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A rare but specific subset of adult AML patients can be defined by the cytogenetically cryptic NUP98–NSD1 fusion gene

Acute myeloid leukemia (AML) is a heterogeneous group of diseases in terms of clinical presentation, genetic alterations and response to treatment. Cytogenetic abnormalities that are identified in 55% of all adult patients with AML enable subclassification and risk stratification. Fusion genes can be detected in 20–25% of all AML cases, and usually are the result of a cytogenetically detectable chromosomal rearrangement. Most recurrent chromosomal alterations are associated with specific morphological and clinical characteristics,1 such as the reciprocal t(8;21)/RUNX1-RUNX1T1, inv(16)/CBFB-MYH11 and t(15;17)/PML-RARA rearrangements, all of which are associated with good prognosis. In contrast, complex aberrations with 3 chromosomal abnormalities are associated with inferior outcome.2 However, even in high-quality cytogenetic preparations, G-banding analysis suffers from an inherent limit in resolution, such that rearrangements <5–10 megabases in size, particularly those involving uniformly pale G-banded regions, may be impossible to be detected. Molecular genetic approaches such as reverse transcriptase–PCR (RT–PCR) have demonstrated that a small percentage of apparently normal karyotypes may harbor cryptic versions of known recurrent translocations, which are generated by submicroscopic insertions or more complex rearrangements.3 The cryptic NUP98–NSD1 fusion involves the nucleoporin gene 98 (NUP98) on chromosome 11p15 and the non-homeobox gene NSD1 in chromosomal band 5q35. NUP98 encodes a 98-kDa protein of the nuclear pore complex, and is known to fuse to at least 21 different fusion gene partners in chromosomal rearrangements of various hematopoietic disorders.4 NSD1 contains two distinct nuclear receptor interaction domains, as well as a SET domain and multiple PHD fingers, both of which are frequently found in transcriptional regulators and may be involved in chromatin remodeling.5, 6 NSD1 is thought to function as both a transcriptional coactivator and a corepressor. The NUP98–NSD1 fusion gene has been shown to induce AML in vivo, which sustains self-renewal of myeloid stem cells in vitro, and enforces expression of the HOXA7, HOXA9, HOXA10 and MEIS1 proto-oncogenes.7 Recently, Hollink et al.8 published a study on NUP98–NSD1 in 293 pediatric and 808 adult cytogenetically normal AML (CN-AML) cases. The NUP98–NSD1 fusion gene has been described in this single study with a frequency of 16.1% in pediatric and 2.3% in adult AML patients with distinct characteristics (for example, mutual exclusiveness with NPM1) and dismal prognosis. The aim of our study was to further investigate the frequency and clinical relevance of the NUP98–NSD1 fusion transcript in 378 adult cases of de novo NPM1 unmutated (NPM1wt) CN-AML. Furthermore, we screened 64 adult AML cases with cytogenetic aberrations involving chromosomes 5 and 11 for the presence of NUP98–NSD1 fusion gene. In addition, we aimed at evaluating this fusion transcript as a target for quantitative RT–PCR (qRT–PCR)-based minimal residual disease (MRD) monitoring. The study design adhered to the tenets of the Declaration of Helsinki and was approved by our institutional review board before its initiation. NUP98–NSD1 fusion transcript analysis was performed on either bone marrow or peripheral blood samples. Presence of NUP98–NSD1 fusion transcript was determined by PCR and validated by direct Sanger sequencing using the following primers: NUP98ex12-F: 5′-GATTTAATACTACGACAGCCACTTT-3′ and NSDex6-R: 5′-GGAACTTACCTTGTGCACC-3′. qRT–PCR was performed by the use of the LightCycler 1.5 System (Roche Diagnostics, Mannheim, Germany). Amplification was performed with the PCR primers described above. Detection probes were as follows: NUP98–NSD1-FL, 5′-AGCTGTGCGGTCAGAGAAGAA-Fluo-3′ and NUP98–NSD1-FLC640, 5′-LCRed-640-GCCTTAGGAAGCCAAGCAAGTGGC-Pho-3′. The expression of the fusion gene NUP98-NSD1 was normalized against the expression of the control gene ABL1 to adjust for the variations in mRNA quality and efficiencies of complementary DNA synthesis. The NUP98–NSD1 mutation levels are given as % NUP98-NSD1/ABL1. Quantification of ABL1 was performed as described previously.9 To analyze the efficiency and sensitivity of NUP98–NSD1 specific assay, we performed serial dilution experiments of NUP98–NSD1-positive diagnostic samples in NUP98–NSD1-negative complementary DNA of individual patients. The mutation status of other genes was as follows: NPM1: n=378, FLT3-ITD: n=378, CEBPA: n=377, MLL-PTD: n=375, RUNX1: n=348, ASXL1: n=371, FLT3TKD: n=344, and WT1: n=366. Female/male ratio was 144/234, and age ranged from 15.7 to 89.6 years (median: 63.7) (Table 1). A NUP98–NSD1 fusion transcript was detected in 8/378 NPM1wt CN-AML cases (2.1%), indicating that it is a rare event in adult CN-AML. Patients with del(5q) or complex aberrations were all negative for NUP98-NSD1. In contrast, the single patient with ins(5;11) (q35;p13p15) harbored a NUP98–NSD1 fusion transcript. We observed significantly higher bone marrow blast counts in NUP98–NSD1-positive cases compared with negative cases (mean blast cells 73% vs 53%, P=0.039). Sex (4 males vs 4 females) and white blood cell count at diagnosis (median number of cells 80.4 vs 28.9; P=0.224) were not different compared with NUP98–NSD1-negative cases, as well as hemoglobin level (median level 8.7 vs 9.4; P=0.323) and platelet count (median number of cells 115.3 vs 94.4; P=0.584) (Table 1). With regard to cytomorphology, NUP98–NSD1-positive cases were restricted to AML M1 (n=5/100), AML M2 (n=2/155) and AML M4 (n=1/74) FAB subgroups (Supplementary Figure 1). In 194 cases immunophenotyping data was available. Cases with the NUP98–NSD1 fusion transcript as compared with those without revealed a more immature phenotype with stronger expression of CD34 (mean number of positive cells: 73±30% vs 41±28%, P<0.001). Furthermore, a stronger expression of HLA-DR was observed (mean number of positive cells: 62±18% vs 38±22%; P=0.058). CN-AML patients with NUP98–NSD1 fusion were younger than the NUP98–NSD1-negative patients (median: 42.3 vs 62.4 years, P<0.001) (Table 1). A distribution according to age decades is depicted in Figure 1a. The age range of NUP98–NSD1-positive cases was 20.9–71.4 years, indicating that NUP98–NSD1 translocation is not restricted to younger age, although it appears to be more frequent in those below 50 years of age. This age-dependent frequency resembles that of the core-binding factor AML (t(8;21)/RUNX1-RUNX1T1 and inv(16)/CBFB-MYH11), which also occurs more frequently in younger patients. Interestingly, other NUP fusion genes such as NUP98–HOXA9 and DEK–NUP214 have also been associated with a younger age.10, 11 NPM1wt and CN-AML NUP98–NSD1-positive cases had a significantly higher frequency of FLT3-ITD and WT1 mutations compared with NUP98–NSD1-negative cases (6/8, 70.0% vs 67/367, 18.1%; P=0.001; and 4/8, 50.0% vs 23/358, 6.4%; P=0.001, respectively). Furthermore, in NUP98–NSD1-positive cases no RUNX1, CEBPA and FLT3-TKD mutations were detectable, but because of the low number of NUP98–NSD1-positive patients, this mutual exclusiveness is not significant. (Table 1). The association of NUP98-NSD1 with the class I aberration FLT3-ITD has previously been shown by Hollink et al.,8 and recent studies have also found other NUP98 fusions to be associated with FLT3-ITD.10, 12 These findings support the hypothesis of multistep AML pathogenesis that may require the cooperation of class I and class II mutations. This theory is supported by the fact that we did not find any coincidence of a NUP98–NSD1 fusion transcript with the class II mutations such as RUNX1 and CEBPA, which again has also been shown by Hollink et al.,8 and also for other NUP98 fusions.10, 12 In addition, our mutation analysis showed that WT1 mutations were significantly associated with the presence of NUP98–NSD1 fusion transcript, implying a critical role of WT1 genetic alteration in cooperation with NUP98–NSD1 in the pathogenesis of this subtype of AML. Mutations of WT1, which normally induces cellular quiescence and differentiation,13 occur in 10% of CN-AML patients and may be associated with drug resistance.14 The strong association between NUP98–NSD1 and WT1 mutations has also been described by others,8, 12 and suggests that these two genes could interact in acceleration of leukemogenesis and in conferring a poor response to chemotherapy. Interestingly, 4/8 NUP98–NSD1-positive patients harbored simultaneously FLT3-ITD and WT1 mutations, thus further emphasizing the assumed poor clinical outcome of NUP98–NSD1-positive AML. The NUP98–NSD1 fusion transcript did not show an impact on overall survival. However, event-free survival (EFS) of NUP98–NSD1-positive patients was significantly worse compared with patients negative for NUP98-NSD1 (median: 1.8 vs 11.0 months; P=0.045, Figure 1b). This is in line with the study of Hollink et al.,8 who reported that 4-year EFS rates of NUP98–NSD1-positive cases were below 10% for the adult cases studied.8 Furthermore, we show that NUP98-NSD1 is a suitable and specific target for MRD monitoring. The sensitivity of NUP98–NSD1-specific assay was 1:10 000 (Supplementary Figure 2). Of the three NUP98–NSD1-positive patients, bone marrow samples were available at different time-points during the clinical course. All the three patients were either refractory to induction chemotherapy or relapsed within 2 years of diagnosis (Figure 1c), thus emphasizing NUP98-NSD1 as a factor for dismal clinical outcome. In conclusion, we were able to show that the NUP98–NSD1 fusion transcript is a rare event in adult NPM1wt CN-AML. However, NUP98–NSD1-positive AML seems to be a distinct entity, as it is associated with younger age, CD34 overexpression and very poor EFS. Another prominent feature of NUP98–NSD1-positive cases is their significantly higher blast counts. The NUP98–NSD1 fusion transcript has been shown to correlate with FLT3-ITD, which is associated with increased blast percentages.15 Our data indicate that NUP98-NSD1 may function as an enhancer of the proliferative effect of FLT3-ITD. To our knowledge, this is the first report of a correlation of a NUP98 fusion with higher blast counts. Furthermore, NUP98-NSD1 seems to be a suitable marker for sensitive PCR-based monitoring of MRD. Thus, our data suggest performing PCR-based screening for NUP98-NSD1 in CN-AML cases that lack NPM1, CEBPA and RUNX1 mutations to identify this subgroup of poor-risk AML patients.

Table 1 Demographics and clinical and molecular characteristics of AML patients according to NUP98–NSD1 fusion transcript
Figure 1
figure1

(a) Histogram representing the percentage of the NUP98–NSD1 positive cases within the different age categories in NPM1wt CN-AML shows a decreasing frequency of NUP98–NSD1 with increasing age. (b) Survival analysis according to NUP98–NSD1 fusion transcript in NPM1wt CN-AML. Kaplan–Meier plot showing EFS of NUP98–NSD1 positive (red) compared with NUP98–NSD1-negative cases (gray). (c) Clinical course of NUP98–NSD1 expression in three different patients with NUP98–NSD1 fusion transcript.

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Acknowledgements

We thank all clinicians for sending samples to our laboratory for diagnostic purposes, and for providing clinical information and follow-up data. In addition, we would like to thank all the co-workers at the MLL (Munich Leukemia Laboratory) for approaching together many aspects in the field of leukemia diagnostics and research. Especially the technical assistance of Nicole Schlenther, who performed NUP98–NSD1-specific qRT-PCR analyses, is greatly appreciated. In addition, we are grateful for the data management support performed by Tamara Alpermann.

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Correspondence to S Schnittger.

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CH, WK, TH, and SS have equity ownership of MLL Munich Leukemia Laboratory GmbH. AF and TA are employed by MLL Munich Leukemia Laboratory GmbH.

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Fasan, A., Haferlach, C., Alpermann, T. et al. A rare but specific subset of adult AML patients can be defined by the cytogenetically cryptic NUP98–NSD1 fusion gene. Leukemia 27, 245–248 (2013). https://doi.org/10.1038/leu.2012.230

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