Letters to the Editor 1186 A translocation t(2;8)(q12;p11) fuses FGFR1 to a novel partner , RANBP2/NUP358, in a myeloproliferative/myelodysplastic neoplasm

Leukemia (2013) 27, 1186–1188; doi:10.1038/leu.2012.286

The eight p11 (8p11) myeloproliferative syndrome (EMS) term is designated in the 2008 World Health Organization Classification of Tumors of Haematopoietic and Lymphoid Tissues1 as myeloid and lymphoid neoplasms with eosinophilia and abnormalities of fibroblast growth factor receptor 1 gene (FGFR1). These aggressive malignancies are characterized by their rare occurrence, heterogeneity, eosinophilia and rapid progression in acute myeloid leukemia. The fibroblast growth factor receptor 1 gene (FGFR1) encodes for a tyrosine kinase receptor for fibroblast growth factor and is known to be fused to different recurrent gene partners, such as TPR2 (1q25), LRRFIP1 (2q37), FGFR1OP1 (6q27), CUX1(ref.3) (7q22), TRIM24 (7q34), CEP110 (9q33), FGFR1OP2 (12p11), CPSF6 (12q15), ZNF198 (13q12), MYO18A (17q23), HERV-K (19q13) and BCR (22q11), reviewed in Jackson et al.4 In all cases the chimeric contains an aberrant tyrosine kinase domain constitutively activated. Treatments, including tyrosine kinase inhibitors based regimen, are often ineffective so that FGFR1 abnormalities are associated with bad prognosis. The few long-term survivors have undergone stem cell allograft. We report here a novel partner gene for FGFR1, identified in a Figure 1. Cytogenetic analysis of the t(2;8)(q12;p11). (a) Partial 63-year old Caucasian female. The patient was referred to our representative G-banded (left) and R-banded (right) karyotype showing both normal 2 and 8 and derivative institution for dyspnea, impaired general condition and spleno- chromosomes of the t(2;8). (b) Fluorescence in situ hybridization megaly without hepatomegaly nor adenopathy. Medical history analysis with whole painting probes for chromosome included a severe stroke following the rupture of a cerebral 2 (green) and 8 (red) on a metaphase, displaying a balanced t(2;8). aneurysm, a mitral valve replacement and an ischemic cardio- (c) Hybridization pattern using BAC RP11-350N15 (green) encom- pathy. Peripheral blood examination showed hemoglobin level passing FGFR1 gene on 8p11, with BAC RP11-84C2 (red) encom- of 101 g/l, a platelet count of 65 Â 109/l and a white blood cell passing NUP358, showing two fusion signals. count of 311 Â 109/l with 44% neutrophils (136 Â 109/l), 3% eosinophils (3 Â 109/l), 2% monocytes (6.2 Â 109/l), 46% promye- locytes, myelocytes and metamyelocytes and 2% circulating Resource Center, Children’s Hospital Oakland Research Institute blasts. Bone marrow aspiration stained with May Gru¨nwald and Dr E Flori, Cytogenetics Laboratory, Strasbourg University Giemsa was hypercellular, with granular hyperplasia, dysgranulo- Hospital). BACs RP11-85M14, RP11-348G16 and RP11-84C2 poiesis and few eosinophils. The diagnosis of myeloproliferative/ showed signals on normal and derivative myelodysplastic syndrome was retained at that time because of chromosomes 2 and 8. Therefore, dual-color co-hybridization of the proliferative features, with dysplasia and very few monocytes BACs RP11-350N15 and RP11-84C2 showed fused signals on in the bone marrow. Molecular analysis did not found any V617F derivative chromosomes 2 and 8 (Figure 1c), narrowing the or exon 12 mutation in JAK2, nor BCR-ABL1 fusion transcripts. breakpoint region. RANBP2 (alias NUP358) gene was the sole Reverse heat giemsa and G-trypsin giemsa banding chromo- known gene in the 2p12.3 region delineated by these probes, and some analysis performed on bone marrow cells showed the we hypothesized that RANBP2 was a likely candidate for an in- following karyotype: 46,XX,t(2;8)(q12;p11)[14]/46,XX[14] (Figure 1a), frame fusion with FGFR1. Thus, total RNA was extracted from bone according to the 2009 International System for Human Cytogenetic marrow cells and a reverse transcription-PCR was designed to Nomenclature.5 Fluorescence in situ hybridization analysis with amplify a putative RANBP2-FGFR1 transcript, using one forward whole chromosome 2 and 8 painting probes (Kreatech, Strasbourg, primer located in exon 16 of RANBP2 from Ma et al.6 and one France) confirmed the t(2;8)(q12;p11) (Figure 1b). Therefore, BAC reverse primer located in exon 9 of FGFR1 from Hidalgo-Curtis RP11-350N15 encompassing the 8p11 FGFR1 gene region was et al.7 hybridized and revealed signals on normal chromosome 8 and We obtained specific amplification of the putative RANBP2/ derivative chromosomes 2 and 8, demonstrating the breakpoint NUP358-FGFR1, which was not obtained in controls. Sequence on 8p11 in FGFR1 gene. analysis of the three PCR products revealed three fusions of In order to refine the breakpoint in chromosome 2, a series of RANBP2 exon 20 with FGFR1 exon 9 (Figures 2a and b). Only one of BACs was hybridized on 2q12: RP11-38C17, RP11-350B7, RP11- them was in-frame that incorporated 27 bp of intron 8 of FGFR1, 443K8, RP11-465O11, RP11-85M14, RP11-348G16, RP11-84C2, the other presented abnormal splicing, and was not productive. RP11-20G1, RP11-471N11, RP11-566O4, RP11-528G9, RP11- The RANBP2-FGFR1 transcript sequence is predicted to encode a 438K19, RP11-368A17, RP11-412A2 and RP11-314L11 (BAC PAC chimeric protein containing the N-terminal portion of the RANBP2

Accepted article preview online 8 october 2012; advance online publication, 26 October 2012

Leukemia (2013) 1172 – 1218 & 2013 Macmillan Publishers Limited Letters to the Editor 1187

Figure 2. Schematic representation of RANBP2/NUP358-FGFR1 fusion transcript and protein. (a) Schematic presentation of structure of RANBP2/ NUP358-FGFR1 fusion variants deduced from complementary DNA sequence (reference sequences: NM_006267.4 for RANBP2/NUP358, NM_000604 for FGFR1). #1 includes 27 bases of FGFR1 intron 8. #2 and #3 lack intron 8 insertion and contain a shorter part of exon 20 of RANBP2 (177 and 96 bp, respectively). (b) Reverse transcription-PCR complementary DNA sequence of the fusion region of the RANBP2/ NUP358-FGFR1. RANBP2 sequences are underlined, FGFR1 sequences are in plain text, and FGFR1 intron sequence are in bold. (c) Schematic representation of RANBP2, FGFR1 and RANBP2-FGFR1 predicted fusion . RANBP2 comprises a leucine-rich region, including a leucine zipper motif (Leu-rich), several zinc finger motifs, -binding domains (numbered black boxes), a cyclophilin—like domain (Cy) and a E3 ligase region. FGFR1 is composed of three Ig-like domains, a transmembrane domain and two tyrosine kinase domains (TK domain). The breakpoints within proteins are indicated by arrows. protein, including the protein interaction leucine zipper portion, and the two tyrosine kinase domains of FGFR1, and was also and the two tyrosine kinase domains of FGFR1 (Figure 2C). The associated with a monocytosis. reciprocal transcript was also expressed, detected using FGFR1 In all fusion proteins involving FGFR1, the constitutive activation forward primer from Soler et al.8 and RANBP2 reverse primer from of the FGFR1 kinase is mediated by the N-terminus homodimer- Ma et al.6 (data not shown). ization motifs of the FGFR1 partners that promote leukemogen- Hydroxyurea and 6-mercaptopurin therapy was rapidly initiated. esis. These motifs in EMS are mainly coiled–coiled domains, and white blood cell count gradually decreased to a nadir of 2.9 Â 109/l various motifs that are also implicated in protein–protein interaction within 1 month. No related human leukocyte antigen-matched donor (Supplementary table). In the fusion transcript described in this study, was identified. Allogenic hematopoietic stem cell transplant from an the portion of RANBP2 includes a leucine zipper motif embedded unrelated donor was not considered because of poor general in a leucine rich region, suggesting similar oncogenic mechanisms. condition. Disease progressed rapidly despite changes in therapy to In addition to FGFR1 activation, FGFR1 partners are thought to subcutaneous cytarabine. Patient died 6 months after the diagnosis. have a specific role in the phenotypic expression of the disease. RANBP2/NUP358 is a component of the complex For instance, patients with t(8;22)(p11;q11) often present with a (NPC), localized at its cytoplasmic side.9 It is the major component chronic myeloid leukemia (CML)-like clinical syndrome, and of cytoplasmic filaments of the pore and is involved in the final patients with t(8;13)(p11;q12), t(8;9)(p11;q33) and t(6;8)(q27;p11) steps of nuclear export. RANBP2/NUP358 protein interacts with are correlated with lymphadenopathy, peripheral blood mono- NUP88 in a NPC subcomplex and the latter also interacts with cytosis and polycythemia.4 The CEP110-FGFR1 fusion protein that other proteins of the NPC, such as NUP214 and NUP98, NUP214 retains the leucine zipper motifs of CEP110 and the catalytic mediating the attachment of NUP358 to the NPC.10 domain of FGFR1 is associated with monocytosis and shares If NUP214 and NUP98 have recurrently been involved in structural similarities with the putative one of RANBP2/NUP358, hematopoietic tumors, RANBP2/NUP358 has mainly been impli- which presented a monocytosis. In mice, the leucine-rich domain cated in a small subset of anaplastic lymphoma kinase (ALK)- of RanBP2 also exhibits strong chaperone activity toward Cox11 positive myofibroblastic tumors, where a NUP358-ALK fusion was proteins, supporting a chaperone role of RanBP2 in the cytosol.14 identified, resulting from a t(2;2)(p23;q13).1,11,12 In these cases, the The role of this protein interaction in the oncogenesis is not complementary DNA contained the first 18 exons of RANBP2/ known, and COX11, to our knowledge, has not been reported to NUP358, encoding the leucine zipper domain that has been proposed be associated with hematological malignancies or solid tumors. to be the site of attachment to the NPC core, and the cytoplasmic Otherwise, in T lymphoblastic leukemia (T-ALL), the NUP214- domain of the receptor tyrosine kinase ALK.6 Recently, the same ABL1 transcripts retain the N-terminal region of NUP214 includ- abnormality was detected in two juvenile myelomonocytic leukemias ing the predicted coiled–coiled domains (that may serve as and one M4 acute myeloid leukemia,13 associated to GM-CSF oligomerization motifs) and the tyrosine kinase domain of ABL1.15 (granulocyte-macrophage colony-stimulating factor) hypersensitivity. NUP98 fusion transcripts often have a different feature, including The RANBP2/NUP358-FGFR1 fusion transcript identified in the case phenyl-alanine (FG) motifs that serve as interaction sites for karyo- herein reported contained the first 20 exons of RANBP2/NUP358, pherins and chromatin interacting homeobox domains of the coding for the same protein domains as in RANBP2/NUP358-ALK, partner gene.

& 2013 Macmillan Publishers Limited Leukemia (2013) 1172 – 1218 Letters to the Editor 1188 In this study, we describe a novel fusion in hematological malig- 3 Wasag B, Lierman E, Meeus P, Cools J, Vandenberghe P. The kinase inhibitor nancies, expanding number of FGFR1 partner and confirming TKI258 is active against the novel CUX1-FGFR1 fusion detected in a patient with the relevance of RANBP2/NUP358 in myeloid neoplasm. The clinical T-lymphoblastic leukemia/lymphoma and t(7;8)(q22;p11). Haematologica 2011; presentation as a myeloproliferative/myelodysplastic disorder was 96: 922–926. not different from other EMS. More cases with chromosome 2 4 Jackson CC, Medeiros LJ, Miranda RN. 8p11 myeloproliferative syndrome: a abnormalities should be explored in order to evaluate the incidence review. Hum Pathol 2010; 41: 461–476. 5 Shaffer LG, Slovak ML, Campbell LJ. An International System for Human Cytogenetic of RANBP2/NUP358 rearrangements in hematological neoplasms. Nomenclature. Karger: Basel, 2009. 6 Ma Z, Hill DA, Collins MH, Morris SW, Sumegi J, Zhou M et al. Fusion of ALK to the Ran-binding protein 2 (RANBP2) gene in inflammatory myofibroblastic tumor. CONFLICT OF INTEREST Genes Chromosomes Cancer 2003; 37: 98–105. The authors declare no conflict of interest. 7 Hidalgo-Curtis C, Chase A, Drachenberg M, Roberts MW, Finkelstein JZ, Mould S et al. The t(1;9)(p34;q34) and t(8;12)(p11;q15) fuse pre-mRNA processing proteins SFPQ (PSF) and CPSF6 to ABL and FGFR1. Genes Chromosomes Cancer 2008; 47: ACKNOWLEDGEMENTS 379–385. 8 Soler G, Nusbaum S, Varet B, Macintyre EA, Vekemans M, Romana SP et al. We thank Dr Elisabeth FLORI for providing part of the BACs. LRRFIP1, a new FGFR1 partner gene associated with 8p11 myeloproliferative syndrome. Leukemia 2009; 23: 1359–1361. C Gervais1,2,3, L Dano1,2,3, N Perrusson4,CHe´lias1,2,3, E Jeandidier2,3, 9 Wu J, Matunis MJ, Kraemer D, Blobel G, Coutavas E. Nup358, a cytoplasmically A-C Galoisy1,3, A Ittel1,2,3, R Herbrecht5, K Bilger5 and exposed nucleoporin with peptide repeats, Ran-GTP binding sites, zinc fingers, a L Mauvieux1,2,3,4 cyclophilin A homologous domain, and a leucine-rich region. J Biol Chem 1995; 1Poˆle de Biologie, Laboratoire d’He´matologie, Hoˆpitaux Universitaires 270: 14209–14213. de Strasbourg, Strasbourg, France; 10 Bernad R, van der Velde H, Fornerod M, Pickersgill H. Nup358/RanBP2 attaches to 2 the nuclear pore complex via association with Nup88 and Nup214/CAN and plays Laboratoire Re´gional de Cytoge´ne´tique He´matologique d’Alsace a supporting role in CRM1-mediated nuclear protein export. Mol Cell Biol 2004; 24: Mulhouse–Strasbourg, Strasbourg, France; 2373–2384. 3 Plateforme Re´gionale INCa de Ge´ne´tique Mole´culaire des Cancers 11 Patel AS, Murphy KM, Hawkins AL, Cohen JS, Long PP, Perlman EJ et al. RANBP2 d’Alsace, Strasbourg, France; and CLTC are involved in ALK rearrangements in inflammatory myofibroblastic 4Laboratoire d’He´matologie Cellulaire, Faculte´ de Me´decine de tumors. Cancer Genet Cytogenet 2007; 176: 107–114. Strasbourg, Strasbourg, France and 12 Chen ST, Lee JC. An inflammatory myofibroblastic tumor in liver with 5De´partement d’Onco-He´matologie, Hoˆpitaux Universitaires de ALK and RANBP2 gene rearrangement: combination of distinct morphologic, Strasbourg, Strasbourg, France, immunohistochemical, and genetic features. Hum Pathol 2008; 39: E-mail: [email protected] 1854–1858. 13 Rottgers S, Gombert M, Teigler-Schlegel A, Busch K, Gamerdinger U, Slany R et al. ALK fusion genes in children with atypical myeloproliferative leukemia. Leukemia 2010; 24: 1197–1200. REFERENCES 14 Aslanukov A, Bhowmick R, Guruju M, Oswald J, Raz D, Bush RA et al. RanBP2 1 Swerdlow SH, Campo E, Harris NL, Jaffe´ ES, Pileri SA, Stein H et al. WHO Classifi- modulates Cox11 and hexokinase I activities and haploinsufficiency of RanBP2 cation of Tumours of Haematopoietic and lymphoid tissues. IARC: Lyon, 2008. causes deficits in glucose metabolism. PLoS Genet 2006; 2:e177. 2 Li F, Zhai YP, Tang YM, Wang LP, Wan PJ. Identification of a novel partner gene, 15 Graux C, Cools J, Melotte C, Quentmeier H, Ferrando A, Levine R et al. Fusion of TPR, fused to FGFR1 in 8p11 myeloproliferative syndrome. Genes Chromosomes NUP214 to ABL1 on amplified episomes in T-cell acute lymphoblastic leukemia. Cancer 2012; 51: 890–897. Nat Genet 2004; 36: 1084–1089.

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Exome sequencing in tracking clonal evolution in multiple myeloma following therapy

Leukemia (2013) 27, 1188–1191; doi:10.1038/leu.2012.287 presents as a highly heterogeneous disease, this introduces a further complexity, requiring each subset of the disease to be evaluated separately in its response to therapy. Multiple myeloma (MM), defined as accumulation of malignant plasma cells in Sequencing the tumor genome using next-generation sequencing the bone marrow, exemplifies such a tumor and exhibits marked (NGS) is providing an unparalleled insight into the pathogenesis disease heterogeneity at presentation.1,2 At the outset, aberrant and progression of the disease. Much of the current focus of chromosomal markers exist in diagnostic MM samples that are NGS in cancer is on defining mutations in the tumor genome at associated with poor prognosis and help in delineating specific disease presentation, and these findings are central to under- disease subsets, including t(4;14) and 1q21 amplification among standing the molecular mechanisms that underlie pathogenesis. others,1–5 suggesting that these markers may be highly relevant to However, additional questions remain about the stability of the defining specific patterns of progression and clonal response to tumor clone under therapeutic pressure and these require a therapy. distinct second wave of NGS analyses. Understanding how each The pivotal NGS study of MM centered on the MM Research tumor evolves following therapy will be the key to delivering Consortium (MMRC) analysis of 38 tumor genomes that identified targeted therapy tailored for individual patients and in develop- key somatic mutations, either at disease presentation or at relapse, ing stratified therapeutic programs. Where a given tumor but not in a paired setting.6 Consequently, the data did not permit

Accepted article preview online 9 October 2012; advance online publication, 13 November 2012

Leukemia (2013) 1172 – 1218 & 2013 Macmillan Publishers Limited