NUP214 fusion genes in acute leukemias: genetic characterization of rare cases

Introduction Alterations of the NUP214 gene (9q34) are recurrent in acute leukemias. Rearrangements of chromosomal band 9q34 targeting this locus can be karyotypically distinct, for example t(6;9)(p22;q34)/DEK::NUP214, or cryptic, in which case no visible change of 9q34 is seen by chromosome banding. Methods We examined 9 cases of acute leukemia with NUP214 rearrangement by array Comparative Genomic Hybridization (aCGH), reverse-transcription polymerase chain reaction (RT-PCR), and cycle sequencing/Sanger sequencing to detect which fusion genes had been generated. Results The chimeras DEK::NUP214, SET::NUP214, and NUP214::ABL1 were found, only the first of which can be readily detected by karyotyping. Discussion The identification of a specific NUP214 rearrangement is fundamental in the management of these patients, i.e., AMLs with DEK::NUP214 are classified as an adverse risk group and might be considered for allogenic transplant. Genome- and/or transcriptome-based next generation sequencing (NGS) techniques can be used to screen for these fusions, but we hereby present an alternative, step-wise procedure to detect these rearrangements.


Introduction
Leukemias are a highly heterogeneous -both phenotypically and pathogeneticallygroup of aggressive neoplasias that affect the production and maturation of bone marrow cells (1,2).They are classified into two major groups: myeloid leukemias that involve more or less mature granulocytes, erythrocytes, megakaryocytes, and/or monocytes, and the lymphoid ones that involve lymphocytes and their precursor cells (3).Based on disease development (classically) or the degree of cellular maturation (currently), leukemias are additionally classified as acute or chronic.Acute myeloid leukemia (AML) is the most common leukemia in adults (4).Depending on phenotypic features such as cellular morphology, but increasingly in later years on which acquired genetic change brought about neoplastic transformation, the various leukemias are subdivided further into entities with distinctive prognostic features dependent on the therapeutic regimens that experience has shown to be preferable (1).For these reasons, one currently pays more attention to (patho) genetic features when diagnosing and treating leukemia patients; a precise understanding of what lies behind leukemogenesis in individual patients has become adamant in clinical state-of-theart medicine.To do this, studies of the leukemic cells with different techniques are necessary.
In this study, we selected for examination 9 acute leukemia cases with rearrangements of chromosomal band 9q34 detected either by Gbanding and/or FISH, but excluding cases displaying the translocation t(9;22)(q34;q11) that characterizes Philadelphia (Ph)-positive leukemias.We used array comparative genomic hybridization (aCGH), reverse-transcription polymerase chain reaction (RT-PCR), and cycle sequencing/Sanger sequencing to identify the fusion genes generated by the different genomic rearrangements.

Material
Nine acute leukemias, from 6 males and 3 females aged between 8 and 78 years (Table 1), were investigated diagnostically in our laboratory during 2011 and 2023.Bone marrow aspirated and/or blood sampleswere found by karyotype/FISH analysis to carry cells with rearrangement of chromosomal band 9q34 other than the t (9;22) specific for Ph-positive leukemias.The study was approved by the Regional Committee for Medical and Health Research Ethics (REK, project number S-0747a; http://helseforskning.etikkom.no.

G-banding and fluorescence in situ hybridization analyses
Hematopoietic cells were short-term cultured, harvested, stained for G-banding analysis, and analyzed cytogenetically as previously described (5,6).The karyotype description followed the recommendations of the International System of Cytogenomic Nomenclature (7).In addition, routine leukemia-relevant FISH analyses were performed according to standard protocols.For acute myeloid leukemia (AML), tests were performed using commercial  Additional FISH investigations to detect rearrangement of the NUP214 locus (9q34) were performed using the Dual Color Break Apart Probe (Zytovision, ZytoLight, Bremerhaven, Germany) on those cases from which suitable material was available.Chromosome preparations were counterstained with 0.2 mg/ml DAPI and overlaid with a 24×50 mm 2 coverslip.Fluorescent signals were captured and analyzed using the CytoVision system (Leica Biosystems, Newcastle, UK).

DNA and RNA extraction and sequencing
Fresh frozen bone marrow or blood was used to extract DNA and RNA.DNA was extracted using the Maxwell 16 extractor (Promega, Madison, Wisconsin, United States) and purified using the Maxwell 16 Cell DNA Purification kit (Promega) according to the manufacturer's recommendations.RNA was extracted using a miRNeasy kit (Qiagen, Hilden, Germany) or the Monarch Total RNA miniprep kit (New England Biolabs, Ipswich, Massachusetts, USA).The concentrations were measured using QIAxel microfluidic UV/VIS spectrophotometer (Qiagen) and Quantus fluorometer (Promega).

Array comparative genomic hybridization
Array Comparative Genomic Hybridization (aCGH) using CytoSure Consortium Cancer + SNP arrays (Oxford Gene Technology, Oxford, UK) was performed to screen for genomic imbalances.Data were analyzed using Agilent Feature Extraction Software (Agilent Technologies; version 10.7.3.1) and CytoSure Interpret Software (Oxford Gene Technology; version 4.9.40).
Copy number aberrations (CNA) were identified using the Circular Binary Segmentation (CBS) algorithm, adding a custommade aberration filter defining imbalances as a region with a minimum of five gained/lost probes (8).Imbalances were scored according to (9); however, also those smaller than 5Mb were considered.Annotations were based on the human reference sequence GRCh37/hg19 (10).

Reverse transcription polymerase chain reaction
One µg of total RNA was reverse-transcribed in a 20 µL reaction volume using iScript Advanced cDNA Synthesis Kit for RT-PCR according to the manufacturer's instructions (Bio-Rad, Hercules, CA, USA).Primers used for PCR reactions are listed in Supplementary Table 3.The PCR amplifications were run on a C-1000 Thermal cycler (Bio-Rad) with an initial denaturation at 94°C for 30 sec followed by 35 cycles at 98°C for 7 sec, 60°C for 30 sec, and a final extension at 72°C for 1 min.Three µL of the PCR product were stained with GelRed (Biotium, Hayward, CA, USA), analyzed by electrophoresis through 1.0% agarose gel, and photographed.The remaining PCR products were purified using the MinElute PCR Purification Kit (Qiagen) and sequenced using an Applied Biosystems SeqStudio Genetic Analyzer system with BigDye ™ Terminator v1.1 Cycle Sequencing Kit (ThermoFisher Scientific, Waltham, MA, USA) according to the company's recommendations.The basic local alignment search tool (BLAST) software (BLAST; https://blast.ncbi.nlm.nih.gov/Blast.cgi;accessed on 15 April 2023) was used for computer analysis of sequence data (11).The BLAT alignment tool and the human genome browser at the University of California, Santa Cruz (UCSC) were also used to map the sequences on the Human GRCh37/hg19 assembly (BLAT; http://genome.ucsc.edu/cgi-bin/hgBlat;accessed on 15 April 2023) (12).

Quantitative RT-PCR analyses
qRT-PCR was carried out to determine the expression level of T-cell leukemia homeobox 3 (TLX3) in T-ALL patients using the CFX96 Touch Real-Time PCR detection system (Bio-Rad Laboratories).Reactions were carried out in triplicate using TaqMan Assays and the TaqMan Universal Master Mix II with UNG (Applied Biosystems, Foster City, CA) according to the manufacturer's protocol.The thermal cycling parameters consisted of 40 cycles of an initial denaturation step of 95°C for 10 minutes, followed by 60°C for 1 min.The primers used for the qRT-PCR reaction were TLX3 (Hs00253271_m1; ThermoFisher) and, as an endogenous control, actin beta (ACTB; Hs01060665_g1, ThermoFisher).Human universal reference total RNA (Clontech, Mountain View, CA) was used as internal reaction control.

Results
Four of the nine leukemias (cases 2, 3, 4, and 5) had t(6;9)(p22;q34) as a sole aberration by G-banding.Case 1 had trisomy 15 in addition to the t(6;9).Altogether, five of the leukemias were diagnosed as AML (cases 1-5).One acute undifferentiated leukemia (AUL; case 7) showed a complex, near-tetraploid karyotype.Cases 8 and 9, diagnosed as near ETP-ALL and T-ALL, showed a normal karyotype and the chromosomal deletion del(11)(q14), respectively.The karyotype from the ALAL-NOS patient (case 6) was not available.All karyotype descriptions as well as FISH profiles are listed in Table 1   The imbalances detected by aCGH analyses are presented in Supplementary Table 2. Genomic imbalances were detected in cases 1, 5, 6, 7, 8, and 9.A total of 69 imbalances were scored (range 1 to 29), with losses being more frequent than gains.
FISH was performed to validate NUP214-rearrangements using a break-apart probe (Table 1).The expression level of TLX3 was investigated in the T-ALL cases.The mean quantification cycle (Cq Mean) of TLX3 was found to be 18.65 and 21.53 for cases 8 and 9, respectively.The Cq Mean for ACTB was 18.90 and 22.37 for cases 8 and 9, respectively (both T-ALL); the human reference control gave a Cq Mean of 20.01.

Discussion
Rearrangements of chromosomal band 9q34 are common and important in the genesis and classification of leukemias ('https:// mitelmandatabase.isb-cgc.org/').The most common gene-fusion result by far is the BCR::ABL1 generated by the t(9;22)(q34;q11) translocation that is specific for chronic myeloid leukemias but also occurs in Ph-positive acute leukemias.Other leukemia-relevant genes mapping to 9q34 include SET and NUP214.The differentiation between these rearrangements has important therapeutic implications.G-banding and/or FISH investigations can hint at gene fusions generated by inter-chromosomal rearrangements, e.g., the t(6;9) leading to DEK::NUP214 fusion; however, this is not the case with cryptic intra-chromosomal rearrangements such as SET::NUP214 and NUP214::ABL1 (all three genes map within 9q34).Furthermore, standard diagnostic FISH analyses using commercial dual fusion probes for BCR::ABL1 (used whenever CML or ALL is suspected) may sometimes show an abnormal signal pattern for ABL1 (mostly the presence of an extra signal) but no fusion with BCR.Next generation sequencing, at both DNA and RNA level, is an effective method to screen for presence of cryptic rearrangements/fusion genes.However, the commercial panels available for diagnostic purposes have specific targets and may therefore miss less common gene-fusions.Furthermore, although the screening ability of NGS technologies is indisputable; its use in standard leukemia diagnostics is still limited.We here present a combination of cytogenetic and molecular methods that can be used in a stepwise manner to identify cryptic rearrangements in neoplastic cells.
Patients with DEK::NUP214 account for around 1% of all AML cases (1) and include both children (mean age 10 years) and adults (mean age 45 years) (29, 30); our data are in accordance with previous literature as DEK::NUP214fusion was present in four adults and a 15-year-old girl with AML.The exact role of DEK:: NUP214 in leukemogenesis is still unknown, but Ageberg et al. showed that it could promote phosphorylation of the eukaryotic translation initiation factor 4E (eIF4E) and activation of the mTOR signaling pathway, increasing myeloid cell proliferation (31,32).
SET::NUP214 was found in two patients, with ALAL-NOS and AUL, respectively, (cases 6 and 7).In both cases, FISH analysis using an ABL1 probe showed only one fluorescent signal on 9q34/ ABL1.The genomic imbalances detected by aCGH included loss of a genomic region at 9q34 with breakpoints inside the abovementioned genes (SET and NUP214).RT-PCR, followed by sequencing, detected the SET::NUP214 fusion gene in both cases.The mechanism by which SET::NUP214 mediates leukemogenesis has not been fully elucidated, but the chimeric gene encodes a fusion protein that inhibits cell apoptosis by cytotoxic T-cells (33).NUP214 showed the same breakpoint (exon 18) in types of fusion (DEK::NUP214 and indicating that a similar pathogenetic mechanism, namely deregulation of the DEK and SET histone binding proteins, could be critical leading to abnormal histone hypoacetylation and silencing of genes important for lineage commitment and hematopoietic differentiation (2).
FISH analysis in case 8 and 9 using a commercial ABL1 probe showed amplification of the gene.Graux et al. (23) were the first to show that, in such cases, the ABL1 gene is involved in a cryptic rearrangement with NUP214 creating from one to multiple extrachromosomal episome(s) containing the NUP214::ABL1 fusion genes not detectable by karyotyping.RT-PCR confirmed the presence of a 9q34 rearrangement resulting in the fusion of exon 29 of NUP214 with exon 2 of ABL1 in case 9, and fusion of exon 31 of NUP214 with exon 2 of ABL1 in case 8.This is the first time that a NUP214::ABL1 fusion gene was found together with a PCM1:: FGFR1 transcript (case 8).The patient was a 23-year-old previously healthy man diagnosed with near, early precursor Tcell leukemia (near ETP-ALL) after 2-3 weeks with night sweats, weight loss, and enlarging lymph nodes on his neck.Upon admission, anemia and thrombocytopenia were noted and leukocytes were increased to 106 x 10 9 /L (normal range 3-10).
The final diagnosis in this case was made based on examination of the bone marrow and a lymph node biopsy.Treatment with dexamethasone, vincristine, doxorubicine, asparginase, methotrexate, and mercaptopurine according to European ALL Together protocol was then started (34), whereas imatinib was added afterwards.The patient became minimal residual disease (MRD) negative and achieved complete remission on day 78.The second gene fusion in this case, PCM1::FGFR1, was also described by Kasbeker et al. (35 ) in a patient suffering from a rare, aggressive myeloid neoplasia with eosinophilia; treatment with futibatinib, an oral selective small molecule inhibitor of FGFR1-4, resulted in complete hematologic and cytogenetic remission (35).The PCM1 gene as partner in the chimera PCM1::JAK2 has been implicated in the pathogenesis of also other acute myeloid/lymphoid neoplasms (36) whereas rearrangements of FGFR1 are rare (37).The use of selective TKIs can potentially affect outcomes in these patients (35,37).Because FGFR inhibitors may be used as first-line therapy for patients with myeloid/lymphoid neoplasms with FGFR1 rearrangement, gene-level diagnosis testing for such aberrations is clinically important (35).Finally, the detection of both NUP214::ABL1 and PCM1::FGFR1 fusions in the near ETP-ALL of case 8 leaves no information as to which fusion was the primary and which was secondary in this leukemia.
Aberrant expression of transcription factor oncogenes such as TLX3 plays a major role in the pathogenesis of T-ALL and defines distinct molecular groups with prognostic significance (38).Our two T-ALL cases were also characterized by aberrant expression of TLX3.T-ALL patients co-expressing TLX3 and NUP214::ABL1 fusion transcript may belong to a subgroup of poor survival (39).However, data are still too limited to conclude.
Finally, some methodological comments on the scoring principles of aCGH analyses may be warranted.Many laboratories, including ours, follow standard guidelines/recommendations (9,40,41) as to the size of which imbalances to score and which not.If one sticks to a requirement of size more than 5 Mb for any imbalance to be paid attention to, one risks overlooking small but important changes.This would have been the case for patients 6, 7, 8, and 9 of our series.Therefore, when determining the scoring of imbalances, each clinical laboratory applies its own professional judgment to the specific circumstances presented by individual patients or specimens, reporting all the imbalances deemed clinically relevant; this is also fundamental to avoid false positives in case of low-quality results.Based on our experience and the results reported here, we recommend checking and reporting all imbalances, irrespective of their size.One should also note which genes are affected so as to identify different pathways leading to the same phenotypic disease, or even identify possible therapeutic approaches associated with the rearrangement of specific genes in other contexts.
In conclusion, the use of different methodological approacheswhenever possible a combination of karyotyping, FISH and molecular methods -is often necessary to detect genomic NUP214 rearrangements in leukemic cells.The identification of a specific NUP214 rearrangement is fundamental to choosing an optimal therapeutic strategy: by way of example, patients with DEK::NUP214 are classified as an adverse risk group within AML and might be considered candidates for allogenic bone marrow transplantation.
research was funded by The Norwegian Radium Hospital (Radiumhospitalets legater).

1
FIGURE 1 Overview of genes mapping on 9q34 and genomic imbalances scored on this chromosomal band.(A) Ideogram of chromosome 9 showing the mapping position of SET, ABL1, and NUP214; (B) loss of 33.62 kb genomic material from case 8. (C) gain of 97.54 kb genomic material from case 9; in addition, loss of 9p is shown.Arrows are pointing to the imbalances at 9q34.

TABLE 1
Overview of clinical diagnostic parameters and molecular analysis of the nine acute leukemias.

Table 1 ;
; Supplementary Table 1.The FISH analyses were those r e c o m m e n d e d b y t h e W H O ( 1 ) , n a t i o n a l ( h t t p s : / / www.helsedirektoratet.no/retningslinjer/maligne-blodsykdommerhandlingsprogram),ALLTogether (ClinicalTrials.govIdentifier: NCT04307576), and NOPHO (2008) protocols (13) as standard for the diagnosis of AML and ALL.Signal patterns detected by FISH did not show any aberrations for any probes except for ABL1 probe (9q34).These FISH data, together with the NUP214 signal pattern, are summarized in Supplementary Table 1.Mutation analysis and flow cytometry data are shown in Supplementary Table