NAP1L1‐MLLT10 is a rare recurrent translocation that is associated with HOXA activation and poor treatment response in T‐cell acute lymphoblastic leukaemia (2015)

MLLT10 (previously AF10) was originally described as a fusion partner of the KMT2A (previously MLL) gene in acute myeloid leukaemia (AML) (Beverloo et al, 1995), and was subsequently found to be recurrently rearranged in both AML and T-acute lymphoblastic leukaemia (T-ALL) (Dreyling et al, 1998). PICALM (previously CALM) is the most frequent MLLT10 translocation partner in T-ALL, where it is found in 6-7% of cases (Ben Abdelali et al, 2013). The list of MLLT10 partners in T-ALL now also comprises NAP1L1 (Zhang et al, 2012), HNRNPH1, DDX3X (Brandimarte et al, 2013) and XPO1 (Bond et al, 2014). While DDX3X-MLLT10 has been estimated to occur in about 3% of adult T-ALL cases (Brandimarte et al, 2014), all other fusion transcripts have to date been described in single cases only, and their precise incidence remains to be defined. The NAP1L1-MLLT10 rearrangement was originally discovered by whole genome sequencing of leukaemic blasts from a child with early thymic precursor (ETP) ALL (Zhang et al, 2012). Diagnostic karyotypes of two T-ALL patients revealed similar t(10;12) translocations (Table 1), leading us to suspect the presence of NAP1L1-MLLT10. Expression of the transcript was confirmed by reverse transcription polymerase chain reaction (RT-PCR) (Fig 1A), and direct sequencing revealed fusion of NAP1L1 exon 14 and MLLT10 exon 4 in both cases (Fig 1B). CD34− CD1a− CD3− cCD3+ CD5+ CD7+ CD4weak CD8− CD10− TCRAB− TCRGD− MPO− CD117− CD13+ CD33+ HLA-DR+ CD34− CD1aweak CD2− CD3+ CD5+ CD7+ CD4+ CD8− CD10+ TCRGD+ CD13− CD33− CD117− Persistent MRD positivity (3%) post-induction and consolidation phases of treatment. Underwent SCT 7 months post-diagnosis. Remained in remission from leukaemia but died 2 years post-SCT due to chronic GVHD. Clinical details of the NAP1L1-MLLT10 patients are shown in Table 1. Strikingly, both were found to have high expression of HOXA9 by quantitative real time RT-PCR (QRT-PCR). Similar to the index case, Patient A had an ETP-ALL-like immunophenotype, with absence of CD1a, and expression of HLA-DR and the CD13 and CD33 myeloid markers (Table 1). Detailed characterization of T-cell receptor gene configuration revealed biallelic rearrangement of TRD and TRG and incomplete rearrangement of the TRB locus, conforming to an immature pre-β-selection immunogenotype (Asnafi et al, 2003). In contrast, Patient B exhibited a mature TCRγδ+ phenotype and absence of expression of myeloid markers. In common with the reported cases of DDX3X-MLLT10 T-ALL (Brandimarte et al, 2014), both cases had NOTCH1-activating mutations. Notably, both patients had poor initial treatment responses, with positive minimal residual disease (MRD). Patient B had persistent MRD positivity throughout induction and consolidation therapy, which necessitated allogeneic stem cell transplantation (allo-SCT) 7 months after diagnosis. Although this resulted in molecular remission, the patient ultimately died of SCT-related complications 2 years later while in remission from leukaemia. The follow-up of Patient A is at an early stage, and latest assessment revealed significant persistent MRD positivity following the consolidation phase. We sought to determine the incidence of the NAP1L1-MLLT10 translocation in T-ALL by screening 565 cases, comprising 141 patients under 16 years of age and 424 adults, using a specific and sensitive (<1%, data not shown) QRT-PCR assay (Fig 1C). As MLLT10 translocations are normally associated with HOXA locus activation (Brandimarte et al, 2013; Bond et al, 2014), we targeted the screening to 173 patients with elevated HOXA9 at diagnosis (‘HOXA High’ in Fig 1C). We detected no further cases of NAP1L1-MLLT10 in patients for whom leukaemic material was available for testing. We therefore estimate the incidence of the translocation in T-ALL to be 0·35% (2/565 patients), and 1·2% of patients with HOXA9/ABL1 > 0·4 (2/173 patients). HOXA overexpression has not previously been described to be associated with NAP1L1-MLLT10. In order to fully characterize HOX transcription in these cases, we performed Taqman Low-Density Array (TLDA) analysis of diagnostic RNA. This revealed specific deregulation of HOXA gene expression in both samples (Fig 1D). These results, along with the predicted retention of the MLLT10 octapeptide-motif leucine zipper (OM-LZ) domain in the fusion protein, suggest that NAP1L1-MLLT10 is likely to recruit the DOT1 Ligand histone methyltransferase and to activate the HOXA locus by a similar mechanism as PICALM-MLLT10 (Okada et al, 2005). It remains unclear how the activities of MLLT10 fusion partners might contribute to T-ALL biology. Prediction of NAP1L1 (Nucleosome assembly protein 1-like1) function is based on its structural resemblance to NAP1, which is thought to be involved primarily in chromatin modulation. NAP1 has been shown to interact directly with histone H2A-H2B dimers in vitro (Okuwaki et al, 2010), suggesting that NAP1L1 might alter the regulation of histone dynamics. It remains to be determined whether the epigenetic effects of NAP1L1 haploinsufficiency and/or activity of a NAP1L1 fusion protein might contribute to a leukaemic phenotype. In summary, NAP1L1-MLLT10 is a rare recurrent translocation in T-ALL and we report an overall incidence of 0·35%. This may be an underestimation, as our QRT-PCR screening method may not have identified patients with novel breakpoints, and lack of diagnostic material precluded screening in 11% of HOXA-overexpressing patients. Reassuringly, all three cases of NAP1L1-MLLT10 described to date exhibited the t(10;12) translocation by conventional karyotyping, and none of our patient cohort had evidence of chromosome 10 or 12 rearrangement. As with other MLLT10 fusions, NAP1L1-MLLT10 is associated with HOXA deregulation and poor early treatment response. Although one of our patients died of transplant-related complications, the prolonged leukaemia-free survival in this case suggests that these patients should be considered for allo-SCT in first remission, particularly in the event of persistent treatment resistance and/or MRD positivity. JB, AT, AC, AT, MM and TF performed and interpreted diagnostic investigations. TM, ME, AC and CS provided clinical care and interpreted clinical data. JB, VA and EAM analysed and collated data and wrote the manuscript. JB is supported by a Kay Kendall Leukaemia Fund Intermediate Research Fellowship. The Macintyre laboratory is supported by the Association Laurette Fugain and the INCa CARAMELE Translational Research and PhD Programmes. The authors report no conflicts of interest.