(P22;Q22) in Acute Myeloid Leukemia Results in Fusion of RUNX1 with the Ubiquitin-Speciﬁc Protease Gene USP42

A novel and cytogenetically cryptic t(7;21)(p22;q22) in acute myeloid leukemia results in fusion of RUNX1 with the ubiquitin-speciﬁc protease gene USP42

K Paulsson1,ANBe´ka´ssy2, T Olofsson3, F Mitelman1, B Johansson1 and I Panagopoulos1

1Department of Clinical Genetics, Lund University Hospital, Lund, Sweden; 2Department of Pediatrics, Lund University Hospital, Lund, Sweden and 3Department of Hematology, Lund University Hospital, Lund, Sweden

Although many of the chromosomal abnormalities in hemato- blastic leukemias (ALLs),3 the t(5;14)(q35;q32), found in logic malignancies are identifiable cytogenetically, some are 10–20% of T-ALLs,3 and the del(4)(q12q12), which is characteristic only detectable using molecular methods. We describe a novel 4 cryptic t(7;21)(p22;q22) in acute myeloid leukemia (AML). FISH, for hypereosinophilic syndrome. Considering the diagnostic, 30RACE, and RT-PCR revealed a fusion involving RUNX1 and prognostic, and biologic impact of leukemia-associated trans- the ubiquitin-specific protease (USP) gene USP42. The genomic locations and fusion genes, it is important to search for other breakpoint was in intron 7 of RUNX1 and intron 1 of USP42. The cryptic rearrangements that may be present in hematologic reciprocal chimera was not detected – neither on the transcrip- malignancies. 0 tional nor on the genomic level – and FISH showed that the 5 The RUNX1 gene (previously AML1, CBFA2), located at part of USP42 was deleted. USP42 maps to a 7p22 region 21q22, was first identified as one of the fusion partners in the characterized by segmental duplications. Notably, 17 kb dupli- 5 cons are present 1 Mb proximal to USP42 and 3 Mb proximal to t(8;21)(q22;q22) in AML-M2. Since then, RUNX1 has been RUNX1; these may be important in the genesis of t(7;21). This is shown to be involved in numerous translocations as well as the second cryptic RUNX1 translocation in hematologic malig- harboring mutations – germ line or acquired – in a substantial nancies and the first in AML. The USPs have not previously proportion of AML cases.6,7 RUNX1 codes for a transcription been reported to be rearranged in leukemias. The cellular factor belonging to the ‘RUNT domain’ gene family, and is a key context in which USP42 is active is unknown, but we here show 8 that it is expressed in normal bone marrow, in primary AMLs, regulator of hematopoiesis. The wild-type RUNX1 forms a and in cancer cell lines. Its involvement in the t(7;21) suggests heterodimer with CBFB – encoded by a gene targeted by the 9 that deregulation of ubiquitin-associated pathways may be inv(16)(p13q22) in AML-M4 – and the active complex pathogenetically important in AML. functions both as a transcriptional activator and as a repressor.8 Leukemia (2006) 20, 224–229. doi:10.1038/sj.leu.2404076; Apart from the t(12;21), which results in an ETV6/RUNX1 fusion published online 15 December 2005 in B-lineage ALL,10 and the recently described t(4;21)(q28;q22), Keywords: acute myeloid leukemia; RUNX1; USP42; cryptic 11 translocation leading to a RUNX1/FGA7 chimera in T-ALL, RUNX1 is typically rearranged through translocations in AML and myelo- dysplastic syndromes (MDSs). To date, five in-frame fusion gene partners have been reported: RUNX1T1 (previously ETO, MTG8, CBFA2T1) at 8q22,12 MDS1/EVI1 at 3q26,13 CBFA2T3 (previously MTG16) at 16q24,14 PRDX4 at Xp22,15 and ZFPM2 Introduction (previously FOG2) at 8q23.16 The translocations resulting in these fusions have all been detectable with G-banding. In the The acquired, clonal chromosomal aberrations frequently found present study, we describe a novel and cytogenetically cryptic in hematologic malignancies are closely associated with t(7;21)(p22;q22) in AML, fusing RUNX1 with the ubiquitin- leukemogenesis. For example, the strong correlations that exist specific protease (USP) gene USP42, which is involved in the between certain balanced rearrangements and specific clinical, ubiquitin pathway.17 This is the second cryptic RUNX1 immunophenotypic, and morphologic patterns strongly indicate translocation described in hematologic malignancies, and the that these fusion gene-generating abnormalities play a major first in AML. It is also the first report of a fusion involving a USP 1 role in the transformation process. However, cytogenetically gene in hematologic malignancies. identifiable chromosomal aberrations are seen only in approximately half of all acute myeloid leukemias (AMLs). This notwithstanding, it has been suggested that primary, balanced Patients, materials, and methods rearrangements – often resulting in chimeric genes – are 2 necessary for malignant hematologic disorders to arise. It Case history should be stressed that the absence of rearrangements does not A previously healthy 7-year-old boy was admitted to the hospital exclude the presence of chromosomal changes because the in March 1995 because of pyrexic tonsillitis and cervical resolution of standard cytogenetics is quite poor. In fact, some adenitis. The clinical examination was unremarkable. The cryptic aberrations, detectable only with molecular methods, peripheral blood values were hemoglobin 102 g/l, white blood have been shown to be quite common, for example, the 9 9 cell count 35.6 Â 10 , with 1.4 Â 10 /l granulocytes and t(12;21)(p13;q22), occurring in 20% of B-lineage acute lympho- 9 24.9 Â 10 atypical monocytic cells, and thrombocytes 69 Â 109/l. The bone marrow (BM) aspirate was hypercellular, Correspondence: Dr K Paulsson, Department of Clinical Genetics, containing blasts difficult to classify morphologically. Flow University Hospital, SE-221 85 Lund, Sweden. E-mail: [email protected] cytometric two-color analysis showed that 75–80% of the BM Received 4 July 2005; revised 10 October 2005; accepted 18 October cells were positive for HLA-DR, CD34, CD33, CD13, CD11c, 2005; published online 15 December 2005 CD7, CD5, and CD4, and partly positive for CD56 but negative Cryptic RUNX1/USP42 fusion in AML K Paulsson et al 225 for B-lymphocytic markers. The diagnosis was AML-M0 with 400 ng of genomic DNA as template, and the amplified aberrant expression of T-lymphocyte-associated markers. The fragments were sequenced (Supplementary Table 2). Attempts chromosome analysis revealed a seemingly normal male were also made to amplify the reciprocal genomic fusion with karyotype. The patient received treatment according to the primer pairs listed in Supplementary Table 2. NOPHO-AML-93 protocol.18 After induction therapy, the BM still contained 25% blasts; three additional chemotherapy blocks were needed to achieve complete remission (CR) (May RT-PCR and Northern blot expression analyses 1995). Allogeneic stem cell transplantation, with an HLA To investigate the expression of USP42, RT-PCR was performed identical sister as a donor, was performed in June 1995. BM (Supplementary Table 2) using cDNA from two normal BM, two relapse occurred in March 2000, at which time the karyotype childhood AMLs (one with a normal karyotype and one with a was 46,XY,t(4;6)(q24;p11),del(5)(q15),t(11;18)(q23;q21)[24]. t(8;21)), and two adult AMLs (one with a complex karyotype and The immunophenotype was essentially the same as at diagnosis. one with an add(7)(p22)) as template. Northern blot analysis was A second CR was achieved with a single cytoreductive performed, utilizing the Human Cancer Cell Line MTN Blot (BD chemotherapy course in April 2000. Then, donor lymphocyte Biosciences Clontech, Palo Alto, CA, USA), containing poly A þ infusions were given on three occasions. The patient is a RNA, on the cancer cell lines HL-60 (AML), HeLa S3, K-562 complete donor chimera, with no signs of the disease on the (CML), MOLT-4 (T-ALL), Raji (Burkitt’s lymphoma), SW480 latest follow-up in April 2005. (colorectal adenocarcinoma), A549 (lung carcinoma), and G-361 (melanoma). A probe for USP42 was obtained by RT-PCR using the primers USP42-127F and USP42-562R (Supplemen- FISH analyses tary Table 2) with K-562 cDNA as template. The probe was The t(7;21) was originally detected with a whole chromosome labeled with 32P using the RediprimeII labelling system paint (WCP) probe for chromosome 7. To characterize the (Amersham, Little Chalfont, UK). translocation, FISH was performed according to standard protocols on BM cells, obtained at diagnosis and relapse. For multicolor-FISH (M-FISH), WCP probes for combined binary Additional cases ratio labeling-FISH were used.19 RUNX1 rearrangements were As the t(7;21)(p22;q22) is not visible with G-banding, we investigated with the LSI AML1/ETO and the LSI TEL/AML1 screened additional cases for the RUNX1/USP42 fusion with RT- probes, covering the whole RUNX1 gene (Vysis, Downers PCR (Supplementary Table 2). These included 25 childhood Grove, IL, USA). BACs and PACs used for mapping of the AMLs with available RNA, regardless of karyotypic features, four chromosome 7 breakpoint were generously supplied by Dr M adult AML-M0 with various chromosome abnormalities, and Rocchi, Bari, Italy (Supplementary Table 1). The fosmids four adult AMLs with 7p aberrations. G248P88689C2 and G248P8048B11, specific for the 50 and 30 parts of USP42, respectively, were also applied (BACPAC Resources, St Oakland, CA, USA). In addition, WCP probes Results for chromosomes 7 and 21 were utilized (Vysis). FISH reveals a translocation of RUNX1 to 7p and narrows down the chromosome 7 breakpoint region 30RACE and gene-specific RT-PCR to 1.2 Mb Total RNA was extracted according to standard methods from The karyotype was normal at the time of diagnosis (Figure 1a); at BM cells, obtained at relapse. First-strand cDNA synthesis was relapse, several abnormalities were identified, none of which performed with the SMART RACE cDNA Amplification kit (BD involved chromosomes 7 or 21. The t(7;21) was first identified Biosciences, San Jose, CA, USA). The GC-RICH PCR System with WCP probes (Figure 1b). M-FISH analysis did not reveal (Roche Diagnostics, Penzberg, Germany) was used for the any additional chromosomal aberrations. FISH with a probe for 30RACE PCR reaction, with the forward primer RUNX1-765F RUNX1 and WCP7 disclosed that a part of this gene was (Supplementary Table 2), specific for exon 4 of RUNX1. The translocated to 7p (Figure 1c). To map the breakpoint on 7p, PCR products were separated by gel electrophoresis, excised BACs and PACs were used (Supplementary Table 1). All BACs/ from the gel, and used as template for a nested PCR reaction PACs proximal to RP11-577O18 hybridized to the der(7), and (Supplementary Table 2). The resulting fragments were se- all distal to RP5-1163J12 to the der(21) (Supplementary Table 1), quenced (Supplementary Table 2) using the BigDye terminator suggesting a 7p22 breakpoint. Analyses of the clones in between v1.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, were difficult to interpret due to the presence of a 100 kb USA) on an ABI PRISM 3100-Avant Genetic Analyzer (Applied inverted duplication in this region,20 involving RP11-577O18 Biosystems). All sequences were tested against reported and RP11-611L7 at 6.6–6.5 Mb and RP5-1163J12 and RP1- sequences (http://www.ncbi.nlm.nih.gov/BLAST/). To confirm 42M2 at 5.7–5.8 Mb from the 7p telomere. Three of these four the RACE findings, nested PCR was performed using forward clones yielded signals on both the der(7) and the der(21), RUNX1-specific primers and reverse USP42-specific primers indicating that the breakpoint was localized between or in near (Supplementary Table 2). In addition, nested PCR was proximity to the duplications (Supplementary Table 1). RP5- performed for the reciprocal fusion transcript (Supplementary 1163J12 hybridized only to the der(7), and may thus have been, Table 2). at least partly, deleted. This was confirmed by use of two fosmids. G248P8048B11, corresponding to the 30 part of USP42, hybridized to the der(7); no signals for the more Genomic fusion telomeric G248P88689C2, specific for the 50 part of this gene, DNA was extracted according to standard methods from BM were detected. The FISH investigations narrowed down the cells, obtained at relapse. To identify the genomic fusion breakpoint region to 1.2 Mb. As the involved sequence breakpoints in RUNX1 and USP42, nested PCR was performed contained more than 20 genes (www.ensembl.org), 30RACE with the GeneAmp XL PCR kit (Applied Biosystems), using was used to detect the fusion partner of RUNX1.

Leukemia Cryptic RUNX1/USP42 fusion in AML K Paulsson et al 226 30RACE and RT-PCR amplifies a RUNX1/USP42 Genomic PCR reveals that the breakpoints occurred fusion mRNA in RUNX1 intron 7 and USP42 intron 1 30RACE was performed with forward primers for RUNX1 The genomic RUNX1/USP42 fusion was amplified, using (Supplementary Table 2) and reverse universal primers. The primers in the surrounding exons (Supplementary Table 2), first PCR resulted in a band of 1.6 kb after gel electrophoresis and sequenced. The analysis disclosed that nucleotide (nt) 4968 (Supplementary Figure 1A), which was used as a template for a of the 6690 bp RUNX1 intron 7 was fused to nt 3006 of the second PCR, resulting in four distinct bands, ranging in size 3948 bp USP42 intron 1, with insertion of one thymine in the from 0.4 to 1.5 kb (Supplementary Figure 1B). The three junction (Supplementary Figure 2B). The genomic breakpoint in shorter fragments were shown to be splice variants of RUNX1. USP42 intron 1 was located in a repetitive LINE/L1 sequence The 1.5 kb fragment contained an in-frame fusion between (http://woody.embl-heidelberg.de/repeatmask/). No homolo- exon 7 of RUNX1 in the 50 position and exon 2 of the USP42 gous sequences were found in the vicinity of the breakpoints. gene in the 30 position (Supplementary Figure 2A). USP42 PCR in order to amplify the reciprocal genomic fusion yielded is present in the PAC RP4-810E6, located at 7p22 between no products. the inverted duplications (Supplementary Table 1). RUNX1 and USP42 are oriented in opposite directions (http:// www.ncbi.nlm.nih.gov/mapview/). Thus, a fusion between USP42 is expressed in normal BM, in primary AMLs, these two genes can occur by a simple translocation between and in eight cancer cell lines 7p and 21q, with the RUNX1/USP42 chimeric mRNA being RT-PCR with USP42-specific primers revealed expression in transcribed from the der(7) locus. RT-PCR with gene-specific normal BM, as well as in four primary AML samples (Figure 2a). primers (Supplementary Table 2) confirmed the RUNX1/USP42 Northern blot analysis of eight cancer cell lines disclosed the fusion, and revealed an additional transcript missing exon 6 of presence of two USP42 transcripts in all cell lines; one 5 kb RUNX1 (Supplementary Figure 1C–D). RT-PCR with primers and one 2 kb (Figure 2b). The first of these may correspond to for the reciprocal fusion did not amplify a USP42/RUNX1 the aAug05 transcript (http://www.ncbi.nih.gov/IEB/Research/ transcript. Acembly/), which is 5093 bp and contains the sequences that

Figure 1 G-banding and FISH ﬁndings. (a) Partial karyotype, showing apparently normal chromosomes 7 and 21. In hindsight, the der(7) is probably to the right and the der(21) to the left. (b) The t(7;21) was discovered using WCP probes for chromosomes 7 (green) and 21 (red). Derivative chromosomes are indicated by arrows. (c)ARUNX1-speciﬁc probe (red) revealed that this gene was translocated to 7p (green). Derivative chromosomes are indicated by arrows. For clarity, the ETV6 probe included in the hybridization is not shown.

Figure 2 Expression analyses of USP42. Transcripts are indicated by arrows. (a) RT-PCR demonstrating expression of USP42 in normal bone marrow and in primary AMLs. M, molecular marker; lanes 1–2, normal bone marrow; lanes 3–6, AMLs; lane 7, K-562; and lane 8, negative control. (b) Northern blot analysis of eight cancer cell lines. Two USP42 transcripts are visible; one approximately 5 kb and one approximately 2 kb.

Leukemia Cryptic RUNX1/USP42 fusion in AML K Paulsson et al 227

Figure 3 Schematic view of retained protein domains in the RUNX1/USP42 chimera. RUNT, RUNT domain; TA, transactivation domain; UCH, ubiquitin carboxyl-terminal hydrolase domain. The fusion protein retains the CBFB- and DNA-binding RUNT domain of RUNX1 and the catalytic UCH domain of USP42. The breakpoint is within the TA domain of RUNX1. hybridize to the USP42 blot probe. The latter transcript may be localization of proteins, and hydrolyze linear and branched a previously not described USP42 splice variant. ubiquitin modifications, including the removal of polyubiquitin from target proteins, thereby saving them from ubiquitin- mediated degradation.17 USP42 contains the conserved cata- Screening for additional AMLs possibly harboring the lytic Cys, His, and Asp/Asn residues that form the catalytic triad cryptic RUNX1/USP42 fusion of the UCH domain of USPs (Figure 3) and can remove ubiquitin Screening for RUNX1/USP42 fusion transcripts with RT-PCR in from Ub-M-b-galactosidase, strongly suggesting that it is a 33 AMLs revealed no additional t(7;21)-positive cases. functional USP.17 The gene is highly expressed in skeletal muscle, liver, and pancreas, more weakly in placenta, brain, and heart, and not at all in ovary, testis, prostate, and thymus.17 Discussion In this study, we show that USP42 is expressed also in normal BM and primary AMLs (Figure 2a). Furthermore, Northern blot We here describe a novel and cytogenetically cryptic t(7;21) analyses revealed USP42 expression in all examined cancer cell (p22;q22) involving the RUNX1 and USP42 genes. This AML lines (Figure 2b). Taken together, USP42 seems to be widely, case had an apparently normal karyotype at diagnosis (Figure 1a); albeit not ubiquitously, expressed in human tissues. the t(7;21) was an unexpected finding while screening pediatric Several members of the USP family have previously been leukemias with FISH for the t(7;12)(q36;p13).21 implicated in tumorigenesis, primarily by being deregulated in FISH analyses to characterize the region surrounding the 7p various neoplasms.24–27 Most notably, Usp18 (previously breakpoint resulted in complex signal patterns (Supplementary Ubp43) levels have been shown to be specifically upregulated Table 1). This was partly due to a 100 kb inverted duplicon in mice expressing the RUNX1/CBFA2T1 chimera,25 and USP33 present on both sides of the actual breakpoint site,20 leading to is overexpressed in B-ALL as compared with T-ALL,26 suggesting crosshybridization of some BACs/PACs (Supplementary Table that deregulation of USPs may be important in leukemogenesis. 1). This crosshybridization was not visible on control slides or Furthermore, a recurrent rearrangement involving USP6 has on the other chromosome 7 homologue in the present case, previously been described in aneurysmal bone cysts (ABC); the most likely because the duplicons were too close (o1 Mb) to be t(16;17)(q22;p13) which places the whole coding sequence of seen as separate signals when residing on the same chromo- USP6 under the transcriptional control of the highly active some. Furthermore, the FISH assays disclosed the presence of CDH11 promotor.27 USP6 is also the 30 part of other fusion noncontinuous deletions in the breakpoint region (Supplemen- genes in ABC, suggesting that its increased expression may be tary Table 1), including deletion of the 50 part of USP42. In line pathogenetically important in this disorder.27 with the latter, we did not detect a reciprocal USP42/RUNX1 RUNX1 is involved in both in-frame and out-of-frame fusions chimera, neither on the transcriptional nor on the genomic level. in myeloid malignancies. All in-frame chimeras have RUNX1 in Deletions surrounding translocation breakpoints have been the 50 position.13–16,28 This results in proteins that retain the reported in several other leukemia-associated rearrangements, CBFB- and DNA-binding RUNT domain but lose the transacti- such as the t(9;22)(q34;q11) in chronic myeloid leukemia vation (TA) domain of RUNX1. Several of these fusions have (CML).22 The 7p region surrounding USP42 is characterized been shown to act as dominant-negative inhibitors of normal by a particularly large number of segmental duplications and RUNX1 function;8 the same is true for some mutations.7 Taken has been described as highly rearrangement-prone.20 Interest- together, there is strong support for a leukemogenic effect ingly, a 17 kb duplicated sequence is present approximately resulting from a dominant-negative function of RUNX1 re- 0.8 Mb proximal to USP42 at 7p22 and 3 Mb proximal to arrangements/mutations. However, gain-of-function is most RUNX1 at 21q22 (http://projects.tcag.ca/humandup/). It is in this likely also involved in translocations. For example, the context noteworthy that a duplicon of 76 kb has previously been RUNX1/CBFA2T1 fusion protein upregulates Usp18 in mice.25 shown to be situated close to BCR and ABL1, the genes involved Like previously described chimeras in myeloid malignancies, in the t(9;22) in CML.23 It is possible that such homologous the t(7;21) places RUNX1 in the 50position, although its intron 7 sequences are of importance in the formation of translocations. breakpoint is 1–2 introns further downstream compared with USP42 contains 25 alternative exons and codes for five these fusions.13–15,28 Thus, the RUNT domain is retained, but protein isoforms (http://www.ncbi.nih.gov/IEB/Research/Acem- the breakpoint is within, rather than 50 to, the TA domain bly/). The encoded protein belongs to the USP family, a large (Figure 3). However, the latter is most likely nonfunctional group of cysteine proteases with variable size and structure and because it is disrupted. Based on previous studies,13–15,28 it with conserved motifs mainly in the catalytic region.17 They are seems likely that the RUNX1/USP42 fusion protein may have a involved in the ubiquitin pathway, which regulates important dominant-negative effect on normal RUNX1 function. Since the functions, such as the activity, degradation, and subcellular present case was an AML-M0, it is also notable that biallelic

Leukemia Cryptic RUNX1/USP42 fusion in AML K Paulsson et al 228 RUNX1 mutations have been reported to be frequent in such 6 Song W-J, Sullivan MG, Legare RD, Hutchings S, Tan X, Kufrin D cases, indicating that the absence of normal RUNX1 may be et al. Haploinsufficiency of CBFA2 causes familial thrombocyto- associated with this particular subtype.29 Furthermore, the penia with propensity to develop acute myelogenous leukaemia. RUNX1/USP42 chimera retains almost the entire USP42 open Nat Genet 1999; 23: 166–175. 7 Osato M. Point mutations in the RUNX1/AML1 gene: another actor reading frame, including the catalytic UCH domain (Figure 3; in RUNX leukemia. Oncogene 2004; 23: 4284–4296. http://www.sanger.ac.uk/Software/Pfam/). Thus, it cannot be 8 Kurokawa M, Hirai H. Role of AML1/Runx1 in the pathogenesis of excluded that the protease is still active. Although the hematological malignancies. Cancer Sci 2003; 94: 841–846. pathogenetic consequences of the t(7;21) resulting from 9 Liu P, Tarle´ SA, Hajra A, Claxton DF, Marlton P, Freedman M et al. USP42 is difficult to interpret, since the specific cellular context Fusion between transcription factor CBFb/PEBP2b and a in which USP42 is active has not been elucidated, the fact that myosin heavy chain in acute myeloid leukemia. Science 1993; 261: 1041–1044. deregulation of several members of the USP family have 10 Golub TR, Barker GF, Bohlander SK, Hiebert SW, Ward DC, Bray- previously been described in neoplasias suggests that USP42 Ward P et al. Fusion of the TEL gene on 12p13 to the AML1 gene 24–27 deregulation may have a leukemogenic effect. RUNX1 has on 21q22 in acute lymphoblastic leukemia. Proc Natl Acad Sci been shown to be subject to ubiquitin–proteasome-mediated USA 1995; 92: 4917–4921. degradation.30 This is interesting considering that some USPs 11 Mikhail FM, Coignet L, Hatem N, Mourad ZI, Farawela HM, El can remove polyubiquitin from target proteins, thereby saving Kaffash DM et al. A novel gene, FGA7, is fused to RUNX1/AML1 in a t(4;21)(q28;q22) in a patient with T-cell acute lymphoblastic them from degradation by the proteasome. It is thus tempting to leukemia. Genes Chromosomes Cancer 2004; 39: 110–118. speculate that fusion with USP42 helps to stabilize the RUNX1 12 Miyoshi H, Kozu T, Shimizu K, Enomoto K, Maseki N, Kaneko Y part of the chimera. This could possibly enhance the putative et al. The t(8;21) translocation in acute myeloid leukemia results in dominant-negative effects of RUNX1/USP42. Taken together, production of an AML1-MTG8 fusion transcript. EMBO J 1993; 12: data from similar fusions and from other USPs in malignant 2715–2721. disorders make it likely that the pathogenetically important 13 Nucifora G, Begy CR, Kobayashi H, Roulston D, Claxton D, outcome of the t(7;21) includes dominant-negative effects of Pedersen-Bjergaard J et al. Consistent intergenic splicing and production of multiple transcripts between AML1 at 21q22 normal RUNX1 function and possibly deregulation of USP42. and unrelated genes at 3q26 in (3;21)(q26;q22) translocations. The present identification of the t(7;21) clearly demonstrates Proc Natl Acad Sci USA 1994; 91: 4004–4008. that lack of cytogenetically detectable chromosomal aberrations 14 Gamou T, Kitamura E, Hosoda F, Shimizu K, Shinohara K, Hayashi does not exclude chimeric genes. Previously, only three truly Y et al. The partner gene of AML1 in t(16;21) myeloid cryptic chromosomal abnormalities resulting in fusion genes malignancies is a novel member of the MTG8 (ETO) family. have been described in AML: the del(11)(q23q23) leading to Blood 1998; 91: 4028–4037. 15 Zhang Y, Emmanuel N, Kamboj G, Chen J, Shurafa M, Van Dyke MLL/ARHGEF12 and MLL/CBL chimeras, respectively,31,32 and 33 DL et al. PRDX4, a member of the peroxiredoxin family, is fused the t(5;11)(q35;p15.5) resulting in a NUP98/NSD1 fusion. to AML1 (RUNX1) in an acute myeloid leukemia patient with However, recent gene expression studies have shown that a t(X;21)(p22;q22). Genes Chromosomes Cancer 2004; 40: AMLs with normal karyotypes display heterogeneous expression 365–370. patterns,34 indicating the presence of many different underlying, 16 Chan EM, Comer EM, Brown FC, Richkind KE, Holmes ML, cryptic primary rearrangements. In the future, molecular Chong BH et al. AML1-FOG2 fusion protein in myelodysplasia. methods such as M-FISH/SKY or array-based comparative Blood 2005; 105: 4523–4526. 17 Quesada V, Dıáz-Perales A, Gutie´rrez-Fernańdez A, Garabaya C, genome hybridization (CGH) may identify more hidden Cal S, Lo´pez-Otıń C. Cloning and enzymatic analysis of 22 abnormalities. novel human ubiquitin-specific proteases. Biochem Biophys Res Commun 2004; 314: 54–62. 18 Lie SO, Abrahamsson J, Clausen N, Forestier E, Hasle H, Hovi L et al. Treatment stratification based on initial in vivo response in Acknowledgements acute myeloid leukaemia in children without Down’s syndrome: results of NOPHO-AML trials. Br J Haematol 2003; 122: 217–225. This study was supported by grants from the Swedish Cancer 19 Tanke HJ, Wiegant J, van Gijlswijk RPM, Bezrookove V, Pattenier Society, the Swedish Children’s Cancer Foundation, and Gunnar H, Heetebrij RJ et al. New strategy for multi-colour fluorescence Nilsson’s Cancer Foundation. in situ hybridisation: COBRA: COmbined Binary RAtio labelling. Eur J Hum Genet 1999; 7: 2–11. 20 Scherer SW, Green ED. Human chromosome 7 circa 2004: a model for structural and functional studies of the human genome. References Hum Mol Genet 2004; 13: R303–R313. 21 Slater RM, v Drunen E, Kroes WG, Weghuis DO, van den Berg E, 1 Rowley JD. Chromosome translocations: dangerous liaisons Smit EM et al. t(7;12)(q36;p13) and t(7;12)(q32;p13) – transloca- revisited. Nat Rev Cancer 2001; 1: 245–250. tions involving ETV6 in children 18 months of age or younger with 2 Johansson B, Mertens F, Mitelman F. Primary vs. secondary myeloid disorders. Leukemia 2001; 15: 915–920. neoplasia-associated chromosomal abnormalities – balanced 22 Kolomietz E, Al-Maghrabi J, Brennan S, Karaskova J, Minkin S, rearrangements vs. genomic imbalances? Genes Chromosomes Lipton J et al. Primary chromosomal rearrangements of leukemia Cancer 1996; 16: 155–163. are frequently accompanied by extensive submicroscopic 3 Johansson B, Mertens F, Mitelman F. Clinical and biological deletions and may lead to altered prognosis. Blood 2001; 97: importance of cytogenetic abnormalities in childhood and adult 3581–3588. acute lymphoblastic leukemia. Ann Med 2004; 36: 492–503. 23 Saglio G, Storlazzi CT, Giugliano E, Surace C, Anelli L, Rege- 4 Gotlib J, Cools J, Malone III JM, Schrier SL, Gilliland DG, Coutre´ Cambrin G et al. A 76-kb duplicon maps close to the BCR gene on SE. The FIP1L1-PDGFRa fusion tyrosine kinase in hypereosino- chromosome 22 and the ABL gene on chromosome 9: possible philic syndrome and chronic eosinophilic leukemia: implications involvement in the genesis of the Philadelphia chromosome for diagnosis, classification, and management. Blood 2004; 103: translocation. Proc Natl Acad Sci USA 2002; 99: 9882–9887. 2879–2891. 24 Graner E, Tang D, Rossi S, Baron A, Migita T, Weinstein LJ et al. 5 Miyoshi H, Shimizu K, Kozu T, Maseki N, Kaneko Y, Ohki M. The isopeptidase USP2a regulates the stability of fatty acid t(8;21) breakpoints on chromosome 21 in acute myeloid leukemia synthase in prostate cancer. Cancer Cell 2004; 5: 253–261. are clustered within a limited region of a single gene, AML1. 25 Liu L-Q, Ilaria R, Kingsley PD, Iwama A, van Etten RA, Palis J et al. Proc Natl Acad Sci USA 1991; 88: 10431–10434. A novel ubiquitin-specific protease, UBP43, cloned from leukemia

Leukemia Cryptic RUNX1/USP42 fusion in AML K Paulsson et al 229 fusion protein AML1-ETO-expressing mice, functions in hemato- 30 Huang G, Shigesada K, Ito K, Wee H-J, Yokomizo T, Ito Y. poietic cell differentiation. Mol Cell Biol 1999; 19: 3029–3038. Dimerization with PEBP2b protects RUNX1/AML1 from ubiquitin– 26 De Pitta` C, Tombolan L, Campo Dell’Orto M, Accordi B, te proteasome-mediated degradation. EMBO J 2001; 20: 723–733. Kronnie G, Romualdi C et al. A leukemia-enriched cDNA 31 Kourlas PJ, Strout MP, Becknell B, Veronese ML, Croce CM, Theil microarray platform identifies new transcripts with relevance to KS et al. Identification of a gene at 11q23 encoding a guanine the biology of pediatric acute lymphoblastic leukemia. Haemato- nucleotide exchange factor: evidence for its fusion with MLL logica 2005; 90: 890–898. in acute myeloid leukemia. Proc Natl Acad Sci USA 2000; 97: 27 Oliveira AM, Perez-Atayde AR, Dal Cin P, Gebhardt MC, Chen C-J, 2145–2150. Neff JR et al. Aneurysmal bone cyst variant translocations upregulate 32 Fu J-F, Hsu J-J, Tang T-C, Shih L-Y. Identification of CBL, a proto- USP6 transcription by promoter swapping with the ZNF9, COL1A1, oncogene at 11q23.3, as a novel MLL fusion partner in a patient TRAP150,andOMD genes. Oncogene 2005; 24: 3419–3426. with de novo acute myeloid leukemia. Genes Chromosomes 28 Roulston D, Espinosa III R, Nucifora G, Larson RA, Le Beau MM, Cancer 2003; 37: 214–219. Rowley JD. CBFA2(AML1) translocations with novel partner 33 Jaju RJ, Fidler C, Haas OA, Strickson AJ, Watkins F, Clark K et al. chromosomes in myeloid leukemias: association with prior A novel gene, NSD1, is fused to NUP98 in the t(5;11)(q35;p15.5) therapy. Blood 1998; 92: 2879–2885. in de novo childhood acute myeloid leukemia. Blood 2001; 98: 29 Preudhomme C, Warot-Loze D, Roumier C, Grardel-Duflos N, 1264–1267. Garand R, Lai JL et al. High incidence of biallelic point mutations 34 Bullinger L, Do¨hner K, Bair E, Fro¨hling S, Schlenk RF, Tibshirani R in the Runt domain of the AML1/PEBP2aB gene in Mo acute et al. Use of gene-expression profiling to identify prognostic myeloid leukemia and in myeloid malignancies with acquired subclasses in adult acute myeloid leukemia. N Engl J Med 2004; trisomy 21. Blood 2000; 96: 2862–2869. 350: 1605–1616.

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