First case report of a NUP98-PMX1 rearrangement in de novo acute myeloid leukemia and literature review.

BACKGROUND: The nucleoporin 98 (NUP98)-paired related homeobox 1 (PMX1) fusion gene, which results from t(1;11)(q23;p15), is rare in patients with acute myeloid leukemia (AML). Currently, only two cases of chronic myeloid leukemia in the accelerated phase or blast crisis and three cases of therapy-related AML have been reported. Here, we first report a patient with de novo AML carrying the NUP98-PMX1 fusion gene. CASE
PRESENTATION: A 49-year-old man diagnosed with AML presented the karyotype 46,XY,t(1;11)(q23;p15)[20] in bone marrow (BM) cells. Fluorescence in situ hybridization analysis using dual-color break-apart probes showed the typical signal pattern. Reverse transcription-polymerase chain reaction (RT-PCR) analysis suggested the presence of the NUP98-PMX1 fusion transcript. The patient received idarubicin and cytarabine as induction chemotherapy. After 3 weeks, the BM aspirate showed complete remission, and the RT-PCR result for the NUP98-PMX1 fusion gene was negative. Subsequently, the patient received three cycles of high-dose Ara-c as consolidation chemotherapy, after which he underwent partially matched (human leukocyte antigen-DP locus mismatch) unrelated allogeneic hematopoietic stem cell transplantation (HSCT). The follow-up period ended on September 30, 2020 (6 months after HSCT), and the patient exhibited no recurrence or transplantation-related complications.
CONCLUSION: This is the first report of a patient with de novo AML carrying the NUP98-PMX1 fusion gene. The reported case may contribute to a more comprehensive profile of the NUP98-PMX1 rearrangement, but mechanistic studies are warranted to fully understand the role of this fusion gene in leukemia pathogenesis.

Background

The nucleoporin 98 (NUP98) gene encodes a 98 kDa protein and is located on chromosome 11p15. NUP98 is part of the nuclear pore complex and regulates the transport of proteins between the cytoplasm and nucleus. Structurally, the N-terminus of NUP98 contains numerous phenylalanine-glycine (FG) and glycine-leucine-phenylalanine-glycine (GLFG) repeats. The first and third functional domains consist of FG and GLFG repeats, respectively. The second domain contains the Gle2-binding sequence, which is the binding site of the RNA export factor RAE1, and the fourth domain is the RNA-binding site [1-3]. Translocations involving the NUP98 gene are rare but play important roles in the initiation and development of hematopoietic malignancies. To date, the NUP98 gene has been implicated in hematopoietic development and found to fuse with more than 30 partner genes and contribute to the onset of leukemia [4]. These partner genes can be separated into two categories, namely homeobox (HOX) and non-homeobox (non-HOX) genes. HOX genes represent a class of transcription factors that share a conserved DNA-binding motif called homeodomain (HD) and include seven clustered “class I” HOX genes (HOXA9, HOXA11, HOXA13, HOXC11, HOXC13, HOXD11, and HOXD13) and five non-clustered “class II” HOX genes (HHEX, GSX2, PRRX1, PRRX2, and POU1F1). Non-HOX genes share a coiled-coil domain and include multiple genes (DDX10, TOP1, and NSD1).

The rearrangement of t(1;11)(q23;p15) involving NUP98 and the class II HOX gene paired related homeobox 1 (PMX1) has been rarely reported thus far. To date, only five cases of NUP98-PMX1 fusion have been reported, involving chronic myeloid leukemia in the accelerated phase, blast crisis, or therapy-related acute myeloid leukemia (AML) (Table 1) [5-8]; no case of de novo AML carrying the NUP98-PMX1 fusion gene has been reported. Furthermore, there is insufficient information regarding the clinical features, appropriate treatment, and outcomes of patients with the NUP98-PMX1 fusion gene.

Table 1

Summary of six cases of leukemia with NUP98-PMX1 rearrangement

Case no	Year	Sex	Age (years)	Primary diagnosis	Current diagnosis	Karyotype	Treatment	Outcome	References
1	1999	M	55	NHL stage III, gastric cancer	t-AML	46,XY,t(1;11)(q23;p15)	Topo II inhibitor; MACOP-B	Dead	[5]
2	2007	M	74	Liposarcoma	t-MDS/t-AML	46,XY,t(1;11)(q23;p15)[17]/46,XY[3]	Doxorubicin, ifosfamide, radiation	Dead	[6]
3	2004	M	42	CML-AP	CML-AP	46,XY,t(1;11)(q21;p15),t(9; 22)(q34;q11)[30]	Hydrea, Myleran	Dead	[7]
4	2004	F	44	Breast carcinoma	t-AML	46,XX,t(1;11)(q25;p15)[17]/46,XX[3]	Adriamycin, Cytoxan, 5-FU, BMT	Dead	[7]
5	2006	M	51	CML-CP	AML (CML BP)	46,XY,t(1;11)(q23;p15),t(9; 22)(q34;q11)	Hydrea; arabinosylcytosine, aclamycin, daunorubicin	–	[8]
6	2020	M	49	De novo AML	De novo AML	46,XY,t(1;11)(q23;p15)[20]	Idarubicin, cytarabine, BMT	Alive	Current report

NHL non-Hodgkin lymphoma, CML chronic myelogenous leukemia, AP accelerated phase, CP chronic phase, AML acute myeloid leukemia t-AML treatment-related acute myeloid leukemia, MDS myelodysplastic syndrome, BP blast phase, MACOP-B methotrexate with doxorubicin, cyclophosphamide, vincristine, prednisone, and bleomycin, 5-FU 5-fluorouracil, BMT bone marrow transplant

This is the first report of de novo AML in a patient carrying the NUP98-PMX1 fusion gene. For a comprehensive understanding of this specific translocation, we have further reviewed the relevant literature.

Case presentation

On August 12, 2019, a 49-year-old man who presented with fever was referred to the Changhai Hospital (Shanghai, China). The peripheral blood counts of this patient were as follows: white blood cells, 34 × 109/L (with 26% blasts); hemoglobin, 74 g/L; and platelets, 92 × 109/L. The bone marrow (BM) aspirate showed that 22.5% of blast cells were positive for peroxidase staining. Immunophenotypic analysis using multiparameter flow cytometry revealed that the blast cells were myeloperoxidase+, cluster of differentiation (CD)13+, CD33+, CD123+, CD34+, CD117+, CD38+, CD11c+, CD64+, CD14+, CD11b+, human leukocyte antigen (HLA)–antigen D related–, cytoplasmic CD79a–, CD7–, cCD3–, CD19–, CD4–, CD10–, CD15–, CD56–, CD2–, and CD16–. The antibodies against CD7, CD19, CD13, CD10, CD14, CD15, CD123, CD38, and CD13 were purchased from BD Biosciences (San Jose, CA, USA), and other antibodies were purchased from Beckman Coulter (Brea, CA, USA). A total of 1 mL freshly isolated whole BM aspirate was collected, of which 400 µL was stained with monoclonal antibodies for 15 min at room temperature. Following red blood cell lysis, BM cells were washed, collected, and analyzed following the manufacturer’s instructions using a FACSAria II instrument (BD Biosciences).

The karyotype of the patient was 46,XY,t(1;11)(q23;p15)[20] (Fig. 1). Fluorescence in situ hybridization (FISH) analysis using dual-color break-apart probes was performed with Isis Software (MetaSystems, Germany). Hybridized chromosome slides were analysed using an epifluorescence microscope Axio imager A2 (Carl Zeiss, Germany). The result showed the typical split signal pattern as split 3'-end (green) and 5'-end (red) probe signals along with a single normal unsplit red-green signal pair (yellow). This indicated that NUP98 was disrupted as a result of translocation (Fig. 2). Then we performed reverse transcription-polymerase chain reaction (RT-PCR) analysis of the BM cells. The sequences of primers were referred to the previously reported of Nakamura et al. [5]. RNA was extracted from the BM cells with the Trizol reagent (15596026, Thermo Fisher Scientific, Inc., Waltham, MA, USA). PCR was performed on the Agilent SureCycler 8800 (Agilent Technology Inc., Santa Clara, CA, USA) according to the manufacturer's instructions. Sequence analysis of this product confirmed the NUP98-PMX1 fusion transcript (Fig. 3). The sequence datasets are available in the Additional file 1. Next-generation sequencing analysis revealed FMS-like tyrosine kinase-3 (FLT3)-internal tandem duplication (ITD) and neuroblastoma RAS (NRAS) mutations. The sequence datasets are available in the Additional file 2. Therefore, the patient was diagnosed with de novo AML with the NUP98-PMX1 fusion gene.

Fig. 1

R-Banded karyogram at diagnosis of the patient. Arrowa indicate t(1;11)(q23;p15) chromosomal abnormalities

Fig. 2

FISH analysis of the patient. FISH analysis by use of the dual-color break-apart probes that showed the typical split signal pattern: split 3'-end (green) and 5'-end (red) probe signals indicated by arrows with a single normal unsplit red-green signal pair (yellow)

Fig. 3

Nucleotide sequences of NUP98-PMX1 fusions. Arrows indicate the fusion points

The patient received idarubicin and cytarabine (idarubicin, 8 mg/m2/day on days 1–3; cytarabine, 100 mg/m2/day on days 1–7) as induction chemotherapy. After 3 weeks, the BM aspirate showed complete remission. The second RT-PCR result for the NUP98-PMX1 fusion gene was negative, and the second mutation analysis showed that FLT3-ITD and NRAS mutations were cleared. Subsequently, the patient received three cycles of high-dose Ara-c (3 g/m2 q12 h on days 1–3) as consolidation chemotherapy. During chemotherapy, BM aspiration was performed before each consolidation therapy, and the NUP98-PMX1 fusion gene was invariably negative. Then, the patient underwent a partially matched (HLA-DP locus mismatch) unrelated allogeneic hematopoietic stem cell transplantation (HSCT) along with myeloablative conditioning with busulfan (3.2 mg/kg/day on days –8 to –6), cyclophosphamide (1.8 g/m2/day on days –5 to –4), and anti-thymoglobulin (8 mg/kg dose divided over 3 days). The patient had received mononuclear and CD34+ cells (6.5 × 108/kg and 2.5 × 106/kg, respectively). The prophylaxis regimen for acute graft versus host disease consisted of cyclosporine A, mycophenolate mofetil, and short-term methotrexate. Engraftment was confirmed on day 14 after HSCT by the peripheral absolute neutrophil count of more than 0.5 × 109/L for 3 consecutive days and platelet count of more than 20 × 109/L for 7 consecutive days. The follow-up period ended on September 30, 2020 (6 months after HSCT), and the patient exhibited no recurrence or transplantation-related complications. The study was conducted according to the guidelines of the Declaration of Helsinki, and the patient provided an informed consent.

Discussion and conclusion

NUP98 gene fusions interfere with the expression of downstream transcription genes and participate in cell proliferation, differentiation, and nucleocytoplasmic exports, thereby promoting myeloid leukemogenesis. Moreover, NUP98 gene fusions co-occur with a set of additional mutations, including FLT3-ITD and other events contributing to increased cell proliferation [9-14]. Although translocations with NUP98 involvement are rare, they are recurrent in different types of leukemia. Generally, the frequency of NUP98 gene fusion has been reported to be less than 5% in adult AML [13, 15, 16].

The presence of NUP98 gene fusions defines a high-risk leukemia subset and has been shown to result in remarkably high induction failure and poor survival [9, 10, 13–21]. Notably, patients with AML harboring NUP98 gene fusions with concomitant FLT3-ITD have a worse prognosis than those without genetic aberrations, and the poor outcomes are determined by the interaction between NUP98 gene fusions and FLT3-ITD [10, 12, 22]. Thanasopoulou et al. [23] found that co-expression of FLT3-ITD increased cell proliferation and maintained self-renewal ability in a NUP98 gene fusion-positive mouse model.

Although NUP98 has a series of functionally diverse partner genes, the most observed NUP98 fusion partners belong to the HOX gene family. The NUP98-HOXA9 gene, resulting from t(7;11)(p15;p15), is the most common fusion gene. Patients with AML harboring the NUP98-HOXA9 rearrangement were found to have poorer overall survival (OS) and relapse-free survival (RFS) than those not harboring this rearrangement, even when patients with low-risk karyotypes were excluded (median OS: 13.5 months vs. 20 months, P = 0.045; median RFS: 6 months vs. 12 months, P = 0.003) [13, 16].

PMX1 is a member of the class II HOX gene family located at 1q23. The function of PMX1 in the hematopoietic system and leukemogenesis remains unknown. Moreover, the formation of the NUP98-PMX1 fusion gene caused by t(1;11)(q23;p15) is rarely reported, and its clinical features and outcomes remain to be clarified. To date, only five cases with NUP98-PMX1 have been reported (Table 1). NUP98-PMX1 juxtaposition was confirmed in this patient using RT-PCR and FISH. As shown in Table 1, five of the six patients (including the patient described here) were male. The median age of patients at diagnosis was 50 years (range 42–74 years). Thus, it seems the t(1;11)(q23;p15) occurs at a higher frequency in older male patients, though more cases are needed to solidify this relationship.

The mechanisms of the NUP98-PMX1 fusion protein underlying leukemogenesis remain unclear. Studies have reported that NUP98-PMX1 but not PMX1 has the ability to impair differentiation and promote proliferation of hematopoietic progenitor cells in vitro [24]. Mice transplanted with NUP98-PMX1-transduced BM cells has a potent effect on induction of myeloproliferative disease [24, 25]. Moreover, the fusion protein might act as an oncogenic transcription factor. It has been shown that the in-frame fusion of PMX1 HD and the N-terminal GLFG repeat of NUP98 results in strong transcriptional activation and PMX1 HD upregulation [5]. Constitutive expression and alteration of the transcriptional activity of PMX1 HD may substantially contribute to myeloid leukemogenesis. This evidence further supports the involvement of NUP98-PMX1 in the occurrence and development of myeloid leukemia.

Since NUP98 gene fusions are now recognized as markers of a high-risk leukemia subset, current treatment paradigms often utilize chemotherapy followed by HSCT during the first complete molecular remission. Our patient with NUP98-PMX1 and concomitant FLT3-ITD achieved molecular remission after induction chemotherapy. Subsequently, he underwent HSCT and was disease-free at the time of the last visit. However, the excellent prognosis of our patient may be due to the relatively short follow-up period.

Since reports of patients carrying the NUP98-PMX1 fusion gene are limited, it is difficult to deduce any conclusions involving the prognostic significance of this gene. This is the first report of a patient with de novo AML carrying the NUP98-PMX1 fusion gene, which may contribute to a more comprehensive profile of this genetic rearrangement. In the future, mechanistic studies are needed to investigate the role of the NUP98-PMX1 fusion gene in leukemia pathogenesis.

Additional file 1. Nucleotide sequences of NUP98-PMX1 fusions by Sanger sequencing.

Additional file 2. The patient’s genome sequencing datasets.