neoplasia. recurringly mutated in AML (n 5 42)werestudiedbytargetedamplicon 1 We set out to characterize the clinical course of AML 13 patients sequencing (Haloplex; Agilent) in all AMLCG AML113 patients with available and to elucidate the underlying spectrum of molecular genetic changes material (16 of 23). The resulting libraries were sequenced in a single run on by exome sequencing, targeted sequencing, and gene expression a MiSeq instrument. Sequence data were aligned to the human reference genome profiling. (version hg19) using BWA.20 Single nucleotide variants and short insertions or deletions were called using VarScan 2 and Pindel, respectively.24,25 In addition, Sanger sequencing of genomic DNA was performed for additional validation of selected mutations. Primer
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