NUP214–ABL1 fusion in childhood T‐ALL

To the Editor: T-cell acute lymphoblastic leukemia (T-ALL) accounts for 15% of pediatric ALL and has been historically associated with a poorer outcome compared to B-ALL; treatment intensification has however allowed marked improvement in outcome in the last two decades.1,2 The identification of new molecular markers could help to guide riskadapted personalized treatments and further improve results in these patients. ABL1 fusion genes can be found in about 8% of T-ALL in adults, and NUP214–ABL1 is the most frequently observed and specific for T-ALL.3 Several breakpoints of NUP214 were observed, ranging from exon 23 and exon 34, which are in-frame fused, usually with exon 2 and rarely with exon 3 of ABL1.4 These lead to the generation of episomes resulting from the extrachromosomal circularization of the coiled-coil domains of the N-terminal region of NUP214, and the tyrosine kinase domains of ABL1 as in BCR–ABL1.5 As NUP214– ABL1 retains the coiled-coil domains to the nuclear pore complex localization, it needs to be amplified to compete with endogenous NUP214 located on the cytoplasmic side,6 and it needs to be associated with other nuclear pore proteins to exert its transforming potential.7 Moreover, additional events such as CDKN2A deletions or TLX1 and TLX3 overexpression are frequently observed associated to NUP214–ABL1 patients and are likely to be required to induce leukemia.4–8 Whereas the frequency of this fusion gene in adult T-ALL patients has been assessed at 3.9%,9 previous studies in pediatric cancers have yielded limited results about its frequency and prognostic value.10 To further define the frequency of NUP214–ABL1 in the pediatric population and to clarify its prognostic significance, we retrospectively analyzed at diagnosis a large cohort of 178 pediatric T-ALL patients diagnosed between 2000 and 2010 and treated with two protocols of the Italian Association of Pediatric Hematology and Oncology (AIEOP): AIEOP-BFM ALL2000 and AIEOP ALL R2006 protocols (NCT00613457). The cohort of 178 patients here analyzed was comparable in terms of main clinical characteristics with the whole ALL 2000/R2006 T-ALL cohort (Table S1). Patients were screened for NUP214–ABL1 by RT-PCR using a mix of forward primers mapping on exons 22, 26, 29, 31, and 34 of NUP214 and one reverse primer mapping on exon 3 of ABL1 (Figure S1). Positive samples were then analyzed by PCR using these primers in separate reactions. Based on single PCR results, samples were next analyzed by Sanger sequencing to assess the exact fusion product. T-ALL cell lines, ALL-SIL and PEER, purchased from DSMZ (Braunschweig, Germany), were used as positive controls (Figure S2). Among the 178 patients analyzed by RTPCR, we found only three positive for NUP214–ABL1 (3/178 = 1.68%) after screening. These three patients showed heterogeneous features (Table S2), and two of them experienced bone marrow relapse, the other one is alive in continuous complete remission 15 years after diagnosis. Both relapsed patients died during chemotherapy treatment, one due to disease progression after stem cell transplantation and the other of a therapy-related adverse event (Table S2). Intriguingly, in the two relapsed patients, the coexistence of two different fusion products was observed for the first time. As shown in Figure 1A,B, in patient #131,we found in-frame fusions between exon 31 ofNUP214 and exon 3 of ABL1, and exon 34 ofNUP214 and exon 2 of ABL1. In patient #169, we observed both exon 26 of NUP214 fused with exon 2 of ABL1, and exon 31 of NUP214 fused with exon 2 of ABL1. In patient #25, who did not relapse, exon 26 of NUP214 is fused with exon 2 of ABL1. We further analyzed the relapse samples of patient #131 (54% blast infiltration) and patient #169 (40%), and we observed that in patient #131, only the 31-3 product was preserved at relapse, whereas the 34-2 was lost (Figure 1C), and patient #169 became negative, losing both 26-2 and 31-2 fusions (Figure 1D). Although, also inour cohort, thenumberofNUP214–ABL1 patients remains too small to assess its prognostic value, these results may suggest that NUP214–ABL1 in the pediatric population may be associated with unfavorable prognosis, similarly to what described in TLX3 good prognosis patients in which the presence of this fusion gene is associated with relapse.8 Also, the concurrent presence of two different NUP214–ABL1 fusions might reflect a general genome instability,11 which could predispose these patients to relapse. Finally, one of the driving interests on identifying NUP214–ABL1-rearranged patients derives from the retention of ABL1 kinase domains in the fusion protein, thus making leukemia cells potentially sensitive to tyrosine kinase inhibitors (TKI) such as imatinib or dasatinib. Interestingly,NUP214–ABL1-induced cell proliferation is dependent on the activity of the SRC family, supporting the indication for theuseofdualABL1/SRC inhibitors, suchasdasatinib or bosutinib as new potential therapeutic approaches for NUP214– ABL1 patients.12 A few papers report treatment with TKI, in particular dasatinib, in NUP214–ABL1 patients with promising results,12–16 but underlining that TKI monotherapy should be avoided due to the possibility of positive selection of NUP214–ABL1-negative clones.13 However, as survival remains quite poor for relapsed T-ALL patients,17 characterized by secondary chemotherapy resistance, a poor outcome, and limited treatment opportunities,18 screening for NUP214– ABL1 is encouraged for relapsed/refractory patients to explore the