BRIEF COMMUNICATION. MLL-AF1q fusion resulting from t(1;11) in acute leukemia. Patients. Cytogenetic studies

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1 Leukemi (1999) 13, Stockton Press All rights reserved /99 $ BRIEF COMMUNICATION MLL-AF1q fusion resulting from t(1;11) in cute leukemi M Busson-Le Conit 1, F Slomon-Nguyen 1, J Hillion 1,2, OA Bernrd 1 nd R Berger 1 1 INSERM U 434 nd CNRS SD 401 No. 434, Pris, Frnce The MLL gene, locted on chromosome bnd 11q23 is fused to different prtner genes s result of vrious chromosoml trnsloctions in hemtopoietic mlignncies. A t(1;11) (q21;q23) resulting in MLL-AF1q fusion gene hs previously been reported. Cytogenetic studies on six cses re reported, including one three-wy trnsloction. FISH nlysis using YAC encompssing the MLL gene nd YAC encompssing the AF1q locus showed splitting in three cses nd two ptients, respectively. PCR nlysis of two cses confirmed tht AF1q is specificlly ssocited with t(1;11)(q21;q23). The MLL-AF1q fusion mrna ws similr to tht previously described in one cse nd involved MLL exon 7 in the other. This study confirms the specific involvement of AF1q in t(1;11) (q21;q23)-positive cute leukemi with monocytic involvement. Keywords: cute myelomonocytic leukemi; trnsloction; t(1;11), FISH, MLL-AF1q Introduction The chromosoml bnd 11q23, where the MLL gene is locted, is nonrndomly rerrnged with bout 30 other bnds in chromosoml bnormlities of mlignnt blood disorders. 1,2 Most of the rerrngements of 11q23 in cute leukemis cluster within region of 8.3 kb nd result in fusion of the MLL gene with prtner gene on the other chromosome. 1 3 Seventeen of these chimeric genes hve been identified to dte Identifiction of other prtners of MLL is now in progress in order to try to understnd the consequences of the fusion of MLL to so mny vrious prtner genes. The incidence of the vrious trnsloctions involving MLL seems to be different for the different types of rerrngement. For instnce, 281 cute lymphoblstic leukemis (ALL) with t(4;11)(q21;q23) were collected by Mitelmn et l 11 while only six cses of cute myeloblstic leukemi (AML) with t(1;11)(q21;q23) were listed in the sme rticle. Two t(1;11)(q21;q23) trnsloctions were shown to recombine the MLL gene on 11q23 with the AF1q on chromosome The contribution of AF1q to the fusion protein is only of 90 mino cids, nd the function of AF1q is not yet known. As exemplified by the t(11;19)(q23;p13) trnsloction, it is known tht different genes (ENL, ELL, EEN) locted on the sme chromosome bnd my be fused with MLL. 8,13,14 Severl genes, AFIq, BCL9 on 1q21 12,15 my be recombined by different trnsloctions seen in hemtopoietic mlignncies. Moleculr techniques re needed to estblish the recurrence of gene fusions in such trnsloctions. We now report three new cses of cute leukemi with t(1;11)(q21;q23), in ddition to three other cses (Nos 1 3) previously published Correspondence: R Berger, U 434 INSERM, IGM, 27 rue Juliette Dodu, Pris, Frnce; Fx: Present ddress: U496 INSERM, Centre Hyem, 1 Ave Clude Vellefux, Pris, Frnce Received 21 September 1998; ccepted 29 October 1998 Mterils nd methods Ptients Clinicl nd hemtologicl dt of six ptients studied in the lbortory re summrized in Tble 1. All were children (four infnts), three with AML-M4 in the FAB nomenclture, two with AML-M5 nd one with AML-M1/M2. Five relpsed nd one ws still in complete remission 15 months fter utogrft. Cytogenetic studies Chromosome studies were performed on bone mrrow nd/or unstimulted blood cultures t dignosis, nd in relpse in five ptients, nd in remission in one. RHG nd/or GTG bnds were obtined nd the kryotype clssified ccording to the Interntionl Nomenclture. Fluorescence in situ hybridiztion (FISH) studies FISH studies were performed on metphse chromosomes in three ptients (Nos 3, 5, 6) using YAC 742f9 (CEPH librry) s probe. This YAC is currently used in the lbortory to detect possible MLL involvement in 11q23 trnsloctions. YAC 978e4 (CEPH librry) which covers the AF1q locus nd gives strong hybridiztion locus on bnd 1q21 in spite of its chimerism ws lso used. More recently, BAC from the CEPH librry, 767A8, identified s contining AF1q sequences ws lso used. Moleculr studies Moleculr studies were performed in two ptients (Nos 5 nd 6). Nucleic cid nlysis: RNA ws isolted using Trizol regent (Gibco BRL, Cergy-Pontoise, Frnce), following the mnufcturer s instructions nd processed using stndrd protocols. 18 PCR experiments: Tking dvntge of the pprent similrity between the present cse nd the chromosoml rerrngement tht led to the isoltion of the AF1q gene, 12 we resoned tht similr moleculr rerrngement could be present. RNA nd then cdna were obtined from blood cells of the ptient (Kit Gibco BRL) nd polymerse chin rection (PCR) ws crried out. The primers used were: CACTCCTAGTGAGCCCAAG (MLL exon 5), CCCAAG- TATCCCTGTAAAACA (MLL exon 6), TCAGCACTCTCTC-

2 Tble 1 Brief communiction Acute leukemis with t(1;11)(q21;q23). Clinicl nd hemtologicl dt 303 Ptient Age/Sex FAB subtype Bone mrrow % Peripherl blood CR Relpse Survivl blst cells 10 9 /l (% blst) Hb g/dl pltelets 10 9 /l 1 (2051) K 4 m/f M5b (79) m 7 m (R) 2 (3097) D 10 m/f M (28) m 13 m (d) 3 (2957) P 3 y/f M1/M (37) m 28 m (d) 4 (3404) C 12 m/f M (58) m 16 m (d) 5 (7365) N 26 m/m M (64) m 6 (94278) M 11 m/f M (37) m () m, months; y, yers; Hb, hemoglobin; CR, complete remission; R, relpse; d, ded; live. utogrft in CR. Cse 2 hs been previously reported (in Ref. 16) nd cses 1, 3 nd 4 (in Refs 17, 18). Tble 2 Acute leukemis with t(1;11). Cytogenetic nd FISH dt Ptient Disese Mteril Kryotype FISH sttus YAC 942f9 YAC978e4 BAC767A7 11 1q 11q+ 11 1q 11q+ 11 1q 11q+ 1 D B24, 48 47,XXXc,t(1;11) [56] R Md/M24 47,XXXc,t(1;11) [75]/47,XXXc [41] 2 D B48 46,XX,t(1;11) [33] R B24,48 46,XX,t(1;11) [34] 3 D B24,48,72 46,XX,t(1;11) [40] R B248 46,XX,t(1;11) [28] 4 D M48;B24,48 46,XX,t(1;11;3)(q21;q23;q21) [24;40] 5 D M24;B24 46,XX,t(1;11) [31;9] R M24 46,XX,t(1;11) [14]/46,XY [3] 6 D M24 46,XX,t(1;11) [18] r M24,48 46,XX [15] D, dignosis; R, relpse; r, remission. B24,48,72, unstimulted blood cell culture for 24, h; Md, M24, M48, bone mrrow, direct exmintion, 24C, 48 h culture. Tble 3 Acute leukemis nd myelodysplstic syndrome with t(1;11)(q21;q23) Type of AL Kryotype Ref. ALL 46,XX,der(11)t(1;11)(q21;q23) 22 ALL-L1 (mixed) 51,XY,+6,+der(11)t(1;11)(q21;q23),+19, +21, ALL 56,XX,+4,+5,+6,+8,+10,+12,+14,+17,+8,+21/ 57,idem,+der(11)t(1;11)(q21;q23) 24 sall (B) 46,XX,der(11)t(1;11)(q21;q23),t(12;17) (p13;q21) 25 AML-M5 46,XX,t(1;11)(q21;q23) 16 AML-M5b 47,XXXc,t(1;11)(q21;q24) 17,18 AML-M4 46,XX,t(1;11)(q21;q23) 17,18 AML-M4 46,XY,t(1;11)(q21;q23) 26 AML-M4 46,XX,t(1;11)(q21;q23)/48,idem,+19, AML-M4 48,XY,t(1;11)(q21;q23),+6, AML-M5b t(1;1)(q21;q23) 29 s-aml 44,XX, 3, 5, 7,del(9)(p13),der(11) t(1;11)(q21;q23),+2mr 30 s-aml 75, 4n,XXYY, 2, 3,+4, 10,der(11) t(1;11)(q21;q23), 30 der(1;11), 12, 14, 14, 15, 15, 16, 17,i(17)(q10),+18, del(20)(q11),del(20),10 25 min AML-M2 46,XX,t(1;11)(q21;q23) 25 AML-M4 46,XY,t(1;11)(q21;q23)/47,XY, smds 47,XY,der(11)t(1;11)(q21;q23),+mr/48,idem,+mr 25 ALL, cute lymphoblstic leukemi; sall, secondry ALL; AML-M4, AML-M5, cute myeloblstic leukemi, M4, M5 in the FAB nomenclture; s-aml, secondry cute myeloblstic leukemi; smds, secondry myelodysplstic syndrome. See Tble 1 for more dt.

3 Brief communiction 304 Results Chromosome studies Rerrngement of bnds 1q21 nd 11q23 ws present in the six ptients (Figure 1), t(1;11)(q21;q23) in five nd t(1;11;3) (q21;q23;q21) in one (Tble 2). A triplo-x constitution ws detected in cse 1. It ws thought to be constitutionl since kryotypes 47,XXX were observed in relpse in ddition to those with 47,XXX,t(1;11). FISH Three hybridiztion signls, one on the norml chromosome 11 nd one ech of the rerrnged chromosomes (1q nd 11q+) were observed on metphse cells using YAC 742f9 s the probe for ptients 3, 5 nd 6. This YAC splitting fvored the existence of the MLL rerrngement. A cler splitting of the signls ws observed using YAC 978e4 s probe in ptients 1, 3 nd 5, suggesting possible rerrngement of AF1q. Similrly, three spots, on norml one nd on the two rerrnged chromosomes, were observed in the metphses of ptient 6 with BAC 767A8, confirming the involvement of AF1q. FISH nlysis ws not performed in other ptients becuse of the shortge of mteril. PCR studies Figure 1 Prtil kryotypes of ptients 1 to 6 (see Tble 1) showing the t(1;11)(q21;q23). R bnds (R) nd G bnds (G). Arrows show the rerrnged chromosomes. CAATGG (MLL exon 7), GGTTCGAAGTCTTGGCCTTA- GAGCA (AF-1qR) corresponding to AF1q sequences. 12 Amplifiction of the frgments ws performed s follows: denturtion ws undertken t 94 C for 5 min in the first cycle, nd for 1 min in the subsequent cycles. Anneling steps were performed for 10 s t 62 C (four cycles), 58 C (four cycles) nd 52 C (30 cycles) nd extension t 72 C for 1 min. Nucleotide sequence ws obtined from commercil source. In order to detect puttive MLL-AF1q fusion trnscripts, we performed PCR nlysis of rndom-primed ptients 5 nd 6 cdna using MLL primers corresponding to exon 5, 6 nd 7, nd AF1q-specific oligonucleotide. As shown in Figure 2, 411 bp frgment ws specificlly mplified from ptient 6 mteril using MLL Ex5 primer (pnel ), 327 bp frgment ws seen using MLL Ex6 (pnel b) nd no mplifiction product using MLL Ex7 primer (pnel c). These result were in good greement with MLL Ex6-AF1q fusion, s previously reported. The frgments mplified using ptient 5 mteril were slight lrger, 543 bp with MLLEx5 primer (pnel ), nd 459 bp using MLL Ex 6 primer (pnel b). In ddition specific 370 bp frgment ws mplified using MLL Ex7 primer (pnel c), indicting tht the fusion could hve occurred downstrem of MLL Ex7. As shown in Figure 3, nucleotide sequence nlysis confirmed n in frme fusion of MLL exon 7 to AF1q, 5 of its puttive trnsltionl strt codon. 12 Discussion The chromosoml bnd 11q23 is frequently rerrnged in mlignnt blood disorders nd MLL gene rerrngements re the most often implicted in cute leukemis. Fusion of MLL with other genes results from vrious chromosoml trnsloctions nd 17 different fusion genes, including MLL selffusion, hve been described in hemtopoietic mlignncies. The incidence of ech trnsloction is vrible due in prt to the reltive difficulty of identifiction with bnding techniques s for exmple, t(6;11)(q27;q23), 20 the pprent vribility of the brekpoints on bnded chromosomes s for t(10;11)(p12;q23), 21 or becuse the incidence of some trnsloctions is relly low. Trnsloction t(1;11)(q21;q23) is n

4 Brief communiction 305 Figure 2 Amplifiction of MLL-AF1q fused trnscripts from t(1;11) ptients. PCR experiments were performed with the sets of primers indicted t the bottom of the figure (see text). The size of the mplified frgment is indicted in bse pirs (bp). The ptient number is indicted t the top of the figure, unrelted cdna (negtive control). Figure 3 Nucleotide sequence of fused MLL-AFIq cdna nd corresponding sequences of norml chromosomes 11 nd 1. Conceptul trnsltion of norml cdna is shown. AF1q sequences pper in lower cse letters. The predicted AF1q trnscriptionl strt codon is underlined. Arrows indicte the fusion point, 3 of MLL exon 7. exmple of such n infrequent rerrngement since only few cses hve previously been reported in cute myeloid nd cute lymphoid leukemi nd myelodysplstic syndrome (Tble 3). Moleculr nlysis of two of these cses, two cute myelomonocytic leukemis in infnts, hs llowed the isoltion of new gene, nmed AF1q, fused with MLL. 12 The AF1q gene encodes 9 kd protein without pprent similrity to ny other protein nd is expressed in norml thymus nd myeloid cells. 12 The presence of n MLL-AF1q fusion gene hs not been described in other t(1;11)(q21;q23) of hemtopoietic mlignncies. The present study confirms the nonrndomness of the specific MLL-AF1q rerrngement s well s the similr mrna fusion pttern s described before. However, the direct proof of MLL involvement in most of the cses previous described is lcking. This involvement in other 1q21 rerrngements of mlignnt prolifertions remins to be studied. Acknowledgements This work ws supported in prt by grnts from The Ligue Ntionle Contre Le Cncer (Comité Ntionl et Comité de Pris), nd the Fondtion Sint-Louis. FS-N is recipient of fellowship from the The Ligue Ntionle Contre Le Cncer. References 1 Bernrd OA, Berger R. Moleculr bsis of 11q23 rerrngements in hemtopoietic mlignnt prolifertions. Genes Chromos Cncer 1995; 13: Young BD, Sh V. Leukemi: the 11q23 prdigm. Cncer Sur 1996; 28: Wring PM, Clery ML. Disruption of homolog of trithorx by 11q23 trnsloctions Leukemogenic nd trnscriptionl implictions. In: Ruscher FJ III, Vogt PK (eds). Chromosoml Trnsloctions nd Oncogenic Trnscription Fctors. Springer: Berlin, 1997, pp Hillion J, le Conit M, Jonveux P, Berger R, Bernrd OA. AF6q21, novel prtner of the MLL gene in t(6;11)(q21;q23), defines forkhed trnscriptionl fctor subfmily. Blood 1997; 90: Borkhrdt A, Bojesen S, Hs OA, Czznig G, Hrbott J, Biondi A, Loncrevic I, Jeger U, Lmpert F. Cloning of trnsloction t(5;11)(q31;q23) identifies new MLL fusion prtner nd the potentil myeloid tumor suppressor gene t 5q31. Blood 1997; 90 (Suppl. I): Id K, Kitbyshi I, Tki M, Noro K, Ymmoto M, Ohki M,

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