A common 93-kb duplicated DNA sequence at 1q21.2 in acute lymphoblastic leukemia and Burkitt lymphom

Short communicationA common 93-kb duplicated DNA sequence at 1q21.2in acutelymphoblastic leukemia and Burkitt lymphomaRoberta La Starza a ,Barbara Crescenzi a ,Valentina Pierini a ,Silvia Romoli a ,Paolo Gorello a ,Lucia Brandimarte a ,Caterina Matteucci a ,Maria Grazia Kropp b ,Gianluca Barba a ,Massimo Fabrizio Martelli a ,Cristina Mecucci a ,*aDepartment of Hematology,University of Perugia,IBiT Foundation,Fondazione IRCCS Biotecnologie nel Trapianto,via Brunamonti 51,06122Perugia,ItalybDepartment of Hematology,‘‘A.Pugliese’’Hospital,Catanzaro,ItalyReceived 16October 2006;received in revised form 17January 2007;accepted 22January 2007AbstractIn three patients with acute lymphoblastic leukemia (ALL)and in another with Burkitt lymphoma (BL),conventional cytogenetics and fluorescence in situ hybridization (FISH),applied singly or in combination,showed 1q duplication in two cases of ALL with hyperdiploid karyotypes,1q dupli-cation resulting from an unbalanced translocation in a third case of ALL,and inv dup(1)(q)in a pa-tient with BL.Centromeric or telomeric breakpoints and extension of the 1q duplicons varied in each case.FISH defined a minimal,common duplicated region of 93kb at band 1q21.2correspond-ing to clone RP11-212K13.In this region three putative oncogenes or tumor suppressor genes havebeen mapped:SF3B4(splicing factor 3b,subunit 4),OTUD7B (OTU domain containing 7B),and MTMR11(myotubularin related protein 11).For the first time,a minimal common 1q21.2dupli-cated sequence has been identified in lymphoid malignancies in a region where putative oncogenes or suppressor genes have been mapped.This finding elucidates the genomic background of ALL and BL with 1q duplication and provides the basis for molecular studies investigating which genes are involved in leukemogenesis or disease progression in these cases.Ó2007Elsevier Inc.All rights reserved.1.IntroductionChromosome 1long arm duplications,or dup(1q),and trisomy 1q are recurrent changes in chronic myeloprolifer-ative disorders,multiple myeloma (MM),acute lympho-blastic leukemia (ALL),and Burkitt lymphoma/leukemia (BL).In chronic myeloproliferative disorders,both a 1q23~q32duplication and an unbalanced 1q translocation with various chromosomes,producing a gain of the entire 1q arm,have been observed [1].In up to 40%of patients with MM,1q12~q32duplications,whole-arm transloca-tions,or jumping translocations are associated with com-plex karyotypes,aggressive disease,and poor prognosis [2].In BL,dup(1q)is the most frequent secondary changeassociated with t(8;14)(q24;q32);it appears to be linked to disease progression rather than pathogenesis [3,4].Whether dup(1q)and trisomy 1q in lymphoid and myeloid malig-nancies underlie the same molecular lesions remains to be established.We characterized dup(1q)in lymphoid malignancies and identified a common 1q21.2duplicated region in three cases of ALL and one case of BL.Our findings provide the basis for designing molecular studies to investigate any putative oncogenes or tumor suppressor genes that could underlie 1q duplication in ALL and BL.2.Materials and methods 2.1.PatientsWe studied three ALL (patients 1e 3)and one BL (pa-tient 4)at diagnosis (Table 1).Conventional cytogenetic and fluorescence in situ hybridization (FISH)analyses were*Corresponding author.Tel.:þ39-075-5783808;fax:þ39-075-5783691.E-mail address:crimecux@unipg.it (C.Mecucci).0165-4608/07/$e see front matter Ó2007Elsevier Inc.All rights reserved.doi:10.1016/j.cancergencyto.2007.01.011Cancer Genetics and Cytogenetics 175(2007)73e 76performed on bone marrow cells(patients1e3)and pleural and ascitic effusion(patient4).2.2.CytogeneticsKaryotypes were obtained on G-banded metaphases af-ter24-and48-hour cultures and were described according to ISCN2005[5].2.3.FISHMetaphase FISH was performed in dual-color experi-ments,as previously described,with34DNA clones for the1q arm mapping from centromere RP11-97A5to telo-mere RP1-160H3(Fig.1A)[6,7].Probe D1Z5(Oncor,Re-snova,Italy)for the centromeric alpha satellite region and probe PUC1.77for heterochromatin were also used[8]. DNA clones for the telomeric region of chromosomes1 (PAC GS-160-H23),2(PAC GS-1011-O17),11(PAC GS-770-G7),and15(PAC GS-124-O5)were used to fully elu-cidate complex changes[9].In each experiment,a minimum offive abnormal metaphase cells were analyzed with afluo-rescence microscope(Provis,Olympus,Milan,Italy)equip-ped with a charge-coupled device camera(Sensys, Photometrics)operated with SmartCapture software(Vysis, Stuttgart,Germany).3.Results3.1.PatientsTwo of the patients were male(1ALL;1BL)and two were female(2ALL).Two ALL patients and the one BL patient were children(5,9,and11years old)and one ALL patient was a46-year-old adult.Clinical and hemato-logical features and cytogeneticfindings are summarized in Table1.3.2.CytogeneticsIn patients1and2,dup(1q)was present in hyperdiploid karyotypes.Patient3had three leukemic clones.A recipro-cal t(1;18)(q21;q12)was common to all.It was isolated in the main clone and associated with del(3q)alone or with an add(2q)in the two related sidelines.Patient4showed a complex karyotype with t(8;14)(q24;q32)and additional changes including an add(1q42).3.3.FISHFISHfindings are summarized in Figure1.FISH with DNA clones for the1q arm confirmed dup(1q)in patients 1and2and detected it in patient4.In patient4,the add(1)(q42)derived from an inv dup(1)(q25q11)and an unbalanced translocation with the15q21~22band,that is,der(1)t(1;15)(q25~31;q21~22)dup(1)(q25q11)(Fig.1B, upper).FISH with PAC GS-160-H23for the1qter region confirmed chromosome1q translocated to chromosome 15:that is,der(15)t(1;15)(q25~31;q21~22).FISH with PAC GS-124-O5for the15qter region showed chromosome 15q translocated to dup(11)(q11q25):that is,der(11) t(11;15)(q25;q21~22)dup(11)(q11q25).FISH with PAC GS-770-G7for the11qter region yielded only one hybrid-ization signal on normal11,suggesting that der(11)was in-volved in an unbalanced translocation(data not shown).In patient3,FISH revealed a duplication of1q11~q21bands that was translocated to chromosome band2q36:that is, der(2)t(1;2)(q11q21;q36)(Fig.1B,lower).FISH with PAC GS-1011-O17for the2qter region confirmed the trans-location as unbalanced(data not shown).In patients1,3,and4,the dup(1q)centromeric break-points fell within heterochromatic sequences.In patient2, the breakpoint was narrowed to between two contiguous clones:that is,to RP11-196G18(proximal)and RP11-212K13(distal).In patients1,2,and4,the telomeric break-points were located distal to ABL2/1q25.2and in patient3 the breakpoint was mapped between two contiguous clones:RP11-212K13(proximal)and RP11-458I7(distal) at band1q21.2(Fig.1A).A minimal duplicated DNA se-quence that was common to all four patients was identified at chromosome band1q21.2.It extends93kb and corre-sponds to clone RP11-212K13.4.DiscussionThe genomic mechanisms and molecular lesions under-lying dup(1q)in lymphoid malignancies remain to be estab-lished.To date,FISH studies have mapped dup(1q) centromeric breakpoints in the centric or pericentric region of chromosome1within,or in close proximity to,theTable1Clinical,hematological,and cytogenetic features of four patients with dup(1)(q)Patient Sex/Age Diagnosis Karyotype1F/9ALL-L156,XX,þX,dup(1q),þ5,þ6,þ9,þ10,þ14,þ17,þ18,þ21,þ22[6]46,XX[7]2M/5ALL-L254,XY,dup(1q),þ5,þ6,þ9,þ10,þ14,þ15,þ17,þ18[15]3F/46ALL-L246,XX,t(1;18)(p11;p11)[3]/46,idem,del(3q)[5]/46,idem,add(2)(q36)[6]/add(2)(q36).ish der(2)t(1;2)(q11q21;q36) 4M/11BL47,XY,add(1)(q42),þ7,t(8;14)(q24;q32),dup(11)(q11q25),add(15)(q25),þ18[15]/add(1)(q42).ish der(1)t(1;15)(q25~31;q21~22)dup(1)(q25q11)Abbreviations:ALL,acute lymphoblastic leukemia;BL,Burkitt lymphoma;F,female;M,male. Starza et al./Cancer Genetics and Cytogenetics175(2007)73e76heterochromatic sequences [10].The present study identi-fied a minimal common duplicated DNA euchromatic sequence in four cases of ALL/BL.Although 1q duplicons in ALL and BL have various sizes with different proximal and distal breakpoints,they share a sequence at band 1q21.2corresponding to clone RP11-212K13and extending for 93kb.Three putative on-cogenes or tumor suppressor genes have been mapped in the 1q21common duplicated region [7].The SF3B4gene,an RNA-binding protein,is one of the four subunits of splicing factor 3B,which is widespread in human tissues,including bone marrow [7].The OTUD7B gene encodes for a cytoplasmic protein and functions as deubiquitinating enzyme and as a negative regulator of the NF k B transcription factor,which controls cell proliferation,apoptosis,and angiogenesis [11].Remarkably,NF k B and the signaling pathway involved in its activation have been shown to be constitutively activated in solid tumors and in lymphoid malignancies [12].The MTMR11gene is a member of the tyrosine phospha-tase family,whose main activity is to dephosphorylate D3-phosphorylated inositol phospholipids.It is expressed in bone marrow cells,spleen,and thymus [13].Mutations or altered expression of three members of the lipid phospha-tase family are found in congenital neuropathies,singly or in combination.MTM1,the first member of this family to be identified,is responsible for X-linked myotubular my-opathy;MTMR2and MTMR13(alias for SBF2)mutations have been detected in two forms of Charcot-Marie-Tooth disease [14e 16].Although none of these three genes has been directly linked to hematological malignancies in humans,so far,it remains to be investigatedwhetherABDNA clone band locus/gene1234D1Z51q11centromerepuc 1.771q12heterochromatin RP11-97A51q21.1LOC440670RP11-544O241q21.1SEC22L1RP11-94I21q21.1LOC400778RP11-337C181q21.1PRKAB2RP11-533N141q21.1BCL9RP11-14N71q21.1LOC440683RP11-403I131q21.1LOC401967RP5-998N211q21LOC440686RP11-277L21q21LOC440688RP11-353N41q21KIAA0493RP11-196G181q21FCGR1ARP11-212K131q21.2MTMR11,OTUD7B,SF3B4RP11-458I71q21.2PLEKHO1,VP54A RP4-790G171q21.2ANP32E RP11-54A41q21.2KIAA0460RP11-235D191q21.2CTSK RP11-316M11q21.2SETDB1RP11-68I181q21.2MGC29891RP11-98D181q21.3SNX27RP11-107M161q21.3FAM16B RP11-182L111q21.3SPRR1B RP11-307C121q22PMVK RP11-29H231q22ASH1L RP11-336K241q22RIT1RP11-139I141q22RHBG RP11-98G71q22MEF2D RP11-66D171q23.1BCAN RP11-107D161q23.1SH2D2A RP11-110J11q23.1ARHGEF11RP11-217A121q23.1IRTA2RP11-404F101q23.2SLAMF1RP11-38C181q23.3LMX1A RP11-152A161q25.2ABL2RP1-160H31q44subtelomereFig.1.(A)FISH findings with 1q11~q44DNA clones in three cases of acute lymphoblastic leukemia and one of Burkitt lymphoma.Red squares indicate DNA clones in three copies,black squares indicate DNA clones in two copies,and white squares indicate DNA clones that were not tested.(B)Upper:FISH with PUC 1.77(green)and clone RP11-212K13(red)in patient 4shows three red e green fusion signals:one on normal chromosome 1and two on der(1)t(1;15)(q25~31;q21~22)inv dup(1)(q25q11)(arrow ).Lower :FISH with PUC 1.77(green)in patient 3gave three hybridization signals,one on normal chromosome 1,one on der(1)t(1;18),and one on add(2)t(1;2)(q11;q36)(arrow ).75 Starza et al./Cancer Genetics and Cytogenetics 175(2007)73e 76deregulation of one or more plays a role in the leukemo-genic process in ALL and BL with1q21duplication.Whatever the molecular events underlying1q21duplica-tion,they seem to be closely correlated to leukemogenic pathways.A constitutional duplication of proximal1q, dup(1)(q11~q22),has been reported in a pediatric patient, with normal phenotype,who developed a T lymphoblastic lymphoma/leukemia[17].Moreover,Ghose et al.[18] showed in mice that duplication of1q11~q32is associated with proliferation of human leukemic B-cell clones and metastasis formation.In conclusion,we identified a minimal common1q21.2 duplicated sequence in a region in which three putative on-cogenes or tumor suppressor genes have been mapped.In elucidating the genomic background of ALL and BL with 1q21duplication,our study provides the basis for designing molecular studies to investigate which,if any,of these genes is involved in lymphoid leukemogenesis or progression.AcknowledgmentsAIRC(Associazione Italiana Ricerca sul Cancro),CNR-MIUR(Consiglio Nazionale delle Ricerche-Ministero per l’Istruzione,l’Universita`e la Ricerca Scientifica);Fonda-zione Cassa di Risparmio,Perugia,Italy;FIRB,Italy;and Associazione‘‘Sergio Luciani,’’Fabriano,Italy. B.C.is supported by a grant from FIRC(Fondazione Italiana Ri-cerca sul Cancro).BAC clones were kindly provided by Dr.Mariano Rocchi(DAPEG Sez di Genetica,University of Bari,Italy);Telomeric DNA clones were kindly pro-vided by Dr.J Flint(Institute of Molecular Medicine,John Radcliffe hospital,Oxford,UK).Authors wish to thank Dr. Geraldine Boyd for assistance in the preparation of the manuscript.References[1]Bench AJ,Cross NCP,Huntly BJP,Nacheva EP,Green AR.Myelo-proliferative disorders.Best Pract Res Clinic Haematol2001;14: 531e51.[2]Hanamura I,Stewart JP,Huang Y,Zhan F,Santra M,Sawyer JR,Hollmig K,Zangarri M,Pineda-Roman M,van Rhee F,Cavallo F, Burington B,Crowley J,Tricot G,Barlogie B,Shaughnessy JD Jr.Frequent gain of chromosome band1q21in plasma cell dyscrasias detected byfluorescence in situ hybridization:incidence increases from MGUS to relapsed myeloma and is related to prognosis and dis-ease progression following tandem stem cell transplantation.Blood 2006;108:1724e32.[3]Berger R,Bernheim A.Cytogenetic studies on Burkitt’s lymphoma-leukemia.Cancer Genet Cytogenet1982;7:231e44.[4]Offit K,Wong G,Filippa DA,Tao Y,Chaganti RSK.Cytogeneticanalysis of434consecutively ascertained specimens of non-Hodgkin’s lymphoma:clinical correlations.Blood1991;77:1508e15.[5]ISCN2005:an international system for human cytogenetic nomen-clature(2005).In:Shaffer LG,Tommerup N,editors.Basel:S.Kar-ger,2005.[6]Dierlamm J,Wlodarska I,Michaux L,La Starza R,Zeller W,Mecucci C,Van den Berghe H.Successful use of the same slide for consecutivefluorescence in situ hybridization(FISH)experi-ments.Genes Chromosomes Cancer1996;16:261e4.[7]Entrez Gene database.Available at:/entrez/query(build36.2).Accessed April16,2007.[8]La Starza R,Aventin A,Falzetti D,Wlodarska I,FernandezPeralta AM,Gonzales-Aguilera JJ,Ciolli S,Martelli MF, Mecucci C.Regions of juxtaposition in unbalanced1q rearrange-ments of malignant hemopathies.Leukemia2001;15:861e3.[9]Knight SJL,Lese CM,Precht KS,Kuc J,Ning Y,Lucas S,Regan R,Brenan M,Nicod A,Lawrie NM,Cardy DLN,Nguyen H, Hudson TJ,Riethman HC,Ledbetter DH,Flint J.An optimized set of human telomere clones for studying telomere integrity and archi-tecture.Am J Hum Genet2000;67:320e32.[10]Busson-Le Coniat M,Salomon-Nguyen F,Dastugue N,Maarek O,Lafage-Pochitaloff M,Mozziconacci M-J,Baranger L,Brizard F, Radford I,Jeanpierre M,Bernard OA,Berger R.Fluorescence in situ hybridization analysis of chromosome1abnormalities in hematopoi-etic disorders:rearrangements of DNA satellite II and new recurrent translocations.Leukemia1999;13:1975e81.[11]Evans PC,Smith TS,Lai M-J,Williams MG,Barker DF,Heyninck K,Kreike MM,Beyaert R,Blundell TL,Kilshaw PJ.A novel type of deubiquitinating enzyme.J Biol Chem2003;278: 23180e6.[12]Karin M,Cao Y,Greten FR,Li Z-W.NF-k B in cancer:from innocentbystander to major culprit.Nature Rev2002;2:301e10.[13]Wishart MJ,Taylor GS,Slama JT,Dixon JE.PTEN and myotubularinphosphoinositide phosphastases:bringing bioinformatics to the lab bench.Curr Opin Cell Biol2001;13:172e81.[14]Laporte J,Hu LJ,Kretz C,Mandel JL,Kioschis P,Coy JF,Klauck SM,Poustka A,Dahl N.A gene mutated in X-linked myotub-ular myopathy defines a new putative tyrosine phosphatase family conserved in yeast.Nat Genet1996;13:175e82.[15]Bolino A,Muglia M,Conforti FL,LeGuern E,Salih MA,Georgiou DM,Christodoulou K,Hausmanowa-Petrusewicz I, Mandich P,Schenone A,Gambardella A,Bono F,Quattrone A, Devoto M,Monaco AP.Charcot-Marie-Tooth type4B is caused by mutations in the gene encoding myotubularin-related protein-2.Nat Genet2000;25:17e9.[16]Azzedine H,Bolino A,Taieb T,Birouk N,Di Duca M,Bouhouche A,Benamou S,Mrabet A,Hammadouche T,Chkili T,Gouider R, Ravazzolo R,Brice A,Laporte J,LeGuern E.Mutations in MTMR13,a new pseudophosphatase homologue of MTMR2and Sbf1,in two families with an autosomal recessive demyelinating form of Charcot-Marie-Tooth disease associated with early-onset glaucoma.Am J Hum Genet2003;72:1141e53.[17]Chan NPH,NG MHL,Cheng SH,Lee V,Tsang KS,Lau TT,Li CK.Hereditary duplication of proximal chromosome1q(q11q22)in a pa-tient with T lymphoblastic lymphoma/leukemia:a family study usingG banding and comparative genomic hybridization.J Med Genet2002;39:79e82.[18]Ghose T,Lee CL,Fernandez LA,Lee SH,Raman R,Colp P.Role of1q trisomy in tumorigenicity,growth,and metastasis of human leuke-mic B-cell clones in nude mice.Cancer Res1990;50:3737e42. Starza et al./Cancer Genetics and Cytogenetics175(2007)73e76。