NUP214-ABL1 amplification in t(5;14)/HOX11L2-positive ALL present with several forms and may have a prognostic significance

HOX11L2 expression, mostly resulting from the chromosomal cryptic translocation t(5;14)(q35;q32), is observed in more than 20% of childhood T-cell acute lymphoblastic leukemia (T-ALL) and in about 13% of adult T-ALL.1,2 The prognostic significance of the genetic abnormality in patients with this subtype of ALL is still controversial, claimed as unfavorable for some groups3,4 or regarded as neutral for others.5,6 Recently, episomal amplification encompassing the ABL1 gene has been found in 2.3 and 4.3% of childhood and adult T-ALL.7 The incidence of ABL1 amplification rose to 5.8% of T-ALL by Graux et al,8 who showed that a new fusion gene, NUP214-ABL1, was included in the episomal amplicon. Moreover, the latter authors showed that the NUP214-ABL1 fusion was mainly present in T-ALL expressing HOX11 or HOX11L2. We hypothesized that the expression of the fusion gene might account for the discrepancies observed in the evolution of individual HOX11L2-positive patients with T-ALL. We report the finding of fluorescence in situ hybridization (FISH) and real-time PCR in a child with T-ALL with a peculiar form of amplification and rapidly fatal evolution.

Case report

GD, a 9-year-old boy was admitted to the hematological unit of the Armand Trousseau hospital (Paris) with a suspicion of leukemia because of fever, erythematous angina, enlarged cervical lymph nodes and hyperleukocytosis. Clinical and radiological examination displayed mediastinal enlargement, hepatosplenomegaly and voluminous mesenteric lymph nodes. Hematologic data confirmed the diagnosis of acute leukemia: in peripheral blood, 302 × 109/l leukocytes with 93% lymphoblasts, 116 × 109/l platelets and hemoglobin 117 g/l. Immunophenotyping revealed an immature T-cell leukemia scoring positive for c-CD3, CD2, CD4, CD5, CD7 and CD8 antigens. All the myeloid antigens tested (CD13, CD14, CD15, CD33, CD117, CD35 and c-MPO) were negative, with the exception of CD33. The child immediately received induction therapy including prednisolone prephase, vincristine 1.5 mg/m2 weekly × 4, cyclophosphamide 1 g/m2 day 8, asparaginase 6000 IU × 8, daunorubicin 40 mg/m2 days 8, 9, 10 and 15 and two intrathecal injections of methotrexate and aracytine. At day 7, he demonstrated corticoid resistance, and at day 21, bone marrow was aplastic with persistence of 83% leukemic cells. No remission could be obtained and the child died 30 days after the diagnosis because of massive intracranial hemorrhage.

Cytogenetic studies were performed, prior to any treatment, on blood cells after 24 h in vitro culture with R-banding technique. The karyotype was 47,XY,+8[26]/46,XY[1]. No structural aberration was detected.

FISH studies

Two series of FISH studies were performed according to usual techniques.9 The first allowed the detection of a t(5;14) translocation with dual-color FISH with whole chromosome 14 painting probe and YAC 885A6, as described previously.1 The chromosomal breakpoints were more precisely located with BAC probes, on chromosome 5 between BACs 45L16 remaining to chromosome 5 and 546B8 translocated to 14q32 (without split), and on chromosome 14 to BAC 2576L4 giving split signals on normal chromosome 14, der(14) and der(5). In the second step, amplification of ABL1 was investigated with the commercial LSI BCR-ABL1 ES dual probe covering 300 kb of ABL1 from intron 5 toward telomere (Abbott, Rungis). Surprisingly, a strong signal was present on the long arm of chromosome 2 in addition to the normal signals on both 9q34 copies. No extrachromosomal hybridization signal could be observed. It was concluded that amplification of ABL1 sequences were located to 2q23–24. Hybridization with whole chromosome 9 painting (WCP) probe also showed a signal on 2q, consistently less strong that the signal obtained with ABL probes alone. The difference of the two signals was evidenced by dual-color FISH with ABL and chromosome 9 WCP probe showing that the signals generated by the ABL probe were larger than those of the WCP probe. To ascertain the involvement of NUP214, dual-color FISH studies were performed with BACs RP11 235J21 (ABL1) and RP11-235F20 and RP11-106L9 (NUP214). Colocalization of ABL1 and NUP214 probes to both chromosomes 9 as well as to chromosome 2q confirmed the amplification of DNA sequences of the two genes (Figure 1).

Figure 1
figure1

FISH to metaphase chromosomes of the patient with probes BAC RP11-235F20 and RP11-10L9 (NUP214) (green) and RP11 235J21 (ABL) (red). Signals green-red on both chromosomes 9 and amplified sequences on 2q. Also note the different sizes of amplified and normal signals in interphase nuclei.

Molecular studies

Molecular studies included TLX3 expression analysis. The leukemia cells expressed high levels of the TLX3 gene in keeping with the presence of t(5;14)(q35;q14). At the time of the observation, the episomal rearrangement NUP214-ABL1 was not yet identified. Retrospective search for the gene fusion was performed as in Graux et al.8 We could amplify a specific PCR fragment of 0.7 kb with primers NUP29F-ABL1R3 and a shorter fragment of 0.45 kb with primers NUP31F-ABL1R0. This result suggests that the breakpoint was located downstream of exon 31 in the NUP214 gene. Real-time quantitative RT-PCR (RQ-PCR) was also performed to quantify the expression of ABL1, described as abnormally high in patients with the NUP214-ABL1 gene fusion. Primers and probe for ABL1 were as in Beillard et al10 and detected both wild-type and rearranged ABL1. Expression of TBP (Transcription Factor IID) gene, a control gene steadily expressed in normal and leukemic cells, was used to normalize the ABL1 expression values. Those for TBP were as in Bièche et al.11 First, we determined ABL1 expression values in a panel of 50 T-ALLs, all tested negative for NUP214-ABL1 rearrangement, and in a pool of normal fetal thymus (Stratagene, Europe, Amsterdam). The expression values of ABL1 gene were moderately heterogeneous among T-ALLs, varying from 0.45- to 2.5-fold expression over TBP (mean=1.2). Higher expression, 2.7-fold value, was observed in the pool of normal fetal thymus. Among the nine TLX3- and two TLX1-positive leukemias included in the T-ALL panel, the quantified ABL1 values varied in the same range, from 0.37 to 2.26 (mean=1.39), thus ruling out a possible role of TLX3 or TLX1 gene products in the transcriptional regulation of the normal ABL1 gene or a specific association between high expression levels of both genes.

As expected, ABL1 was significantly overexpressed in the recorded case (8.1-fold increase over TBP expression). This value was also significantly higher than that estimated in three other NUP-ABL1-positive T-ALLs (data not shown).

Discussion

Discrepancies about the prognostic value of t(5;14)(q35;q32)/HOX11L2-positive ALL, on the one hand, and the report of amplification of a new fusion NUP214-ABL1 gene, on the other hand, prompted us to search for such amplification in patients with t(5;14). As amplification of NUP214-ABL may be related with a poor evolution of the disease, it was logical to examine cells of patients with unfavorable outcome. Study of leukemic cells of a child with t(5;15)/HOX11L2-positive T-ALL revealed the presence of amplified NUP214 and ABL sequences located to the long arm of one chromosome 2. This morphologic pattern of amplification of NUP214-ABL1 was not reported previously.8 In the reported observations, the amplicons resulted from the formation of episomes, not seen in the present case. It is generally admitted that the first step of genomic amplification of genomic DNA results from episome formation.12,13 In latter steps, the episomes may be included within double minute (dmin) chromosomes or homogeneously staining or abnormally staining regions (HSR or ABR).14 Two mechanisms for the formation of amplicons have been proposed, conservative in which the original DNA sequence remains at its normal location onto the chromosome, and nonconservative in which the original sequence is extracted from its normal chromosomal location.15 With the first mechanism, FISH with appropriate probes shows hybridization to the normal loci of sequences from which the amplicons derive, and with the second mechanism, only one locus is marked with the probe. Whereas the second mechanism is involved in the materials studied by Graux et al,8 the conservative mechanism is implied in the case here reported. Moreover, the localization of the amplified NUP214-ABL1 sequences on chromosome 2q21 might be favored by the peculiar structure of band 2q21 where an alphoid DNA sequence, conserved in human and great ape chromosomes, is located.16 We examined two other children with t(5;14)T-ALL, one with amplification of NUP214-ABL1 in its episomal form who died from his leukemia two and half years after the diagnosis, and the second without genomic amplification who remains in continuous complete remission 18 months after the diagnosis. Whether the mechanism of amplification has any impact to its biological consequences is still to be determined.

Whatever the case, the detection of the amplified DNA NUP214-ABL1 fusion may be important for the treatment of the patients, since it appears that imatinib mesylate (Gleevec) is potentially efficient.8 We therefore conclude that search for the NUP214-ABL1 rearrangement, which is cryptic in its episomal form, must now be systematically included in the exploration of patients with t(5;14)/HOX11L2-positive ALLs. Such studies should also be carried out in HOX11-positive T-ALL patients since this leukemia subtype can also be associated with NUP214-ABL1 amplification.8

References

  1. 1

    Bernard OA, Busson-Le Coniat M, Ballerini P, Mauchauffé M, Della Valle V, Monni R et al. 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 2002; 15: 1495–1504.

    Article  Google Scholar 

  2. 2

    Berger R, Dastugue N, Busson M, van den Akker J, Pérot C, Ballerini P, et al., on behalf of the Groupe Français e Cytogénétique Hématologique (GFCH). t(5;14)/HOX11L2-positive T-cell acute lymphoblastic leukaemia. A collaborative study of the Groupe Français de Cytogénétique Hématologique (GFCH). Leukemia 2003; 17: 1851–1857.

    CAS  Article  Google Scholar 

  3. 3

    Ballerini P, Blaise A, Busson-Le Coniat M, Su XY, Zucman-Rossi J, Adam M et al. HOX11L2 expression defines a clinical subtype of pediatric T-ALL associated with poor prognosis. Leukemia 2002; 100: 991–996.

    CAS  Google Scholar 

  4. 4

    Ferrando AA, Neuberg DS, Staunton J, Loh ML, Huard C, Raimondi SC et al. Gene expression signatures define novel oncogenic pathways in T cell acute lymphoblastic leukemia. Cancer Cell 2002; 1: 75–87.

    CAS  Article  Google Scholar 

  5. 5

    Cavé H, Suciu S, Preudhomme C, Poppe B, Robert A, Uyttebroeck A, et al., for the EORTC-CLG. Clinical significance of HOX11L2 expression linked to t(5;14)(q35;q32), of HOX11 expression, and of SIL-TAL fusion in childhood T-cell malignancies: results of EORTC studies 58881 and 58951. Blood 2004; 103: 442–450.

    Article  Google Scholar 

  6. 6

    Mauvieux L, Leymarie V, Helias C, Perrusson N, Falkenrodt A, Lioure B et al. High incidence of Hox11L2 expression in children with T-ALL. Leukemia 2002; 16: 2417–2422.

    CAS  Article  Google Scholar 

  7. 7

    Barber KE, Martineau M, Harewood L, Stewart M, Cameron E, Strefford JC et al. Amplification of the ABL gene in T-cell acute lymphoblastic leukemia. Leukemia 2004; 18: 1153–1156.

    CAS  Article  Google Scholar 

  8. 8

    Graux C, Cools J, Melotte C, Quentmeier H, Ferrando A, Levine R et al. Fusion of NUP214 to BL1 on amplified episomes in T-cell acute lymphoblastic leukemia. Nat Genet 2004; 36: 1084–1089.

    CAS  Article  Google Scholar 

  9. 9

    Le Coniat M, Romana SP, Berger R . Partial chromosome 21 amplification in a child with acute lymphoblastic leukemia. Genes Chromosomes Cancer 1995; 14: 204–209.

    CAS  Article  Google Scholar 

  10. 10

    Beillard E, Pallisgaard N, van der Velden VH, Bi W, Dee R, van der Schoot E et al. Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patients using ‘real-time’ quantitative reverse-transcriptase polymerase reaction (RQ-PCR) – a Europe against cancer program. Leukemia 2003; 17: 2474–2486.

    CAS  Article  Google Scholar 

  11. 11

    Bièche I, Onody P, Laurendeau I, Olivi M, Lidereau R, Vidaud M . Real-time reverse transcription-PCR assay for future management of ERBB2-based clinical applications. Clin Chem 1999; 45: 1148–1156.

    PubMed  Google Scholar 

  12. 12

    Carroll SM, DeRose ML, Gaudray P, Moore CM, Needham-Vendevanter DR, von Hoff DD et al. Double minute can be produced from precursors derived from a chromosomal deletion. Mol Cell Biol 1988; 8: 1525–1533.

    CAS  Article  Google Scholar 

  13. 13

    Carroll SM, Gaudray P, DeRose ML, Emery JF, Meinkoth JL, Nakkim E et al. Characterization of an episome produced in hamster cells that amplify a transfected CAD gene at high frequency: functional evidence for a mammalian replication origin. Mol Cell Biol 1987; 7: 1740–1750.

    CAS  Article  Google Scholar 

  14. 14

    Wahl GM . The importance of circular DNA in mammalian gene amplification. Cancer Res 1989; 49: 1333–1340.

    CAS  PubMed  Google Scholar 

  15. 15

    Ruiz JC, Wahl GM . Chromosomal destabilization during gene amplification. Mol Cell Biol 1990; 10: 3056–3060.

    CAS  Article  Google Scholar 

  16. 16

    Baldini A, Ried T, Shridhar V, Ogura K, D'Aiuto L, Rocchi M et al. An alphoid DNA sequence conserved in all human and great ape chromosomes: evidence for ancient centromeric sequences at human chromosomal region 2q21 and 9q13. Hum Genet 1993; 90: 577–583.

    CAS  Article  Google Scholar 

Download references

Author information

Affiliations

Authors

Corresponding author

Correspondence to R Berger.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Ballerini, P., Busson, M., Fasola, S. et al. NUP214-ABL1 amplification in t(5;14)/HOX11L2-positive ALL present with several forms and may have a prognostic significance. Leukemia 19, 468–470 (2005). https://doi.org/10.1038/sj.leu.2403654

Download citation

Further reading

Search

Quick links