Wang et al. BMC Pediatrics(2024) 24:547
https://doi.org/10.1186/s12887-024-05007-3

BMC Pediatrics

RESEARCH

Open Access

NUP98::NSD1 and FLT3/ITD co-expression isan independent predictor of poor prognosis
in pediatric AML patients
Jing-wen Wang1,2, Yu-Li1,2, Xing-Ge Yang3* and Lu-Hong Xu1,2*
Abstract
Objective Patients who carry NUP98::NSD1 or FLT3/ITD mutations are reported to have poor prognosis. Previous
studies have confidently reported that the poor outcome in younger AML patients is owning to dual NUP98::NSD1and FLT3/ITD positivity, with a high overlap for those two genetic lesions. In this study, we assessed the prognostic
value of the presence of both NUP98::NSD1 and FLT3/ITD in pediatric AML patients.
Methods We screened a large cohort of 885 pediatric cases from the COG-National Cancer Institute (NCI) TARGETAML cohort and found 57 AML patients with NUP98 rearrangements.
Results The frequency of NUP98 gene fusion was 10.8% in 529 patients. NUP98::NSD1 fusion was the most common
NUP98 rearrangement, with a frequency of 59.6%(34 of 57). NUP98::NSD1 -positive patients who carried FLT3/
ITD mutations had a decreased CR1 or CR2 rate than those patients carried FLT3/ITD mutation alone (P = 0.0001).
Moreover, patients harboring both NUP98::NSD1 fusion and FLT3/ITD mutation exhibited inferior event-free survival(EFS, P < 0.001) and overall survival (OS, P = 0.004) than patients who were dual negative for these two genetic lesions.
The presence of only NUP98::NSD1 fusion had no significant impact on EFS or OS. We also found that cases with highFLT3/ITD AR levels ( > = 0.5) with or without NUP98::NSD1 had inferior prognosis. Multivariate analysis demonstrated
that the presence of both NUP98::NSD1 and FLT3/ITD was an independent prognostic factors for EFS (hazard ratio:
3.2, P = 0.001) in patients with pediatric AML. However, there was no obvious correlation with OS (hazard ratio: 1.3,
P = 0.618). Stem cell transplantation did not improve the survival rate of cases with NUP98 fusion or NUP98::NSD1 AML
in terms of EFS or OS.
Conclusion Presence of both NUP98::NSD1 and FLT3/ITD was found to be an independent factor for dismal prognosis
in pediatric AML patients. Notably, lack of FLT3/ITD mutations in NUP98::NSD1 -positive patients did not retain its
prognostic value.
Keywords NUP98::NSD1, Nucleoporin gene 98, FLT3/ITD, Pediatric, Acute myeloid leukemia (AML)

Jing-wen Wang and Yu-Li Contributed equally to this work.
*Correspondence:
Xing-Ge Yang
13783186162@163.com
Lu-Hong Xu
xulhong@mail.sysu.edu.cn

1

Department of Pediatrics, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen
University, Guangzhou, China
2
Key Laboratory of Malignant Tumor Gene Regulation and Target Therapyof Guangdong Higher Education Institutes, Sun Yat-Sen University,
Guangzhou, China
3
Department of Pediatrics, The First Affiliated Hospital of Henan Universityof Science and Technology, Luoyang, Henan, China© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0
International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you
give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified thelicensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to thematerial. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation
or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://
creativecommons.org/licenses/by-nc-nd/4.0/.

Wang et al. BMC Pediatrics(2024) 24:547

Introduction
As the second most common pediatric hematologic
malignancy, acute myeloid leukemia (AML) is a groupof diseases with various clinical presentations, genetic
alterations, and responses to treatment [1]. Cytogenetic
and molecular abnormalities significantly contribute to
the risk stratification of pediatric acute myeloid leukemia
patients and serve as crucial prognostic factors [2]. The
expert consensus recommended by the 2017 European
Leukemia Network supports inclusion of the nucleoporin 98 fusion gene and its associated molecular genetic
abnormalities in AML risk stratification to guide treatment [3]. NUP98 has been detected with more than 30
partner genes [4–7]in human leukemia, including themost common gene, NUP98::NSD1 [8–10]. Patients whocarry NUP98::NSD1 gene fusions have dismal outcomes[9, 11–13]. Leukemic blasts frequently carry the FLT3/
ITD mutation accompanying the NUP98::NSD1 genefusion [5, 12]. FLT3/ITD mutation in pediatric AML
patients results in poor prognosis according to previous reports [14–18]. Though the clinical value of dual
NUP98::NSD1 and FLT3/ITD anomalies in younger AML
patients has been described [12]. This study comprehensively analyzed the clinical significance of these two
genetic lesions in pediatric AML patients younger than
18 years of age and their impact on prognosis.
MethodData sources

The TARGET dataset contains necessary data on AML
patients under 18 years of age (https://ocg.cancer.gov/
programs/target/data-matrix). 885 pediatric patientswere screened who were diagnosed between1996 and2010, with the last years of follow-up until 2015. AML
diagnosis and risk classification were performed based on
the Children’s Oncology Group (COG) guidelines [19].
AML subtypes were divided based on French-AmericanBritish (FAB) classifications. The AML treatment protocols used were AAML03P1, AAML0531, and CCG-2961.
Patients in the first complete remission underwent stem
cell transplantation (SCT).
Statistical evaluation

Descriptive statistics were used to summarize the clinical and biological features of the patients. For categoricalvariables, chi-square analysis and Fisher’s exact test wereadopted; for continuous variables, the nonparametricMann-Whitney U test was adopted. The study utilized
the following criteria to evaluate clinical outcomes: complete remission (CR), event-free survival (EFS), and overall survival (OS). CR is achieved when the bone marrow
has less than 5% blasts at the end of induction. EFS was
defined as the time between diagnosis and the first event,including induction failure, recurrence, or any cause ofPage 2 of 11

death. OS was defined as the time between diagnosis anddeath from any cause. EFS and OS were assessed andcompared by the Kaplan-Meier method and the log-rank
test. Multivariate Cox regression analysis was adopted toevaluate the independent impact of prognostic factors
on OS and EFS. Hazard ratios (HRs) of multivariate Cox
regression are presented with 95% confidence intervals.
A P−value less than 0.05 indicated statistically significance. The data were evaluated with the SPSS software
version 25.0 (IBM Corporation, Armonk, NY, USA).

Results
Clinical and molecular characteristics of NUP98 fusion
genes

We screened a large pediatric cohort from the COGNational Cancer Institute (NCI) TARGET AML cohort.
A total of 885 AML pediatric patients were included in
the TARGET database. The fusion gene status of 356 children was unknown, which was considered missing data
to be excluded, so 529 patients were ultimately included
as the research subjects (Supplementary Table 2 /Table
2.1). Fifty-seven had NUP98 rearrangements, for a totalfrequency of 10.8%. In this study, we identified six kinds
of NUP98 fusion genes. The most common NUP98fusion gene was NUP98::NSD1 (59.6%, 34 of 57), followed
by NUP98/KMD5A (17.5%, 10 of 57), NUP98/HOXD13(7%, 4 of 57), NUP98/HMGB3, NUP98/HOXA9, and
NUP98/PHF23 (5.3%, 3 of 57).
Comparison of the clinical and molecular characteristics of NUP98-positive and NUP98-negative patients
revealed no significant differences in gender, medianage, or median WBC count at diagnosis (Table 1).
However, the NUP98::NSD1 fusion gene patients hadgreater median WBC of 100.8 × 10^9/L than did the
NUP98::NSD1 negative patients (P = 0.002) (Table 2).
Regarding cytomorphology status, there were significant differences between the NUP98-positive group andthe NUP98-negative group (50.9% vs. 2.3%, P < 0.001)(Table 1).
Compared with NUP98-negative patients, NUP98positive patients more often exhibited the FAB M7 phenotype (10.5% vs. 2.3%, P = 0.001). We also found that
NUP98 fusion gene cases highly overlapped with FLT3/
ITD-positive cases (45.6% vs. 6.1%, P < 0.001) (Table 1)
and that NUP98::NSD1fusion gene cases are highly
overlapped with FLT3/ITD positivity (64.7% vs. 6.7%,
P < 0.001) (Table 2).
Prognosis of NUP98 fusion genes carriers

We initially evaluated the impact of NUP98 fusion gene
positivity on prognosis. In terms of treatment response(Table 1), 80.1% (378 of 472)of NUP98-negative patientsachieved a favorable CR in course 1 which was greaterthan the 59.6% (34 of 57) NUP98-positive patients

Wang et al. BMC Pediatrics(2024) 24:547

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Table 1 Characteristics of study population according to NUP98
gene fusion status

Total
ProtocolAAML03P1, N (%)
AAML0531, N (%)
CCG-2961, N (%)
GenderMale, N (%)
Female, N (%)
Median age at
diagnosis (years,
range)Median WBC
×10^9/L (range)
FAB type, N (%)
M0
M1
M2
M4
M5
M6
M7
Treatment
response
CR status at theend of course1,
N (%)
CR status at theend of course2,
N (%)
HSCT in CR1
FLT3/ITD
Positive, N (%)
Negative, N (%)
WT1 mutation
Positive, N (%)
Negative, N (%)
NPM1mutation
Positive, N (%)
Negative, N (%)
Cytogenetic
statusNormal, N%
Abnormal, N%
Risk group
Low
Standardhigh

All patient NUP98+

NUP98-

529

57

472

55
438
36

5 (8.8%)47 (82.4%)
5 (8.8%)270259
8.9 (0-17.9)

34 (59.6%)
23 (40.4%)
9.8 (1.0-17.6)

50 (10.6%) 0.670
391 (82.8%) 0.942
31 (6.6%)
0.533
0.169
236 (50%)236 (50%)
8.5 (0-17.9) 0.293

40.5(0.2–610)

53.5 (1.1-447.3) 39 (0.8–610) 0.160

8
36
139
143
117
4
17

1 (1.8%)8 (14.0%)
11 (19.3%)
14 (24.6%)
7 (12.3%)
4 (7.0%)6 (10.5%)

7 (1.5%)28 (5.9%)128 (27.1%)
129 (27.3%)
110 (23.3%)
0 (0%)
11 (2.3%)

Table 2 Characteristics of study population according to
NUP98::NSD1 gene fusion statusP-value

0.874
0.028
0.205
0.657
0.058
< 0.001
0.001

412

34 (59.6%)

378 (80.1%) < 0.001

462

38 (66.7%)

424 (89.8%) < 0.001

51

11 (19.3%)

40 (8.5%)

0.009

55
471

26 (45.6%)
31 (54.4%)

29 (6.1%)< 0.001440 (84.7%) < 0.001

32487

15 (26.3%)
42 (73.7%)

17 (3.6%)< 0.001445 (94.3%) < 0.001

8511

3 (5.3%)54 (94.7%)

5 (1.1%)0.014
457 (96.8%) 0.412

40479

29 (50.9%)
22 (38.6%)

11 (2.3%)< 0.001457 (96.8%) < 0.001

242
238
41

4 (7.0%)30 (52.6%)
19 (33.3%)

238 (50.4%) < 0.001
208 (44.1%) 0.402
22 (4.7%)< 0.001

Data for FAB classification, FLT3/ITD, NPM1, WT1 mutation, cytogenetic status andrisk group were missing for some patients

NUP98::NSD1 +Total, N (%)
ProtocolAAML03P1
AAML0531
CCG-2961
GenderMale, N (%)
Female, N (%)
Median age at diagnosis (years, range)
Median WBC ×10^9/L(range)
FAB type, N (%)
M0
M1
M2
M4
M5
M6
M7
Treatment response
CR status at the end ofcourse1 N (%)
CR status at the end ofcourse2, N (%)
HSCT in CR1
FLT3/ITD
Positive, N (%)
Negative, N (%)
WT1 mutation
Positive, N (%)
Negative, N (%)
NPM1 mutation
Positive, N (%)
Negative, N (%)
Cytogenetic status
Normal, N%
Abnormal, N%
Risk group
Low
StandardhighP-value34

NUP98::NSD1
495

2(5.9%)
29(85.3%)3(8.8%)

53(10.7%)409(82.6%)33(6.7%)

0.373
0.690
0.629

24(70.6%)
10(29.4%)
10.3(1.2–17.6)

246(49.7%)249(50.3%)
8.5 (0-17.9)

0.018
0.018
0.169

100.8(1.1-447.3)

36 (0.8–610)

0.002

0
3(8.8%)
7(20.6%)
10(29.4%)
4(11.8%)2(5.9%)
3(8.8%)

8(1.6%)
33(6.7%)132(26.7%)
133(26.9%)113(22.8%)2(0.4%)
14(2.8%)

0.455
0.629
0.436
0.747
0.133
< 0.0010.055

15(44.1%)

397(80.2%)< 0.00117(50.5%)445(89.9%)< 0.0017(20.6%)44(8.9%)0.025

22(64.7%)
12(35.3%)33(6.7%)459(92.7%)< 0.001< 0.00113(38.2%)
21(61.7%)19(3.8%)466(94.1%)< 0.001< 0.0010(0.0%)
34(100%)8(1.6%)
477(96.4%)

0.455
0.258

19(55.9%)
10(29.4%)21(4.2%)469(94.7%)< 0.001< 0.0011(2.9%)
14(41.2%)
16(47.1%)

241(48.7%)224(45.3%)25(5.1%)< 0.001
0.644< 0.001

Data for FAB classification, FLT3/ITD, NPM1, WT1 mutation, cytogenetic status andrisk group were missing for some patients(P < 0.001). After the second course of induction therapy,
CR was achieved in 38 of 57 (66.7%) patients with NUP98
gene fusion and in 424 (89.8%) of 472 patients without
NUP98 gene fusion (P < 0.001).
A total of 885 AML pediatric patients were includedin the TARGET database, and 57 patients with NUP98
fusion gene positivity were analyzed in this study. The

Wang et al. BMC Pediatrics(2024) 24:547

fusion gene status of 356 children was unknown, which
was considered missing data; thus, 529 patients were
included as the research subjects (Supplementary Table
2 /Table 2.1). Survivors were followed up for a medianof 5.6 years among all 529 patients with and without the
NUP98 fusion gene positivity. As shown in Fig. 1(A),patients harboring a NUP98 fusion gene had an inferior
EFS (32.4%) compared with wild-type patients (49.2%,
P = 0.002). Figure 1(B) illustrated a trend toward worse
OS in NUP98 fusion gene patients(48.1%)than in NUP98
wild-type patients(67.0%, P = 0.006).
Stratification analysis of FLT3/ITD and NUP98 fusion genes

NUP98 fusion gene patients had a high frequency of
45.6% (26 of 57) in cooperating genetic alterations with
FLT3/ITD mutation. In the 55 patients with positive
FLT3/ITD mutation, the CR rate 1 in patient who carried
an NUP98 gene fusion was lower at 34.6% (9 of 26) compared to the rate of 79.3% (23 of 29) observed in patients
without NUP98 gene fusion (P = 0.0008), as well the CR
rate 2 in dual positive of NUP98 gene fusion and FLT3/
ITD was 42.3%(11/26)compared with cases without
NUP98 gene fusion 89.7% (26 of 29) (P = 0.0002)(Supplementary Table 1).
As shown in Fig. 2 and Supplementary Table 3, withrespect to the FLT3/ITD mutation, patients with NUP98positive and FLT3/ITD mutation had the worst prognosis with a 5-year OS of 41.4 ± 10.3% and a 5-year EFS
of 13.5 ± 7.0% compared with patients with wild-type
FLT3/ITD without the NUP98 gene fusion patients (OS68.0 ± 2.3%, P = 0.002; EFS 50.3 ± 2.4%, P < 0.001) (Supplementary Fig. 1A, B). According to the NUP98 gene fusion

Page 4 of 11

status and FLT3/ITD status, the survival curves weregrouped into 4 subgroups (Fig. 2). In FLT3/ITD wild-type
cases, NUP98-positive patients did not significantly affect5-year OS (54.0 ± 9.1% NUP98 + vs. 68.0 ± 2.3% NUP98; P = 0.188) or EFS (48.0 ± 9.0% NUP98 + vs. 50.3 ± 2.4%
NUP98-; P = 0.862) (Supplementary Fig. 1.1A, B).
According to the NUP98 gene fusion status, FLT3/ITDpositive patients had a negative impact on the 5-year
EFS rate (13.5 ± 7.0% NUP98 + vs. 31.0 ± 8.6% NUP98-;
P = 0.008) but not on the 5-year OS rate (41.4 ± 10.3%
NUP98 + vs. 55.0 ± 9.3% NUP98-; P = 0.133) (Supplementary Fig. 1.2A, B). A tending negative impact was shown
by FLT3/ITD positive patients on 5-year EFS (31.0 ± 8.6%
FLT3/ITD positive vs. 50.3 ± 2.4% FLT3/ITD wild-type;
P = 0.056) in NUP98 negative patients, but not on 5-year
OS (55.0 ± 9.3%FLT3/ITD positive vs. 68.0 ± 2.3% FLT3/
ITD wild-type; P = 0.242) (Supplementary Fig. 1.3A, B).
Stratification evaluation of FLT3/ITD in NUP98::NSD1
patients

Among 26 patients positive for both NUP98 gene fusion
and FLT3/ITD, 84.6% (22 of 26) of patients harbored
NUP98::NSD1 and FLT3/ITD. The prognostic effect of
NUP98::NSD1 in patients who carried FLT3/ITD wasthen evaluated. The CR rate after the first course of induction therapy in those with both NUP98::NSD1 and FLT3/
ITD was 27.3% (6 of 22) compared with 78.8% (26 of 33)
for FLT3/ITD mutation patients without NUP98::NSD1(P = 0.0001).The CR rate after second course of induction therapy in patients carrying NUP98::NSD1 andFLT3/ITD was 36.4% (8 of 22) compared with 87.9%(29Fig. 1 Survival curves of pediatric AML patients with and without NUP98 gene fusion. A Probability of EFS for all patients with and without NUP98 genefusion. B Probability of OS for all patients with and without NUP98 gene fusion

Wang et al. BMC Pediatrics(2024) 24:547

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Fig. 2 Survival curves of pediatric AML patients according to combined NUP98 gene fusion and FLT3/ITD status. A Probability of EFS for AML patients. B
Probability of OS for AML patients. Pairwise comparison data provided in Supplementary Figs. 1-1.3 and Supplementary Table 3of 33)for patients with FLT3/ITD mutation but without
NUP98::NSD1 (P = 0.0001) (Supplementary Table 1).
As shown in Fig. 3(A), in all 529 patients, patients harboring NUP98::NSD1 had an inferior EFS (26.1 ± 7.6%)
compared with patients who were NUP98::NSD1 -negative (48.9 ± 2.3%, P < 0.001). Additionally, NUP98::NSD1
patients displayed a trend toward worse OS(50.1 ± 8.9%)
compared
to
NUP98::NSD1
negativepatients(66.0 ± 2.2%, P = 0.029) (Fig. 3(B)). In analysis restricted
to 57 NUP98 fusion genes patients, there were no significant differences in OS between NUP98::NSD1(+) and
NUP98::NSD1(-) patients, with EFS being poor in both(26.1 ± 7.6% vs. 41.9 ± 10.5%, P = 0.026) (Fig. 3(C, D)).
When examining only patients who were
NUP98::NSD1 positive and harbored FLT3/ITD mutation, we found unfavorable 5-year OS (41.7 ± 11.0%) and5-year EFS (13.6 ± 7.3%) outcomes compared with thoseof FLT3/ITD wild-type cases without NUP98::NSD1(OS 67.1 ± 2.3%, P = 0.004; EFS 50.1 ± 2.4%, P < 0.001)(Fig. 4B/ Supplementary Fig. 2A, B). Survival curves
were generated based on NUP98::NSD1 status and
FLT3/ITD status (Fig. 4(A, B) and Supplementary
Table 4), revealing that NUP98::NSD1 patients had no
impact on 5-year EFS (50.0 ± 14.4% NUP98::NSD1 + vs.
50.1 ± 2.4%NUP98::NSD1 -; P = 0.705) or OS (65.6 ± 14.0%
NUP98::NSD1 + vs. 67.1 ± 2.3% NUP98::NSD1 -; P = 0.900)
in FLT3/ITD-negative patients (Supplementary Fig. 2.1A,
B). FLT3/ITD-positivity had a negative impact on 5-year
EFS (29.0 ± 8.0% FLT3/ITD-positive vs. 50.1 ± 2.4% FLT3/
ITD-wild-type; P = 0.025) in NUP98::NSD1-negative

patients but not on 5-year OS (53.0 ± 8.9% FLT3/ITDpositive vs. 67.1 ± 2.3% FLT3/ITD-wild-type; P = 0.190)(Supplementary Fig. 2.2A, B).
Overall, FLT3/ITD mutation patients had a negative
impact on 5-year EFS (13.6 ± 7.3% NUP98::NSD1 + vs.
29.0 ± 8.0% NUP98::NSD1 -; P = 0.005) but not on
5-year OS (41.7 ± 11.0% NUP98::NSD1 + vs. 53.0 ± 8.9%
NUP98::NSD1 -; P = 0.147) according to NUP98::NSD1
status (Supplementary Fig. 2.3A, B).
Stratification evaluation of the thresholds FLT3/ITD AR and
NUP98::NSD1

As illustrated in Fig. 5(A, B), patients harboring
NUP98::NSD1 gene fusion with a high FLT3/ITD AR had
poor prognosis with a 5-year OS of 33.3 ± 13.6%, however,there was no significant difference compared with the55.6 ± 16.6% in patients with low FLT3/ITD AR. The reason may be the small sample size.
In analysis restricted to high FLT3/ITD AR, patients
had poor 5-year EFS prognosis, regardless of thepresence or absence of NUP98::NSD1 (23.1 ± 11.7%
NUP98::NSD1 + vs. 26.5 ± 9.9% NUP98::NSD1 -, P = 0.214).
Figure 5(C, D) indicated that patients with high FLT3/
ITD AR and NUP98::NSD1 gene fusion positivity had ashorter 5-year OS of 33.3 ± 13.6%, which was not significantly different from the 55.5 ± 11.1% of patients without
NUP98::NSD1 fusion.
As illustrated in Fig. 5(E, F), for patients with low FLT3/
ITD AR, NUP98::NSD1 had no impact on prognosis, witha 5-year OS of 55.6 ± 16.6% compared with 48.6 ± 14.8%
for patients without NUP98::NSD1 (P = 0.796).

Wang et al. BMC Pediatrics(2024) 24:547

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Fig. 3 Survival curves of pediatric AML patients with and without NUP98::NSD1. A Probability of EFS for all patients with and without NUP98::NSD1. B
Probability of OS for all patients with and without NUP98::NSD1. C Probability of EFS for NUP98 positive patients with and without NSD1 gene fusion. D
Probability of OS for NUP98 positive patients with and without NSD1 gene fusion

The impact of SCT in pediatric AML patients carrying
NUP98 gene fusion

Among the 57 NUP98-positive cases, only 11 patients
received allogeneic SCT, and data were available for 40
patients. Evaluation of the effect of SCT on NUP98 genefusion patients revealed that the therapy did not have asignificant effect on the clinical outcome with respect
to 5-year EFS (36.4 ± 14.5% SCT vs. 43.0 ± 9.5% no SCT,
P = 0.962) or OS (50.5 ± 15.8% SCT vs. 53.1 ± 9.6% no SCT,

P = 0.923) rates when compared with chemotherapy (noSCT) Fig. 6(A, B).
The survival curses depicted In Fig. 6(C, D), demonstrated that SCT had no significant effect on the 5-year
EFS (42.9 ± 18.7% of patients with SCT vs. 40.0 ± 14.6% of
patients with non-SCT, P = 0.717) or OS (68.6 ± 18.6% of
patients with SCT vs. 65.6 ± 14.0% of patients with nonSCT, P = 0.802) compared to chemotherapy (no SCT) in
NUP98::NSD1 patients.

Wang et al. BMC Pediatrics(2024) 24:547

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Fig. 4 Survival curves of pediatric AML patients according to combined NUP98::NSD1 gene fusion and FLT3/ITD status. A Probability of EFS for AML patients. B Probability of OS for AML patients. Pairwise comparison data provided in Supplementary Figs. 2-2.3 and Supplementary Table 4

Multivariate analysis of prognostic factors for overall andevent-free survival in pediatric AML patients

Multivariable Cox regression analysis including
NUP98::NSD1+/FLT3/ITD+, NUP98::NSD1+/FLT3/ITD, NUP98::NSD1-/FLT3/ITD+, NPMI status, WT1 status, age > 10years, WBC, and risk group was performed
to determine whether NUP98::NSD1+/FLT3/ITD + isan independent prognostic factor and concluded that
NUP98::NSD1+/FLT3/ITD + is an independent prognostic factor for poor EFS (HR = 3.2, 95% CI: 1.6–6.5,
P = 0.001), though it did not have a significant impact
on OS. Regardless of NUP98::NSD1+/ FLT3/ITD -or
NUP98::NSD1-/ FLT3/ITD + status, EFS and OS were
not affected. As expected, we showed WT1mutation was
significantly associated with inferior EFS and OS. Moreover, a WBC count > = 100 × 10^9/L, but not OS, had asignificant negative influence on EFS. Conversely, neither
NPM1 mutation nor risk group had significant impact on
EFS or OS (Table 3).

Discussion
Cytogenetic and molecular abnormalities play significant
roles in the risk stratification of pediatric acute myeloid
leukemia patients and are important prognostic factors[2]. Previous studies have shown that expression of the
NUP98 fusion genes in hematopoietic cell lines resultsin poor prognosis [6, 7, 11, 20, 21]. The frequency of
the NUP98 fusion gene presence in pediatric AML wasreported to be 10.8% in this large cohort, which is consistent with previous studies [22]. Several prior reports
have shown that NUP98-positive patients have higher

WBC counts, more frequent FAB-M4/M5 phenotypes,
and more FLT3/ITD mutations, especially among thosewith NUP98::NSD1 gene fusion patients [5, 9], where
we found similar results. However, we observed that
NUP98-positive patients tended to have a normal cytogenetic status. In this study, patients harboring the NUP98
fusion gene had a lower CR rate. Furthermore, our analysis demonstrates that the presence of the NUP98 fusion
gene leads to poorer EFS and OS outcomes. Considering that the presence of NUP98 fusion gene has a highoverlap with FTL3/ITD mutation, we aimed to investigate whether expression of FTL3/ITD in patients with
NUP98-fusion proteins is required for poor prognosis.
Patients with NUP98 gene fusion co-expression and
FLT3/ITD mutation had an inferior EFS of 13.5% compared with the 31.0% in patients with FLT3/ITD mutation alone. Moreover, we found that patients harboring
NUP98 fusion gene only had favorable outcome for EFS
of 48.0%, and similar to dual negative of FLT3/ITD and
NUP98 gene fusion of EFS 50.3%. Based on our findings,
the dismal prognosis of NUP98-positive patients might
be due to the interaction of the FLT3/ITD mutation.
Consistent with the findings of a previous study [22],NUP98::NSD1 gene fusion was the most common
NUP98 rearrangement in pediatric AML patients, occurring in 59.6% of 57 cases. Additionally, NUP98::NSD1
gene fusion and FLT3/ITD mutation are potential key
factors for dismal prognosis of AML patients [11]. Notably, patients who are positive for both FLT3/ITD and
NUP98::NSD1 experience poor outcomes [23]. This study
unequivocally demonstrated that patients carried FLT3/

Wang et al. BMC Pediatrics(2024) 24:547

Page 8 of 11

Fig. 5 Survival curves of pediatric AML patients according to combined NUP98::NSD1 gene fusion and FLT3/ITD status. A Probability of EFS for NUP98::NSD1
gene fusion with FLT3/ITD AR status. B Probability of OS for NUP98::NSD1 gene fusion with FLT3/ITD AR status. C Probability of EFS for high FLT3/ITD AR withor without NUP98::NSD1 gene fusion. D Probability of OS for high FLT3/ITD AR with or without NUP98::NSD1 gene fusion. E Probability of EFS for low FLT3/
ITD AR with or without NUP98::NSD1 gene fusion. F Probability of OS for low FLT3/ITD AR with or without NUP98::NSD1 gene fusion

Wang et al. BMC Pediatrics(2024) 24:547

Page 9 of 11

Fig. 6 Survival curves of pediatric AML patients according to NUP98 gene fusion and stem cell transplantation (SCT) status. A Probability of EFS for
NUP98 gene fusion patients according to SCT status. B Probability of OS for NUP98 gene fusion patients according to SCT status. C Probability of EFS for
NUP98::NSD1 patients according to SCT status. D Probability of OS for NUP98::NSD1 patients according to SCT status

ITD mutations and NUP98::NSD1 had significantly worse
outcomes, with a 5-year OS of 41.7% and a 5-year EFS of
13.6%, than NUP98::NSD1 -negative patients with wildtype FLT3/ITD (OS, P = 0.004; EFS, P < 0.001). These
outcomes also worse than those of patients with FLT3/
ITD positivity alone, who had a 5-year EFS of 29.0%,
but 5-year OS was not affected. Patients positive for
NUP98::NSD1 alone have a favorable outcome, similar to
those who are negative for both NUP98::NSD1 and FLT3/
ITD. This study demonstrated that patients who carryboth NUP98::NSD1 gene fusion and FLT3/ITD mutation
have poor outcomes.
In conclusion, this study indicated that outcome may
not be significantly impacted by the presence of only
NUP98::NSD1 or NUP98 gene fusion in the absence of
FLT3/ITD mutation.
Compared with FLT3/ITD positive, a high FLT3/ITD
AR status predicted poor prognosis. A previous study
reported that the negative impact of FLT3/ITD even withhigh AR might not be as severe as previously publishedWang et al. BMC Pediatrics(2024) 24:547

Page 10 of 11

Table 3 Multivariate analysis of prognostic factors for overall andevent-free survival in 529 pediatric AML patients
Variables

NUP98::NSD1 (+)/FLT3/ITD(+)
NUP98::NSD1 (-)/FLT3/ITD(+)
NUP98::NSD1 (+)/FLT3/ITD(-)
Age > 10years
high risk group
NMP1 mutation
WT1 mutation
WBC > = 100 × 10^9/L

Overallsurvival(OS)HR(95%CI) P
value
1.3(0.5,3.2) 0.618
1.1(0.5,2.5) 0.824
0.8(0.2,2.4) 0.641
1.1(0.8,1.6) 0.420
1.2(0.5,2.7) 0.712
1.0(0.3,3.2) 0.990
1.8(1.0,3.2) 0.040
1.5(1.1,2.2) 0.013

Event-freesurvival(EFS)
HR(95% PCI)value
3.2(1.6,6.5) 0.001
1.5(0.8,2.9) 0.170
1.0(0.4,2.5) 0.971
0.9(0.7,1.2) 0.662
0.9(0.5,1.8) 0.840
1.0(0.4,2.7) 0.991
1.7(1.1,2.8) 0.020
1.6(1.2,2.1) 0.001

in the absence of NUP98::NSD1 fusion [24]. In this study,
we observed that NUP98::NSD1 gene fusion cases concomitant high FLT3/ITD AR led to worse OS, though thiswas not statistically significant compared with those low
FLT3/ITD AR cases. However, high FLT3/ITD AR cases
led to worse survival for EFS of 23.1% and 26.5% with orwithout NUP98::NSD1 gene fusion, respectively.
Several prior reports have shown that patients whocarry both NUP98::NSD1 gene fusion and FLT3/ITD
mutation and who undergo SCT have an improved prognosis compared with those who receive chemotherapy
alone [12, 25]. In this study, SCT did not improve the EFS
or OS of pediatric patients with NUP98 gene fusion or
NUP98::NSD1.
This study is the first to analyze the prognostic significance of NUP98 gene fusion or NUP98::NSD1 in FLT3/
ITD pediatric AML patients. These findings clearlyshow that patients who carry both FLT3/ITD and
NUP98::NSD1 have poor outcomes. Co-expression of
FLT3/ITD and NUP98::NSD1 was identified as an independent factor for dismal outcomes, while positivity for
NUP98::NSD1 alone was not a significant prognostic factor. The presence of NUP98::NSD1 gene fusion and FLT3/
ITD mutation could identify a high risk subgroup. Moreover, those two genetic lesions with the presence of highFLT3/ITD AR appears to lead to worse prognosis, witha low EFS rate of 23.1% and OS rate of 33.3%. A larger
sample is needed for further study. Multivariable analysis showed that WT1 mutation was significantly associated with inferior EFS and OS, and NPM1 mutation had
no significant impact on EFS or OS. We did not further
analyze the interactions among FLT3-ITDs, WT1 and
NUP98, which is a limitation of our study. To further
clarify the impact of these 2 genetic lesions on the prognosis of pediatric AML patients, WT1 mutations, NPM1
mutations, or more cytogenetic and molecular abnormalities with highly overlapping expression levels should
be included in subsequent studies to confirm the predictors with variable effects on the prognosis. Based onprior studies, we cannot conclude that underwent SCT

benefits patients with these 2 genetic lesions. Therefore,
quickly detecting genetic variants early and using FLT3ITD inhibitors as soon as possible may improve the prognosis of these children.
In summary, presence of both NUP98::NSD1 andFLT3/ITD was found to be an independent factor for dismal prognosis in pediatric AML patients. Notably, lack of
FLT3/ITD mutations in NUP98::NSD1 -positive patients
did not retain its prognostic value.
Abbreviations
AML	Acute Myeloid Leukemia
SCT	Stem Cell Transplantation
CR	Complete Remission
EFS	Event-Free Survival
OS	Overall Survival
FAB	French-American-British

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12887-024-05007-3.
Supplementary Material 1
Supplementary Material 2
Supplementary Material 3
Supplementary Material 4
Supplementary Material 5
Supplementary Material 6
Supplementary Material 7
Supplementary Material 8
Supplementary Material 9
Acknowledgements
Thanks are due to TARGET researchers as well as all study participants for
public data.
Author contributions
L.H.X, X.G.Y and J.W.W participated in project design, analysis, interpretation,and manuscript drafting. J.W.W, Y.L obtained, assembled data analyzed andinterpreted the data. All authors contributed to the article and approved thesubmitted version.
Funding
No funding.
Data availability
Data is provided within the manuscript.

Declarations
Ethics approval and consent for publication
The studies involving human participants were reviewed and approved by
TARGET Publications Committee. Written informed consent to participate inthis study was provided by the participants’ legal guardian/next of kin.
Consent for publication
Not applicable.
Clinical trial information
COG studies AAML0531, AAML03P1, and CCG2961 were registered at theUnited States clinical trial registry and www.clinicaltrials.gov as #NCT00372593

Wang et al. BMC Pediatrics(2024) 24:547(Study Registration Date: 2006-09-06), #NCT00070174 (Study Registration
Date: 2003-10-03) and #NCT00002798 (Study Registration Date: 2000-11-24),
respectively.

Page 11 of 1114.

Competing interests
The authors declare no competing interests.
15.
Received: 17 March 2024 / Accepted: 12 August 2024

16.
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