Letters to the Editor 1179 6 Meijerink JP, Raemaekers JM, Mensink EJ. New type of t(14;18) in a 9 Limpens J, Stad R, Vos C, de Vlaam C, de Jong D, non-Hodgkin’s lymphoma provides insight in molecular events in van Ommen GJ et al. Lymphoma-associated translocation early B-cell differentiation. Br J Haematol 1995; 91: 630–639. t(14;18) in blood B cells of normal individuals. Blood 1995; 85: 7 Tsujimoto Y, Gorham J, Cossman J, Jaffe E, Croce CM. The t(14;18) 2528–2536. translocations involved in B-cell neoplasms result 10 Staudt LM. A closer look at follicular lymphoma. N Engl J Med from mistakes in VDJ joining. Science 1985; 229: 1390–1393. 2007; 356: 741–742. 8 Summers KE, Goff LK, Wilson AG, Gupta RK, Lister TA, Fitzgibbon 11 Roulland S, Navarro JM, Grenot P, Milili M, Agopian J, Montpellier J. Frequency of the Bcl-2/IgH rearrangement in normal individuals: B et al. Follicular lymphoma-like B cells in healthy individuals: a implications for the monitoring of disease in patients with follicular novel intermediate step in early lymphomagenesis. J Exp Med lymphoma. J Clin Oncol 2001; 19: 420–424. 2006; 203: 2425–2431.

Ectopic expression of the HLXB9 is associated with an altered nuclear position in t(7;12) leukaemias

Leukemia (2009) 23, 1179–1182; doi:10.1038/leu.2009.15; four out of seven patients with the t(7;12) (patient nos. 1, 2, 5 published online 12 February 2009 and 7) and in none of the patients without the t(7;12) (patients nos. 8–10, Figures 1a and b). There was no evidence for the presence of the reciprocal transcript ETV6–HLXB9 in any The t(7;12) rearrangement involving ETV6 has been found in patient. The NOM1 gene was expressed in two t(7;12) patients Bone-third of acute myeloid leukaemia in early childhood.1,2 In (case nos. 1 and 2) and in one case without the t(7;12) (patient a proportion of these cases, the homeobox gene HLXB9 has no. 10, Figure 1f). There was no evidence for the presence of a been identified as the fusion partner at the transcript level.3 In possible fusion transcript NOM1–ETV6 in any of the analyzed this study, we analyzed 10 paediatric patients with acute patients. myeloid leukaemia. Of these, seven (patient nos. 1–7) carried a A growing number of studies indicate that the higher-order t(7;12)(q36;p13), whereas three patients (nos. 8–10) were used chromatin arrangement is related to gene expression.6 Altera- as controls as they lacked abnormalities of either tions of the nuclear architecture, including localization and or 12 (see Table 1). Expression analysis was carried out on all compaction of large DNA regions, their replication timing and patients to investigate the presence of the specific transcripts the relative expression levels are emerging factors in human ETV6, HLXB9, NOM1, the HLXB9–ETV6 fusion and its diseases, including cancer.7 The aim of this study was to reciprocal ETV6–HLXB9. Furthermore, all patients were studied investigate whether the altered expression pattern of the HLXB9 for the presence of a possible NOM1–ETV6 fusion (for primers, gene would correspond to an altered radial nuclear location of see Supplementary Material). The expression data obtained by the 7q36 fragment (containing HLXB9) translocated on the RT–PCR are reported in Figure 1. The ETV6 gene was expressed der(12). We designed a fluorescence in situ hybridization assay in all patients (data not shown). The HLXB9 gene was expressed that uses combinations of probes specific for the 7q36 and in all seven patients with the t(7;12) (patients nos. 1–7) and in 12p13 regions, and can differentiate the derivative chromo- one of the three patients without the t(7;12) (patient no. 10, somes from the wild-type homologues in the interphase nuclei Figure 1e). The fusion transcript HLXB9–ETV6 was detected in (see Figure 2).

Table 1 Clinical and cytogenetic data of the patients analyzed in this study

Pt Age/sex Disease Karyotype HLXB9–ETV6 Reference fusion

1 3 months/F AML-M0 47,XX,t(7;12)(q36;p13),+19 Positive Present study 2 4 years/F AML 48,XX,t(7;12)(q36;p13),+8,+19 Positive Present study 3 9 months/M AML 48,XY,t(7;12)(q36;p13),+8,+19 Negative Pt 7 in Tosi et al.1 4 5 months/F AML-M1 47,XX,t(7;12)(q36;p13),+19 Negative Pt 3 in Tosi et al.4 Pt 2 in Tosi et al.1 5 3 months/M AML-M0 47,XY,t(7;12)(q36;p13),+19 Positive Pt 5 in Tosi et al.4 Pt 3 in Tosi et al.1 6 7 months/F AML 46,XX,der(7)t(7;12)(q22;p13)del(7)(q22q36) Negative Pt 1 in Tosi et al.1; Tosi et al.5 7 5 months/M AML-M4 48,XY,t(7;12)(q36;p13),+8,+19 Positive Pt 6 in Tosi et al.4 Pt 4 in Tosi et al.1 8 5 months/M AML-M5 16B46,XY,?der(10)(q),add(11)(q23),inc[cp14] Negative Present study 9 8 months/M AML NAa Negative Present study 10 9 months/M AML-M1 45–46,XY,À14,+mar[cp2]/46,XY Negative Present study Abbreviations: AML, acute myeloid leukaemia; F, female; FISH, fluorescence in situ hybridization; M, male; Pt, patient. aThe karyotype was not available for this patient. However interphase FISH has shown a translocation involving the MLL gene and RT–PCR has detected the presence of an MLL–AF9 fusion. Patient 2 has been analyzed at relapse. Patient 6 was reported previously as myelodysplastic syndrome. The revised diagnosis is AML. Patients 8–10 have been used as controls for this study.

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Figure 1 Expression analysis. (a and b) The RT–PCR products obtained using primers specific for the HLXB9–ETV6 fusion (see Supplementary material). Agarose gel electrophoresis show two bands (size: 603 and 503 bp) detected in patients 1, 2, 5 and 7. (c and d) Nucleotide and amino- acid sequences corresponding to the above DNA fragments, respectively. The vertical gray line indicates the junction between the HLXB9 and ETV6 sequences. The marker is FX174/HaeIII (Promega, Southampton, UK). (e) The RT-PCR products obtained using primers specific for HLXB9. The 484-bp band corresponds to an HLXB9 transcript in patients nos. 1–7 and 10. (f) The RT–PCR products obtained using primers specific for NOM1. The 843-bp band corresponds to a NOM1 transcript in patients nos. 1, 2 and 10.

Using an earlier-described procedure,8 we localized the HLXB9 ing as a consequence of a specific chromosomal translocation. gene at the nuclear periphery (see Figure 3, left part), a nuclear In this case, the radial nuclear position of HLXB9 is altered after compartment largely composed of compact, and generally non- juxtaposition to an actively transcribed region. The ETV6 gene, expressed, chromatin. This peripheral localization was consistent that is expressed ubiquitously, is normally localized in the inner in cells of healthy individuals and in leukaemic cells not portion of the nucleus (see Figure 3). On the other hand, HLXB9, harbouring the t(7;12). Our fluorescence in situ hybridization that is not transcriptionally active after the developmental stages experiments on the t(7;12) cell nuclei showed a shift in the radial of embryonic life, is normally localized toward the nuclear position of the translocated HLXB9 allele (in 7q36), from the periphery.8 We have shown here that the translocated HLXB9 nuclear periphery to a more internal nuclear location (compare 7t is brought to a more internal position by its translocation with 7n data in Figure 3). The difference is statistically extremely partner ETV6 in t(7;12) leukaemic cells, in which the HLXB9 significant (Po0.0001). However, the non-translocated HLXB9 expression is activated. allele maintained a peripheral location as observed in the controls The chromosome region that includes ETV6 is shown to be an (see 7n data in Figure 3). active gene-rich area, suggesting that at least some of these From our findings and earlier reports,2 it is apparent that a may be upregulated by positive cis elements. This might position effect mechanism triggers the expression of HLXB9 in mean that the juxtaposition of the HLXB9 gene to the down- the t(7;12) leukaemias. However, this is the first study that stream region of the ETV6 gene causes an ectopic interaction correlates aberrant gene expression with altered gene position- between active cis elements (known to be capable of acting over

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Figure 2 Schematic representation of the regions spanning the chromosome 7 and 12 breakpoints and probes used for fluorescence in situ hybridization (FISH). (a) Ideograms of 7 and 12 with details of the localization of the probes used for FISH. On the left of each chromosome ideogram, the chromosomal sub-bands of the 7q36 and 12p13 bands are indicated. On the right of the ideograms, the cosmid clones used as probes and their position along the genomic sequence are indicated. (b–d) Images of representative nuclei from normal (b) and t(7;12) carrying cells (c and d) hybridized with the 227e6/2G8 (green/red signals) (b and c) and 148a4/179A6 (green/red signals) (d) probe pairs, using previously reported conditions.1 In (c), the probe combination allows the visualization of the der(12) as a dual-colour signal, whereas the single green signal indicates the normal chromosome 7 and the single red signal indicates the normal chromosome 12. In (d), the probe combination allows the visualization of the der(7) as a dual-colour FISH signal, whereas the single red signal indicates the normal chromosome 12 and the single green signals indicate the normal chromosome 7 (slightly larger) and the der(12) (smaller, resulting from the split signal of the 148a4 probe, spanning the breakpoint in 7q36). Nuclei were counter-stained with 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI). Scale bar: 2 mm.

Figure 3 Radial nuclear location of the HLXB9 and ETV6 loci on normal and translocated chromosomes. The radial nuclear location of each locus was defined statistically by the median value (and relative confidence interval, CI) of the 2D nuclear location of the hybridization signals, according to an earlier-described procedure.8 C1, C2 and Pt-8 have been used as controls for the study. C1, and C2 indicate healthy individuals and Pt-8 indicates a patient with leukaemia lacking the t(7;12) rearrangement (see Table 1). Pt-1, Pt-2, Pt-3 and Pt-4 indicate patients with leukaemia carrying the t(7;12) (see Table 1). The healthy controls have been investigated for the following loci: CF14 (mapping at 7q31), D12S158 (mapping at 12p13.3), HLXB9 (mapping at 7q36.3) and ETV6 (mapping at 12p13), whereas the leukaemic controls and the t(7;12) patients were studied for the HLXB9 and ETV6 loci only. 7n indicates the non-translocated HLXB9 allele; 12n indicates the normal ETV6 allele, 7t indicates the HLXB9 allele translocated onto the chromosome 12 and detected as a fusion signal 227e6/2G8, 12t indicates the telomeric end of the ETV6 allele detected as a fusion signal 148a4/179A6 (see Figure 2). Pt-4 was analyzed with the probe combination 227e6/2G8 only, because of the limited amount of cell material available. Median values 40.65 indicate peripheral loci, whereas median values o0.65 indicate internal loci.

Leukemia Letters to the Editor 1182 large distances) and the HLXB9 promoter, both derepressing and Hematology and Oncology, Justus-Liebig University, Giessen, Germany and upregulating its transcription. Activation of the HLXB9 expres- 7 sion through cis elements could occur soon after breakage and MRC Department of Molecular Haematology, Weatherall reunion of the DNA filaments, with concurrent repositioning of Institute of Molecular Medicine, University of Oxford, Oxford, UK the chromatin at the nuclear level. This would render certain E-mail: [email protected] areas of the genome more exposed to the transcription 8These authors contributed equally to this work. machinery, resulting in a stable overexpression of HLXB9. Further work will focus on the study of epigenetic modifications occurring in these translocation cases. This will aim to support References the circumstantial evidence provided here for a position effect mechanism, and will also provide mechanistic information to 1 Tosi S, Hughes J, Biondi A, Scherer SW, Nakabayashi K, Harbott J complement our observations on nuclear positioning and gene et al. Heterogeneity of the 7q36 breakpoints in the t(7;12) involving expression. ETV6 in infant leukemia. Genes Chromosomes Cancer 2003; 38: 191–200. 2 von Bergh AR, van Drunen E, van Wering ER, van Zutven LJ, Acknowledgements Hainmann I, Lonnerholm G et al. High incidence of t(7;12)(q36;p13) in infant AML but not in infant ALL, with a dismal This work was supported in part by the Leukaemia Research Fund outcome and ectopic expression of HLXB9. Genes Chromosomes Cancer 2006; 45: 731–739. UK (EB and ST). The authors would like to thank Dr Joanna 3 Beverloo HB, Panagopoulos I, Isaksson M, van Wering E, van Bridger, CCCB, Brunel University, for valuable comments and Drunen E, deKlein A et al. Fusion of the homeobox gene HLXB9 and critical reading of this manuscript. the ETV6 gene in infant acute myeloid leukemias with the t(7;12)(q36;p13). Cancer Res 2001; 61: 5374–5377. 4 Tosi S, Harbott J, Teigler-schlegel A, Haas OA, Pirc-Danoewinata H, Harrison CJ et al. t(7;12)(q36;p13), a new recurrent translocation E Ballabio1,2,8, CD Cantarella3,8, C Federico3, P Di Mare3,4, involving ETV6 in infant leukemia. Genes Chromosomes Cancer G Hall5, J Harbott6, J Hughes7, S Saccone3 and S Tosi1 2000; 29: 325–332. 1Division of Biosciences, Centre for Cell and Chromosome 5 Tosi S, Giudici G, Mosna G, Harbott J, Specchia G, Grosveld G Biology and Brunel Institute of Cancer Genetics and et al. Identification of new partner chromosomes involved in fusions Pharmacogenomics, Brunel University, West London, UK; with the ETV6 (TEL) gene in haematologic malignancies. Genes 2 Chromosomes Cancer 1998; 21: 223–229. Nuffield Department of Clinical Laboratory Sciences, John 6 Cremer T, Cremer C. Chromosome territories, nuclear architecture and Radcliffe Hospital, Oxford, UK; 3 gene regulation in mammalian cells. Nat Genet 2001; 2: 292–301. Department of Animal Biology ‘M. La Greca’, University of 7 Bickmore WA, van der Maarel SM. Perturbations of chromatin Catania, Catania, Italy; 4 structure in human genetic disease: recent advances. Hum Mol Department of Paediatrics, University of Catania, Genet 2003; 12 (Review issue 2): 207–213. Catania, Italy; 8 Federico C, Cantarella CD, Di Mare P, Tosi S, Saccone S. The radial 5 Paediatric Haematology/Oncology Unit, Oxford Children’s arrangement of the human chromosome 7 in the lymphocyte cell Hospital, John Radcliffe Hospital, Oxford, UK; nucleus is associated with chromosomal band gene density. 6Oncogenetic Laboratory, Department of Pediatric Chromosoma 2008; 117: 399–410.

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

The CLLU1 expression level is a stable and inherent feature of the chronic lymphocytic leukemia clone

Leukemia (2009) 23, 1182–1186; doi:10.1038/leu.2009.16; CLLU1 expression level was shown to be associated with an published online 12 February 2009 increase of 7% in the relative risk of early death.4 Others have since then confirmed the prognostic significance of CLLU1 in Although most leukemias harbor a common genetic aberration, CLL.5,6 The function of the CLLU1 gene is unknown. The gene is useful for the classification and subgrouping of the disease, no not conserved in other species and even though several of the common molecular event leading to chronic lymphocytic CLLU1 splice variants contain a putative open reading frame of leukemia (CLL) has been described. CLLU1 was identified in a 121 amino acids, we have not yet been able to convincingly comparative screen of patients with and without somatic detect expression of the putative CLLU1 in CLL patient hypermutations in the variable region of their immunoglobulin samples. Thus, we do not know whether CLLU1 plays a role in the receptor heavy chain genes (IgVH). The expression of CLLU1 was pathogenesis of CLL, or whether its expression is a reflection of shown to be highly upregulated in patients without somatic other events in the CLL cells. hypermutations. Subsequent analysis showed that CLLU1 up- The goal of this study was to further investigate the biological regulation can be detected only in CLL cells, but not in any other properties of the CLLU1 mRNAs. We first developed specific tissue, cell line or in samples from patients with other QRT-PCR primer sets to measure the relative expression levels of hematological malignancies.1,2 In earlier studies we found CLLU1 the different CLLU1 splice variants in samples from 30 CLL to have independent prognostic significance in CLL.3,4 When patients (Primer and probe sequences are in Supplementary CLLU1 was analyzed as a continuous variable, a doubling of the Table 1.) (Figure 1a). The expression pattern of cDNA4/cDNA5

Leukemia