Leukemia (2006) 20, 224–229 & 2006 Nature Publishing Group All rights reserved 0887-6924/06 $30.00 www.nature.com/leu ORIGINAL ARTICLE 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 approxi- mately 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.