Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Leukemia (2002) 16, 2417–2422 2002 Nature Publishing Group All rights reserved 0887-6924/02 $25.00 www.nature.com/leu High incidence of Hox11L2 expression in children with T-ALL L Mauvieux1,2, V Leymarie1, C Helias1, N Perrusson2, A Falkenrodt1, B Lioure3, P Lutz4 and M Lessard1 ,2 1 Laboratoire d’Hématologie, Hôpitaux Universitaires de Strasbourg, Strasbourg, France; 2Laboratoire d’Hématologie Cellulaire, Université Louis Pasteur, CNRS EA 3431, Strasbourg, France; 3Département d’Onco-hématologie, Hôpital Hautepierre, Strasbourg, France; and 4Service d’Onco-hématologie Pédiatrique, Hôpital Hautepierre, Strasbourg, France The orphan homeobox gene HOX11L2 was previously found to be transcriptionally activated as a result of the t(5;14)(q35;q32) translocation in three T-ALL cases. We now tested by RT-PCR Hox11L2 expression in 23 consecutive cases of T-ALL (15 children aged 0.8–14 years, eight adults aged 17–55 years) and as control 13 B-ALL patients from a single institution. Hox11L2 expression was undetectable in all patients with B-ALL, nor in adults with T-ALL. Nine children (60% of the cases), all boys, expressed Hox11L2. Blast cells from most of the latter patients carried surface CD1a, CD10 and not CD34 antigens, in contrast to the other children. FISH , M-FISH and IPM-FISH analysis failed to detect a t(5;14)(q35;q32) in one of them, which suggests a possible distinct genetic mechanism in Hox11L2 expression induction. Hence, Hox11L2 expression seems to be the most frequent abnormality in childhood T-ALL to date, comparable to the t(12;21) in child B-ALL. Leukemia (2002) 16, 2417–2422. doi:10.1038/sj.leu.2402709 Keywords: T-ALL; childhood; Hox11L2; t(5;14)(q35;q32); FISH Introduction Recurrent chromosomal abnormalities are detectable in about 50% of T-ALL using conventional cytogenetics,1 and can be divided into two main categories: deletions and translocations. Detectable deletions, as for example 9p deletions,2 lead to inactivation of the tumor suppressing genes p15 and p16, or invisible deletions on the short arm chromosome 1 activate the transcriptional regulatory factor Tal1.3 Translocations carry regulatory sequences of TCR genes close to target genes, leading to their inappropriate expression during T cell development. This is notably the case for t(11;14)(p13;q11), which juxtaposes LMO2 and TCR␣/␦ or TCR,4,5 t(10;14)(q24;q11), in which the homeoprotein gene Hox11 is placed under the control of the TCR␣/␦ gene,6,7 and t(1;14)(p32;q11) which joins TAL1 with TCR segments on der(14).8 Conventional cytogenetic techniques fail to identify all abnormalities, due to the frequent poor quality of chromosome banding in T cell proliferations, which display a ‘normal karyotype’. Innovating techniques (FISH, multi-FISH and spectral karyotype) have been of considerable interest in analysis of those difficult cases. Recently, we observed a new recurrent cryptic translocation in about 22% of T-ALL in the first series described,9,10 the t(5;14)(q35;q32). This translocation, cryptic using the conventional cytogenetics (R or G banding), is only detectable by FISH and/or multi-FISH analysis, correlated with the expression of the developmental gene HOX11L2, which is localized closed to breakpoints on chromosome 5. This transcriptional regulator, closely related to Hox11, is critical for the development of the ventral medullary respiratory center, Correspondence: M Lessard, Laboratoire d’Hématologie, Hôpital Hautepierre, Avenue Molière, 67085 Strasbourg Cedex, France; Fax: (33) (0)3 88 12 75 55 The first two authors participated equally in this work Received 25 March 2002; accepted 24 June 2002 and its deficiency in KO mice results in a syndrome resembling congenital central hypoventilation.11 As Hox11L2 expression in T-ALL is thought be the specific consequence of t(5;14)(q35;q32) translocation, we analyzed Hox11L2 expression by RT-PCR in a panel of consecutive T-ALL with available frozen material, in order to evaluate its incidence (independently of cytogenetic data), and the possible correlations with immunophenotype data. Materials and methods Patients Cryopreserved material was obtained after informed consent in 23 consecutive (from 1994 to 2001) T-ALL patients followed-up in Hôpital Hautepierre, Strasbourg, France and retrospectively studied. Eight were adults (17–55 years old, six male, two female), and 15 were children: 11 male, four female, aged from 10 months to 14 years. Diagnosis of T-ALL was made according to the morphological and cytochemical criteria of the French–American–British classification and by immunophenotyping. All children were included in EORTC therapeutic trials. Three patients (Nos 2, 3 and 7) have already been described.9 The control group consisted of 13 B-ALL (three adults and 10 children). Cytogenetics Cytogenetic studies were performed on bone marrow or on blood blast cells if bone marrow was not available. Cells were cultured using three different modes: a 17 h overnight incubation with Colcemid at low concentration (10 g/10ml of RPMI 1640 medium), and 24 h and 48 h cultures with FRDU synchronization in order to improve the quality of the banding. RHG banding techniques were applied in every case.12 Each case was analyzed using a dual-color chromosome paint, following the instructions of the manufacturer (DNACoat; Appligene Oncor, Illkirch, France). Chromosome 5 probe was labeled with Spectrum Green (SG) and chromosome 14 probe was labeled with Spectrum Orange (SO). In order to confirm and precisely define the breakpoints, unique sequence probes were also hybridized: YAC 885A6 (5q35)(CEPH library), BAC 45L16 and BAC 546B8 (5q35) (kindly provided by R Berger, U434 INSERM-CEPH, Paris, France) and IgH (14q32)(LSI IGHc/IGHv dual-color break apart probe, Vysis, Voisins le Bretonneux, France). YAC 885A6, BAC 45L16 and BAC 546B8 were amplified by AluPCR, labeled with digoxigenin and detected with a rhodamine anti-digoxigenin antibody. IgH probe (14q32) was used according to the manufacturer’s instructions. The first three cases were identified using IPM-FISH and published elsewhere (for details see Refs 9 and 13). In all cases, at least 20 mitosis were studied. Patient No. 8 was further studied using Hox11L2 expression in children T-ALL L Mauvieux et al 2418 g of RNA was subsequently reverse transcribed using SuperscriptII reverse transcriptase. The resulting cDNA was PCR amplified (30 cycles, denaturation at 94ºC during 30 s, annealing at 60ºC during 30 s, extension at 72ºC during 30 s), using Hox11L2 primers derived from an already published report10 (No. 005: 5′-GCGCATCGGCCACCCCTACCAGA-3′, No. 006 5′-CCGCTCCGCCTCCCGCTCCTC-3′). Specific amplification of Hox11L2 was confirmed by direct sequencing of PCR products with internal primers: No. 011: 5′AACCGGACGCCGCCCAAGCG-3′ and No. 012: 5′GCCTCCCGCTCCTCCGCCGTCT-3′, using dRhodamine Dye Terminator Mix on ABI 3100 sequencing apparatus. The expression of Hox11L2 was assayed in the control group of 13 B-ALL by RT-PCR, from previously extracted total RNA. cDNA quality was assessed for T-ALL and B-ALL samples using -actin primers (UA 5′-ATCATGTTTGAGACCTTCAA3′, AL 5′-CATCTCTTGCTCGAAGTCCA-3′). interphase nuclei FISH in 250 nuclei using YAC 885A6 probe and in 300 nuclei using BAC45L16 and BAC 546B8. For image acquisition of dual-color and multi-FISH, an epifluorescence microscope Leica DMR-XA (Leica Microsystemes, Rueil-Malmaison, France) fitted with Leica special filters was used, according to Speicher et al14 and Eils et al.15 Immunophenotyping At diagnosis, mononuclear cell fractions containing more than 90% leukemic cells were isolated from blood and/or bone marrow samples by centrifugation on Ficoll–Hypaque. Surface and intracytoplasmic antigens were detected by flow cytometry (FACScan or FACScalibur, Becton Dickinson, Le Pont de Claix, France) using labeled specific monoclonal antibodies (Table 1) and negative isotypic controls. Immunophenotypic features of the patients’ blast cells were classified using the European Group for the Immunological Characterization of Leukemias (EGIL) recommendations.16 Briefly, EGIL T-I (proT ALL) was defined by the presence of CD7, T-II (pre-T ALL) by that of CD2 and/or CD5 and/or CD8, T-III (cortical T-ALL) by CD1a, and T-IV (mature T-ALL) by surface CD3 and lack of CD1a. Bi-phenotypic ALL was defined according to the EGIL scoring system based on the expression of myeloid and lymphoid antigens.16 Statistical analysis Statistical analysis was performed using Fischer’s exact test on Instat software package (GraphPad Software, San Diego, CA, USA). Survival curves and Mann–Whitney non-parametric test were performed on Prism software (GraphPad Software). Molecular studies Results HOX11L2 expression was studied in the 23 T-ALL cases using RT-PCR. Briefly, total RNA was extracted from cryopreserved samples which contained more than 90% of blast cells (from blood sample in six cases, from bone marrow in the remaining 17 cases) using Tri-reagent. RNA quality was assessed by transillumination under UV after agarose electrophoresis. One Table 1 No. Children 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Adults 16 17 18 19 20 21 22 23 Hox11L2 expression in T-ALL Hox11L2 expression was undetectable in the nine adult T-ALL patients under study (Table 1). Conversely, blast cells from nine of the 15 children analyzed expressed Hox11L2, as Immunophenotype of 23 patients with T-ALL Sex Age EGIL CD1a CD4 CD8 CD10 CD34 CD2 CD3 CD5 CD7 TCR␣ TCR␥␦ cCD3 cTCR␣ cTCR␥␦ Hox11L2 M M M M M M M M M M M F F F F 1.5 7 6 8 4 13 7 5 5 7 11 14 2 0.8 6 III III III III III IV III III II II IVb III IV II IVb 21 87 93 83 90 0 79 95 10 0 0 48 0 0 0 84 87 68 63 3 5 85 79 0 85 5 3 3 9 5 0 91 83 92 14 8 96 95 0 1 2 72 18 23 66 88 3 75 90 0 0 77 90 0 0 0 0 10 0 0 57 4 0 0 0 0 5 0 25 81 89 42 ND ND 2 0 91 96 90 94 98 97 96 94 90 5 98 65 0 78 10 9 6 24 6 85 96 0 0 3 95 93 89 0 78 97 92 89 92 85 92 97 85 86 89 94 93 94 92 71 96 94 98 93 95 93 97 96 92 91 89 92 90 95 75 ND 5 5 ND 0 0 ND 0 ND ND 7 14 ND ND 5 ND 1 1 ND 0 0 ND 0 ND ND 91 0 ND ND 60 98 93 96 98 96 96 98 95 97 95 ND 98 ND 97 79 ND 34 57 0 ND ND 0 59 ND ND ND 99 ND ND ND ND 1 0 0 ND ND 38 5 ND ND ND 0 ND ND ND + + + + + + + + + − − − − − − F F M M M M M M 21 55 21 23 41 24 17 25 III II III bi-P III III NA III 69 15 30 8 95 68 ND 72 4 1 7 2 86 1 34 2 4 84 2 38 97 16 56 96 6 0 93 60 96 47 ND 43 15 70 0 6 0 0 ND 0 15 2 1 6 96 2 97 96 8 2 1 21 0 7 87 82 77 94 93 74 96 99 96 72 83 94 95 80 81 96 95 96 ND ND ND ND ND 1 55 72 ND ND ND ND ND 0 0 0 50 95 91 89 24 98 ND 99 9 ND ND ND ND 62 ND 84 0 ND ND ND ND 0 ND 0 − − − − TdT, terminal deoxynucleotide transferase; cCD3 or cTCR, cytoplamic CD3 or TCR; NA, not applicable; ND, not done. Leukemia − − − − Hox11L2 expression in children T-ALL L Mauvieux et al shown for five patients in Figure 1. Sequencing of PCR products proved the correct amplification of Hox11L2 mRNA. Hence, blast cells from 82% of boys in this short series (and none of the four girls) displayed Hox11L2 expression. In the five HOX11L2-positive children, in whom appropriate material was available for cytogenetic studies, no chromosomal abnormality was observed using conventional cytogenetic analysis (data not shown). Identification of a cryptic t(5;14)(q35;q32) in three patients (patient Nos 2, 3, 7)9 correlated with Hox11L2 expression by RT-PCR, in contrast to patient No. 8, in whom FISH (using BAC 45L16, BAC 546B8 and YAC885A6) and multi-FISH failed to detect the translocation. In the control B-ALL group, Hox11L2 expression was undetectable in all three adults and 10 children studied (data not shown), in agreement with the previously reported absence of t(5;14)(q35;q32) by FISH analysis in 10 B-ALL cases.10 Immunophenotype Proliferating cells from the T-ALL patients could be assigned to subgroup T-II of the EGIL classification in five cases, T-III in 13 cases and T-IV in three cases. Both T lymphocytic and myeloid markers were displayed in one case (No. 23), which was classified as bi-phenotypic. In another case (No. 22), CD1a was not tested, and was not classified. Hence, not surprisingly,17 the subgroup T-III (which is defined by the expression of CD1a regardless of the presence of other T cell markers including membrane CD3) predominated (57% of cases). In children (and not in adults), Hox11L2 expression by RT-PCR correlated with that of CD1a (P = 0.04) and CD10 (P = 0.044) on the blast cell surface. In five of the nine cases with Hox11L2 expression, the blast cells were double-positive (DP) for CD4 and CD8 (Table 2). They expressed CD10 in four of five cases and not CD34, which suggests that a differentiation arrest could have occurred at the DP stage (and earlier in patient No. 1 (CD34- and CD4-positive) and No. 5 and No. 6 (double-negative (DN)). Altogether, staining for CD34 was (weakly) positive in two of nine patients with Hox11L2 expression and CD10 was detected in five of nine cases. Conversely, among patients with no detectable Hox11L2 mRNA, Figure 1 RT-PCR analysis of HOX11L2 expression in T-ALL samples. (a) a specific HOX11L2 fragment amplified from several samples. H20: no template control. (b) Beta-actin control amplification. staining for CD10 was consistently negative, no blast cells were DP and CD34 was present in three of the four cases studied for this marker (Table 2). None of the other T cell markers was discriminative with respect to Hox11L2 positivity (Table 1). In adult patients, a common CD1a expression (by most cells in four patients, half of them in one and a minority in the other three patients studied) did not correlate with Hox11L2 expression (undetectable). CD10 was commonly detected and the DP phenotype was rare. As in children, CD10 antigenpositive blast cells of adults were CD34 negative. 2419 Clinical evolution of children and HOX11L2 expression All 15 children with T-ALL were included in EORTC protocols and followed BFM treatments, except one infant patient (No. 14) who followed an infant pilot protocol. Six were classified as intermediate risk, and nine as very high risk (VHR) following EORTC recommendations. HOX11L2 expression did not correlate with age (Hox11L2-positive cases: median age 6 years, HOX11L2-negative cases: median age 6.5 years, P = 0.90), nor white blood cell count at diagnosis (P = 0.688). Two deaths were observed. One toxic death occurred in first remission (case No. 3, Hox11L2 positive), and the other death (case No. 13, Hox11L2 negative) occurred during first relapse, 1 year after autograft. All other patients are alive, and remain in clinical remission. As depicted in Figure 2, survivals were not significantly different in the two groups (P = 0.9, log-rank test, median follow-up 25.5 months), in contrast with a previous report.18 Nevertheless, HOX11L2 expression strongly correlated with male sex (P = 0.011, Fischer’s exact test), but was not very different from the male to female ratio generally observed in T-ALL (P ⬎ 0.05). Discussion We initially reported three cases of T-ALL with t(5;14)(q35;q32) and expression of the developmental gene Hox11L2.10 Hox11L2 belongs to a distinct family of orphan homeobox genes including HOX11, HOX11L1 and HOX11L2. These three genes harbor a threonine in the third helix of the homeodomain, which confers specific DNA binding properties.19,20 Ectopic expression of HOX11 in T-ALL is associated with translocations implicating TCR genes (t(10;14) and t(7;10) for TCR ␣ and TCR , respectively).6,20 In T-ALL with t(5;14)(q35;q32), HOX11L2 expression is probably under the influence of the CTIP2 (BCL11B) gene,10 located on chromosome 14, several hundreds of kilobases centromeric to the main breakpoint area. The latter gene is strongly expressed in the thymus,10 and is potentially implicated in T cell differentiation. CTIP2(BCL11B) is closely related to CTIP1 (BCL11A),21 and both are structurally related to the EVI9 oncogene.22 We now extend our previous findings and show that blast cells from nine of 15 children, all boys (82% of boys), and no adult T-ALL patients expressed Hox11L2 at the mRNA level. In all previously described cases, there was only one female with T-ALL who expressed Hox11L2,10 suggesting a clear correlation with male sex, that remains unexplained. However, in our series, sex ratio of HOX11L2-positive cases was not statistically different from that generally observed in T-ALL (male/female = 3).18 Although FISH study could be performed only in a few Leukemia Hox11L2 expression in children T-ALL L Mauvieux et al 2420 Table 2 Summary of children’s characteristics No. Sex Age WBC (× 109/l) CD1a CD4/CD8 CD10 CD34 Hox11L2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 M M M M M M M M M M M F F F F 1.5 7 6 8 4 13 7 5 5 7 11 14 2 0.8 6 68 38 77 113 146 208 51 9 848 307 4 117 257 540 248 + + + + + − + + − − − + − − − CD4SP DP DP DP DN DN DP DP DN CD4SP DN CD8SP (CD8)SP (CD8)SP CD8SP + − + + − − + + − − − − − − − + −a − − − − − − − + −a + + + ND ND − + + + + + + + + + − − − − − − FISH + + + − − a Weakly positive. WBC, white blood cells count at diagnosis; SP, single positive; DP, double positive; DN, double negative; (CD8), CD8 expression by a minority of the cells. Figure 2 patients. Survival curves of HOX11L2-positive and -negative cases, patient No. 8 illustrates the possibility of Hox11L2 expression in the absence of t(5;14)(q35;q32). Hox11L2 expression was confirmed in another sample of this patient’s blast cells, and cytogenetic analysis was also confirmed in several ways (5q35 BAC45L16, YAC885A6 and YAC546B8 FISH probes, multi-FISH (24 Xcyte probes, Metasysystems) and IPM FISH (IRS-PCR DNA probes). Chromosome 5q35 interphase nuclei FISH (YAC 885A6, BAC45L16 and BAC 546B8) did not detect any translocation in this patient, and no abnormality could be detected using conventional cytogenetics. Hence, Hox11L2 activation might occur independently of the t(5;14) translocation, as previously reported for HOX11, possibly expressed without cytogenetically visible rearrangements of chromosome 10.7,10 Another unknown oncogenic event or submicroscopic abnormalities, different from the cryptic t(5;14), might also occur in chromosome 5 (microdeletions, submicroscopic inversions) and lead to the transcriptional activation of the gene. The second hypothesis was documented for Tal-1 gene.3,23,24 Its deregulation resulted from a cytogenetically undetectable interstitial deletion of chromosome 1.25,26 Such discrete abnormalities might juxtapose Hox11L2 and potentially activate sequences, which may Leukemia be numerous in band 5q32, in view of the large number of active genes in this genomic region. Ongoing molecular characterization of chromosomal breakpoints may pinpoint the mechanisms implicated in Hox11L2 expression. In our series, neither t(5;14)(q35:q32) nor Hox11L2 expression was associated with other cytogenetic abnormalities. The high incidence of chromosomal deletions in T-ALL (probably secondary abnormalities) suggests that previously published cases displaying a chromosomal deletion may harbor the cryptic t(5;14)(q35:q32). We observed a correlation between Hox11L2 gene activation and CD1a (and usually CD10) expression, and an inverse one with CD34. Although the number of patients under study is small, it is quite striking that blast cells from the only two boys in whom Hox11L2 mRNA was undetectable carried CD34, as this is not expected in T-ALL.27 This is in agreement with previous reports on an inverse correlation between CD34 and CD10 in T-ALL.28 In the normal pediatric thymus, the expression of CD1a and CD10 increases concomitantly with the loss of CD34 during thymocyte maturation.29 Physiologically, CD1a expression is restricted to cortical thymocytes, where TCR rearrangements take place. Illegitimate rearrangements in CD34−CD1a+ CD10+ DP thymocytes may juxtapose Hox11L2 and activating sequences, leading to T-ALL. Cloning of chromosomal breakpoints is required to search for an implication of recombinasespecific sequences in the translocation process. If HOX11L2 expression correlated strongly with male sex, no association was observed with age, white blood cell count at diagnosis and, more importantly, with outcome. These observations contrast with previous reports,18 where HOX11L2 expression was associated with poor survival. Moreover, incidence of HOX11L2 expression was lower in this study. These discrepancies may be explained by the age of the patients (not detailed in Ref 18), and the different therapeutic protocol used. In our study, 13/15 children are alive in clinical remission, without any relapse. These therapeutic results prevent any statistically relevant observation about the HOX11L2 role in clinical outcome. From a practical point of view, the finding of DP CD1a and CD10-positive blast cells appears to be predictive of Hox11L2 expression in childhood T-ALL. Further studies are necessary Hox11L2 expression in children T-ALL L Mauvieux et al to evaluate the respective incidence of t(5;14)(q35;q32) and Hox11L2 expressions, which appear to occur independently in certain cases. It will require the use of several methods. Indeed, spectral karyotypic analysis of a T-ALL series failed to detect the cryptic t(5;14)(q35;q32) in childhood T-ALL.30 It is worth mentioning that HOX11L2 was not represented on the microarrays used in T-ALL studies,18,31–33 stressing the limits of microarray diagnostic strategies. Cytogenetic, molecular and immunological studies should be combined in larger series, in order to confirm the correlation between cytogenetic abnormalities, phenotype and Hox11L2 expression, and to specify its prognostic significance. Acknowledgements We thank J-L Preud’homme for critical reading of the manuscript, M-P Gaub for providing material for B-ALL patients, JP Bergerat for adult patients material, D Cherif and J AurichCosta for providing IPM-FISH chromosomal probes. References 1 Schneider NR, Carroll AJ, Shuster JJ, Pullen DJ, Link MP, Borowitz MJ, Camitta BM, Katz JA, Amylon MD. New recurring cytogenetic abnormalities and association of blast cell karyotypes with prognosis in childhood T-cell acute lymphoblastic leukemia: a Pediatric Oncology Group report of 343 cases. Blood 2000; 96: 2543–2549. 2 Heerema NA, Sather HN, Sensel MG, Liu-Mares W, Lange BJ, Bostrom BC, Nachman JB, Steinherz PG, Hutchinson R, Gaynon PS, Arthur DC, Uckun FM. Association of chromosome arm 9p abnormalities with adverse risk in childhood acute lymphoblastic leukemia: a report from the Children’s Cancer Group. Blood 1999; 94: 1537–1544. 3 Delabesse E, Bernard M, Landman-Parker J, Davi F, Leboeuf D, Varet B, Valensi F, Macintyre EA. Simultaneous SIL-TAL1 RT-PCR detection of all tal(d) deletions and identification of novel tal(d) variants. Br J Haematol 1997; 99: 901–907. 4 Dong WF, Billia F, Atkins HL, Iscove NN, Minden MD. Expression of rhombotin 2 in normal and leukaemic haemopoietic cells. Br J Haematol 1996; 93: 280–286. 5 Lowsky R, DeCoteau JF, Reitmair AH, Ichinohasama R, Dong WF, Xu Y, Mak TW, Kadin ME, Minden MD. Defects of the mismatch repair gene MSH2 are implicated in the development of murine and human lymphoblastic lymphomas and are associated with the aberrant expression of rhombotin-2 (Lmo-2) and Tal-1 (SCL). Blood 1997; 89: 2276–2282. 6 Hatano M, Roberts CW, Minden M, Crist WM, Korsmeyer SJ. Deregulation of a homeobox gene, HOX11, by the t(10;14) in T cell leukemia. Science 1991; 253: 79–82. 7 Salvati PD, Ranford PR, Ford J, Kees UR. HOX11 expression in pediatric acute lymphoblastic leukemia is associated with T-cell phenotype. Oncogene 1995; 11: 1333–1338. 8 Bash RO, Crist WM, Shuster JJ, Link MP, Amylon M, Pullen J, Carroll AJ, Buchanan GR, Smith RG, Baer R. Clinical features and outcome of T-cell acute lymphoblastic leukemia in childhood with respect to alterations at the TAL1 locus: a Pediatric Oncology Group study. Blood 1993; 81: 2110–2117. 9 Helias C, Leymarie V, Entz-Werle N, Falkenrodt A, Eyer D, AurichCosta J, Cherif D, Lutz P, Lessard M. Translocation t(5;14)(q35;q32) in 3 cases of childhood T-cell acute lymphoblastic leukemia: a new recurring and cryptic abnormality. Leukemia 2002; 16: 7–12. 10 Bernard OA, Busson-LeConiat M, Ballerini P, Mauchauffe M, Della Valle V, Monni R, Nguyen Khac F, Mercher T, Penard-Lacronique V, Pasturaud P, Gressin L, Heilig R, Daniel MT, Lessard M, Berger R. A new recurrent and specific cryptic translocation, t(5;14)(q35;q32), is associated with expression of the Hox11L2 gene in T acute lymphoblastic leukemia. Leukemia 2001; 15: 1495–1504. 11 Shirasawa S, Arata A, Onimaru H, Roth KA, Brown GA, Horning S, Arata S, Okumura K, Sasazuki T, Korsmeyer SJ. Rnx deficiency results in congenital central hypoventilation. Nat Genet 2000; 24: 287–290. 12 Webber LM, Garson OM. Fluorodeoxyuridine synchronization of bone marrow cultures. Cancer Genet Cytogenet 1983; 8: 123– 132. 13 Aurich-Costa J, Vannier A, Gregoire E, Nowak F, Cherif D. IPMFISH, a new M-FISH approach using IRS-PCR painting probes: application to the analysis of seven human prostate cell lines. Genes Chromosomes Cancer 2001; 30: 143–160. 14 Speicher MR, Gwyn Ballard S, Ward DC. Karyotyping human chromosomes by combinatorial multi-fluor FISH. Nat Genet 1996; 12: 368–375. 15 Eils R, Uhrig S, Saracoglu K, Satzler K, Bolzer A, Petersen I, Chassery J, Ganser M, Speicher MR. An optimized, fully automated system for fast and accurate identification of chromosomal rearrangements by multiplex-FISH (M-FISH). Cytogenet Cell Genet 1998; 82: 160–171. 16 Bene MC, Castoldi G, Knapp W, Ludwig WD, Matutes E, Orfao A, van’t Veer MB. Proposals for the immunological classification of acute leukemias. European Group for the Immunological Characterization of Leukemias (EGIL). Leukemia 1995; 9: 1783– 1786. 17 Crist WM, Shuster JJ, Falletta J, Pullen DJ, Berard CW, Vietti TJ, Alvarado CS, Roper MA, Prasthofer E, Grossi CE. Clinical features and outcome in childhood T-cell leukemia-lymphoma according to stage of thymocyte differentiation: a Pediatric Oncology Group Study. Blood 1988; 72: 1891–1897. 18 Ferrando AA, Neuberg DS, Staunton J, LOH ML, Huard C, Raimondi SC, Behm MG, Pui C-H, Downing JR, Gilliland DG, Lander ES, Golub TR, Look AT. Gene expression signatures define novel oncogenic pathways in T cell acute lymphoblastic leukemia. Cancer Cell 2002; 1: 75–87. 19 Dear TN, Sanchez-Garcia I, Rabbitts TH. The HOX11 gene encodes a DNA-binding nuclear transcription factor belonging to a distinct family of homeobox genes. Proc Natl Acad Sci USA 1993; 90: 4431–4435. 20 Kennedy MA, Gonzalez-Sarmiento R, Kees UR, Lampert F, Dear N, Boehm T, Rabbitts TH. HOX11, a homeobox-containing T-cell oncogene on human chromosome 10q24. Proc Natl Acad Sci USA 1991; 88: 8900–8904. 21 Avram D, Fields A, Pretty On Top K, Nevrivy DJ, Ishmael JE, Leid M. Isolation of a novel family of C(2)H(2) zinc finger proteins implicated in transcriptional repression mediated by chicken ovalbumin upstream promoter transcription factor (COUP-TF) orphan nuclear receptors. J Biol Chem 2000; 275: 10315–10322. 22 Satterwhite E, Sonoki T, Willis TG, Harder L, Nowak R, Arriola EL, Liu H, Price HP, Gesk S, Steinemann D, Schlegelberger B, Oscier DG, Siebert R, Tucker PW, Dyer MJ. The BCL11 gene family: involvement of BCL11A in lymphoid malignancies. Blood 2001; 98: 3413–3420. 23 Breit TM, Beishuizen A, Ludwig WD, Mol EJ, Adriaansen HJ, van Wering ER, van Dongen JJ. tal-1 deletions in T-cell acute lymphoblastic leukemia as PCR target for detection of minimal residual disease. Leukemia 1993; 7: 2004–2011. 24 Borkhardt A, Repp R, Harbott J, Keller C, Berner F, Ritterbach J, Lampert F. Frequency and DNA sequence of tal-1 rearrangement in children with T-cell acute lymphoblastic leukemia. Ann Hematol 1992; 64: 305–308. 25 Francois S, Delabesse E, Baranger L, Dautel M, Foussard C, Boasson M, Blanchet O, Bernard O, Macintyre EA, Ifrah N. Deregulated expression of the TAL1 gene by t(1;5)(p32;31) in patient with T-cell acute lymphoblastic leukemia. Genes Chromosomes Cancer 1998; 23: 36–43. 26 Stock W, Westbrook CA, Sher DA, Dodge R, Sobol RE, WursterHill D, Davey FR, Larson RA, LeBeau MM, Aplan PD, Frankel SR, Stewart CC, Bloomfield CD. Low incidence of TAL1 gene rearrangements in adult acute lymphoblastic leukemia: A cancer and leukemia group B study (8762). Clin Cancer Res 1995; 1: 459–463. 27 Cascavilla N, Musto P, D’Arena G, Ladogana S, Matera R, Carotenuto M. Adult and childhood acute lymphoblastic leukemia: clinico–biological differences based on CD34 antigen expression. Haematologica 1997; 82: 31–37. 2421 Leukemia Hox11L2 expression in children T-ALL L Mauvieux et al 2422 Leukemia 28 Thomas X, Archimbaud E, Charrin C, Magaud JP, Fiere D. CD34 expression is associated with major adverse prognostic factors in adult acute lymphoblastic leukemia. Leukemia 1995; 9: 249–253. 29 Terstappen LW, Huang S, Picker LJ. Flow cytometric assessment of human T-cell differentiation in thymus and bone marrow. Blood 1992; 79: 666–677. 30 Rowley JD, Reshmi S, Carlson K, Roulston D. Spectral karyotype analysis of T-cell acute leukemia. Blood 1999; 93: 2038–2042. 31 Ramaswamy S, Tamayo P, Rifkin R, Mukherjee S, Yeang CH, Angelo M, Ladd C, Reich M, Latulippe E, Mesirov JP, Poggio T, Gerald W, Loda M, Lander ES, Golub TR. Multiclass cancer diagnosis using tumor gene expression signatures. Proc Natl Acad Sci USA 2001; 98: 15149–15154. 32 Getz G, Levine E, Domany E. Coupled two-way clustering analysis of gene microarray data. Proc Natl Acad Sci USA 2000; 97: 12079–12084. 33 Golub TR, Slonim DK, Tamayo P, Huard C, Gaasenbeek M, Mesirov JP, Coller H, Loh ML, Downing JR, Caligiuri MA, Bloomfield CD, Lander ES. Molecular classification of cancer: class discovery and class prediction by gene expression monitoring. Science 1999; 286: 531–537.