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TOP2B)Ina Patient with Acute Myeloid Leukemia with a New T(3;11)(P24;P15) Karin Nebral,1Helmut H

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TOP2B)Ina Patient with Acute Myeloid Leukemia with a New T(3;11)(P24;P15) Karin Nebral,1Helmut H Human Cancer Biology NUP98 Is Fused to Topoisomerase (DNA) IIB 18 0 kDa (TOP2B)ina Patient with Acute Myeloid Leukemia with a New t(3;11)(p24;p15) Karin Nebral,1Helmut H. Schmidt,2 Oskar A. Haas,1and Sabine Strehl1 Abstract Purpose: The nucleoporin 98 kDa (NUP98) gene has been reported to be fused to 17 different partner genes in various hematologic malignancies with11p15aberrations. Cytogenetic analysis of an adult de novo acute myelogenous leukemia (M5a) revealed a t(3;11)(p24;p15), suggesting rearrangement of NUP98 with a novel partner gene. Experimental Design: Fluorescence in situ hybridization (FISH) was used to confirm the involvement of NUP98 in the t(3;11)(p24;p15). Selection of possibleNUP98 partner genes was done by computer-aided analysis of the 3p24 region using the University of California Santa Cruz genome browser. Fusion gene ^ specific FISH and reverse transcription-PCR analyses were done to verify the presence of the new NUP98 fusion. Results: FISH analysis using a NUP98 -specific clone showed a split signal, indicating that the NUP98 gene was affected by the translocation. Of the genes localized at 3p24, TOP2B was selected as a possible fusion partner candidate gene. Dual-color fusion gene ^ specific FISH and reverse transcription-PCR analysis verified that NUP98 was indeed fused to TOP2B.Inaddition to reciprocal NUP98-TOP2B and TOP2B-NUP98 in-frame fusion transcripts, an alternatively spliced out-of-frame TOP2B-NUP98 transcript that resulted in a premature stop codon was detected. Analysis of the genomic breakpoints revealed typical signs of nonhomologous end joining resulting from error-prone DNA repair. Conclusions: TOP2B encodes a type II topoisomerase, which is involved in DNA transcription, replication, recombination, and mitosis, and besides TOP1, represents the second NUP98 fusion partner gene that belongs to the topoisomerase gene family.This finding emphasizes the important role of topoisomerases in malignant transformation processes. One of the most promiscuous fusion partner genes involved in (3–5). NUP98 consists of two NH2-terminal glycine-leucine- hematopoietic malignancy–associated chromosomal rear- phenylalanine-glycine (GLFG) repeat domains (usually called rangements is the recently identified NUP98 gene at 11p15, FG repeats) that flank a GLEBS-like motif, which is a RAE1 bind- which to date has been discovered to fuse to 17 different ing site, and a COOH-terminal ribonucleoprotein-binding partners (1). NUP98 fusions occur in de novo and therapy- motif (6). related myeloid as well as lymphoid malignancies (1, 2). The most frequently observed fusion partners of NUP98 NUP98 encodes a 98 kDa protein that is an important belong to the homeobox family of transcription factors and component of the nuclear pore complex, which mediates include the class I homeobox genes HOXA9, HOXA11, nucleocytoplasmic transport of proteins and RNA. It resides HOXA13, HOXC11, HOXC13, HOXD11, and HOXD13,as predominantly on the nucleoplasmic side of the nuclear well as the nonclustered class II homeobox genes PRRX1/PMX1 pore complex, but was also found at the cytoplasmic face as and PRRX2 (1, 7). The HOX family genes contain a conserved well as dispersed within the nucleus, moving between these helix-turn-helix motif, the homeodomain, which is fused to the different localizations in a transcription-dependent manner NH2-terminal FG-rich region of NUP98 in all NUP98-HOX chimeric proteins reported thus far (1). NUP98-HOX fusions may deregulate expression of HOX target genes by transcrip- tional transactivation via NUP98 GLFG repeat–mediated Authors’ Affiliations: 1Children’s Cancer Research Institute, Vienna, Austria and 2Division of Hematology, Department of Internal Medicine, University Medical recruitment of the CBP/p300 complex, and thus might Center, Graz, Austria influence HOX gene–regulated hematopoiesis (8, 9). The Received 1/20/05; revised 5/17/05; accepted 6/20/05. non-HOX NUP98 fusion partners compose a heterogeneous Grant support: ‘‘O« sterreichische Kinderkrebshilfe’’ and the research program group of genes that are associated with a wide range of ‘‘Genome Research for Health’’of the Austrian Ministry of Education, Science, and biological functions. In contrast to the HOX fusion partner Culture (GEN-AU Child, GZ 200.071/3-VI/2a/2002). The costs of publication of this article were defrayed in part by the payment of page genes, they have not been recognized to play any specific or charges. This article must therefore be hereby marked advertisement in accordance unique role in hematopoiesis. These fusion partners include with 18 U.S.C. Section 1734 solely to indicate this fact. DDX10, RAP1GDS1, TOP1, PSIP2/LEDGF, NSD1, WHSC1L1/ Requests for reprints: Sabine Strehl, Children’s Cancer Research Institute, NSD3, FN1, and ADD3 (1, 2, 10). An intriguing feature of Kinderspitalgasse 6, A-1090 Vienna, Austria. Phone: 43-1-40170-449; Fax: 43-1- 40170-437; E-mail: [email protected]. all non-HOX partners is their propensity to adopt a coiled-coil F 2005 American Association for Cancer Research. conformation (1). Most of the non-HOX NUP98 fusion doi:10.1158/1078-0432.CCR-05-0150 partners are either transcriptional coactivators or factors that www.aacrjournals.org 6489 Clin Cancer Res 2005;11(18) September 15, 2005 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. Human Cancer Biology indirectly contribute to transcriptional control. Thus, oligomer- which spans the TOP2B gene. Probes were differentially labeled by nick ization via the coiled-coil domain and/or the transactivation translation: the NUP98 probe with digoxigenin-11-dUTP and the potential of the NUP98 GLFG repeats might confer aberrant TOP2B clone with biotin-16-dUTP (Roche Diagnostics, Vienna, transcriptional properties to the NUP98 fusions. This hypoth- Austria). Slides for fluorescence in situ hybridization (FISH) were esis is supported by the nuclear localization of the NUP98 prepared from the methanol/acetic acid–fixed cell suspension used for cytogenetic analysis and FISH was done as previously described (14). fusion proteins and oligonucleotide array expression analysis All samples were evaluated using an Axioplan fluorescence microscope (9, 11). Furthermore, the NUP98 fusions might contribute to (Zeiss, Vienna, Austria) equipped with the appropriate filter sets for leukemogenesis by triggering aberrant nucleocytoplasmic trans- FITC, Cy3, and 4V,6-diamidino-2-phenylindole, and images were taken port (8). with a CCD camera (Photometrix, Tucson, AZ) using the IPLab software In this study, we describe the identification of the novel (Vysis, Inc., Stuttgart, Germany). NUP98 partner gene TOP2B in a case of an adult de novo acute Reverse transcription-PCR analysis. Total RNA was extracted from myelogenous leukemia with a t(3;11)(p24;p15). TOP2B is the peripheral blood cells obtained at diagnosis of the patient and from second topoisomerase that fuses to NUP98, and with all non- peripheral blood cells of a healthy individual using the Qiagen RNeasy HOX partners shares the high propensity to adopt a coiled-coil Mini Kit (Qiagen, Inc., Vienna, Austria) according to the recommen- conformation. dations of the manufacturer. RNA was reverse transcribed using ran- dom hexamers and 200 units of Moloney murine leukemia virus re- verse transcriptase (Invitrogen, Lofer, Austria) at 42jC for 60 minutes. Materials and Methods The NUP98-TOP2B fusion transcript was detected using NUP98ex12 and TOP2Bex28 primers, and the reciprocal TOP2B-NUP98 using Case history. A 63-year-old male suffered from thrombocytopenia TOP2Bex24 and NUP98ex14 primers (Table 1). Expression of the nor- of unclear genesis and continuous difficulty swallowing. After biopsy of mal TOP2B allele was detected using TOP2Bex24 and TOP2Bex28 the pharynx, immunohistochemistry revealed an extramedullary primers. All reverse transcription-PCR reactions were done using Hot myeloid cell infiltrate with a French-American-British acute myeloge- Start Taq Polymerase (Qiagen) and an initial activation step at 95jC nous leukemia M5a phenotype. The blast cells were negative for the for 14 minutes. Cycling conditions for all reverse transcription-PCR B-cell markers CD20 and CD21, immunoglobulin n and E, as well as reactions were as follows: denaturation at 95jC for 30 seconds, anneal- for the T-cell markers CD45RO and CD3, and CD15, CD30, and S-100; ing at 63jC for 30 seconds, and elongation at 72jC for 60 seconds but positive for CD43 and CD68. Bone marrow and peripheral blood carried out for 40 cycles, followed by a final extension at 72jC for showed 80% and 99% blast cells, respectively, positive for the myeloid 7 minutes. Sequencing of the PCR products was done by MWG Biotech markers HLA-DR, CD13, and CD33, as well as CD68. Cytogenetic (Ebersberg, Germany) and VBC-Genomics (Vienna, Austria). analysis revealed a 46,XY,t(3;11)(p24;p15) karyotype. Induction Long-range PCR. The genomic breakpoints of the der(11) and therapy with Ara-C (Cytosar-U), amsacrine, and thioguanine failed to der(3) chromosomes were identified by long-range PCR of genomic significantly reduce the tumor load. After additional high-dose DNA using primers NUP98ex13 and TOP2Bex26, and TOP2Bin25-5 chemotherapy with Ara-C and novantrone, the patient achieved and NUP98in13-4, respectively (extension at 68jC for 5 minutes). complete histologic remission but relapsed 15 months after diagnosis Amplification of NUP98 and TOP2B normal alleles was done using and showed a 90% blast cell infiltration in the bone marrow. The blast NUP98in13 and NUP98in13-4, and TOP2Bin25-5 and TOP2Bin25-4 cells were resistant to Ara-C and topotecan (Hycamtin) and the patient primers, respectively (Table 1). died 20 months after initial diagnosis. Conventional and molecular cytogenetics. Cytogenetic analysis was done on GTG-banded metaphases and karyotypes were described Results and Discussion according to the International System for Human Cytogenetic Nomenclature (ISCN, 1995; ref. 12). Detection of the NUP98-TOP2B Cytogenetic analysis revealed a t(3;11)(p24;p15) transloca- rearrangement was done using PAC 1173K1 (13) in combination with tion in a patient with acute myelogenous leukemia M5a.
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