# MutatedWT1,FLT3-ITD, andNUP98-NSD1 Fusion in Various Combinations Define a Poor Prognostic Group in Pediatric Acute Myeloid Leukemia

Authors: Naghmeh Niktoreh; Christiane Walter; Martin Zimmermann; Christine von Neuhoff; Nils von Neuhoff; Mareike Rasche; Katharina Waack; Ursula Creutzig; Helmut Hanenberg; Dirk Reinhardt
Journal: Journal of Oncology
 (2019)

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## Abstract

Acute myeloid leukemia is a life-threatening malignancy in children and adolescents treated predominantly by risk-adapted intensive chemotherapy that is partly supported by allogeneic stem cell transplantation. Mutations in theWT1 gene andNUP98-NSD1 fusion are predictors of poor survival outcome/prognosis that frequently occur in combination with internal tandem duplications of the juxta-membrane domain ofFLT3 (FLT3-ITD).To re-evaluate the effect of these factors in contemporary protocols, 353 patients (<18 years) treated in Germany with AML-BFM treatment protocols between 2004 and 2017 were included. Presence of mutatedWT1 andFLT3-ITDin blasts (n=19) resulted in low 3-year event-free survival of 29% and overall survival of 33% compared to rates of 45-63% and 67-87% in patients with only one (onlyFLT3-ITD; n=33,onlyWT1 mutation; n=29) or none of these mutations (n=272). IncludingNUP98-NSD1 and high allelic ratio (AR) ofFLT3-ITD (AR ≥0.4) in the analysis revealed very poor outcomes for patients with co-occurrence of all three factors or any of double combinations. All these patients (n=15) experienced events and the probability of overall survival was low (27%). We conclude that co-occurrence ofWT1 mutation,NUP98-NSD1, andFLT3-ITDwith an AR ≥0.4 as triple or double mutations still predicts dismal response to contemporary first- and second-line treatment for pediatric acute myeloid leukemia.


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## Body

## 1. Introduction

Pediatric acute myeloid leukemia (AML) is a rare and heterogeneous disorder, for which continuous improvement of risk-adapted treatment approaches over the last 30 years has led to overall survival rates of approximately 70% [1, 2]. In current pediatric AML treatment protocols, cytogenetic abnormalities of the leukemic blasts at initial diagnosis are important indicators for risk group stratification and treatment assignment [1, 2]. Approximately, 25% of pediatric patients have AML blasts with a normal karyotype, but even these cases often harbor somatic mutations in genes such asWILMS TUMOR 1(WT1),NPM1,NRAS, KRAS,Fms-like tyrosine kinase 3 (FLT3), and/orc-KIT/CD117[1, 2].TheWT1 gene is located on chromosome 11, has ten exons and four zinc finger domains, and functions as a transcription factor and master regulator of tissue development [3]. Within normal hematopoiesis,WT1 has two distinct roles: in early stages, it mediates quiescence of primitive progenitor cells, and later, WT1 expression is important for differentiation towards the myeloid lineage [4]. In AML,WT1 mutations are present in approximately 10% of patients and predominantly located in exons 7 and 9, which contain the DNA-binding zinc finger domains of the protein. The majority of these mutations are out-of-frame deletion/insertions or premature termination codons that will lead to truncated proteins with altered functional consequences for the cells [5]. If these truncated proteins are stable, they might have dominant negative effects by partially blocking the wild-type WT1 protein; if unstable, the diminished WT1 protein levels may lead to haploinsufficiency [5]. Nevertheless, it has been clearly established that the occurrence ofWT1 mutations in AML blasts with normal karyotypes is associated with adverse clinical outcomes in adult [6–9] as well as pediatric patients [10, 11].SomaticWT1mutations in AML blasts often co-occur with other genetic aberrations, most frequently with an internal tandem duplication in the juxta-membrane domain of the tyrosine kinase receptorFLT3 (FLT3-ITD) [5]. Classified as type-I or proliferating mutation,FLT3-ITDsare present in 10-15% of pediatric AML cases and lead to poor clinical outcomes [12–14]. We previously demonstrated in a cohort of 298 pediatric patients withde novoAML treated before 2004 on AML-BFM protocols that the combination ofFLT3-ITD and mutatedWT1 is associated with even worse survival [10]. Comparably, an independent study from the Children’s Oncology Group (COG) in a cohort of 842 children withde novoAML showed that the poor prognostic impact ofWT1mutations depends on theFLT3-ITD status [11]. These two pediatric studies confirmed earlier findings in adults that first established the adverse prognostic impact of bothWT1andFLT3-ITDmutations [15, 16].Two additional prognostic indicators inFLT3-ITD-positive AML cases established in the last few years are the mutational burden in each patient defined as the ratio between mutant and wild-typeFLT3-ITD alleles (allelic ratio, AR) [12, 17, 18] and the co-occurrence ofFLT3-ITD with a cytogenetically cryptic translocation of chromosomes 5 and 11 or t(5;11)(q35;p15) [19]. This translocation leads to fusion of thenucleoporin (NUP98) gene on chromosome 11 and the gene for nuclear receptor binding SET-domain protein 1 (NSD1) of chromosome 5 (NUP98-NSD1). As the breakpoints for the NUP98 gene are often not detected by classical cytogenetic due to its terminal localization at 11p15, it has been described in AML cases with a “normal” karyotype [20]. Importantly, this rare recurrent aberration is mutually exclusive with other recurrent translocations and more prevalent in pediatric AML, in which it is associated with the presence ofFLT3-ITD and poor survival outcomes [21, 22].In the present study, we re-evaluated the role of mutations inWT1,FLT3-ITD, and theNUP98-NSD1 translocation as prognostic factors in two contemporary pediatric treatment protocols by analyzing their association with co-occurring genetic and cytogenetic aberrations and by determining their clinical significance and influence on treatment outcome. Thereby, we were able to define a group of high-risk patients for which the efforts for salvage/second line treatment largely failed.


## 2. Materials and Methods

From April 2004 to May 2017, 841 patients aged 0–18 years withde novo AML (excluding FAB M3 and Down Syndrome) were treated in Germany according to the AML-BFM 04 trial (ClinicalTrials.gov Identifier: NCT00111345) or the AML-BFM 2012 registry and trial (EudraCT number: 2013-000018-39) (Figure 1(a)). Both trials were approved by the ethical committees and institutional review boards of university hospitals of Münster and Hannover and an informed consent was obtained from each patient or their legal guardians before the beginning of treatment. Standard procedures for the diagnosis of AML were carried out by the German AML-BFM reference laboratory as previously described [23–25]. This included mutation analysis inWT1,FLT3-ITD,NPM1,NRAS, andc-KIT by Sanger and/or next-generation sequencing or GeneScan analysis. In 353 patients (42%), sufficient material and clinical data were available for further analysis. As a confirmation, material fromWT1 and/orFLT3-ITDpositive and negative cases was re-analyzed by next-generation sequencing (NGS) using the TruSight Myeloid Panel (Illumina)[26] with median read counts forWT1andFLT3-ITD of around 4,200 and 6,000 reads, respectively, as we described previously [27]. In addition, the allelic ratio ofFLT3-ITDtoFLT3wild-type was calculated via GeneScan analysis [13] and the expression ofNUP98-NSD1 was analyzed in 246 out of 353 patients with available material by real-time quantitative PCR using previously described primers [19]. Initial analysis demonstrated that the selected cohort was representative for all patients treated between 2004 and 2017 on the AML-BFM protocols for features such as gender, age, AML subtype, initial cytogenetics, and preliminary, early response to treatment (data not shown).Figure 1
Study flowchart and patient characteristics. (a) Study flowchart outlining the process of patient recruitment in the data analysis. (b)WT1 mutations often co-occurred withFLT3-ITD and other genetic aberrations. AML-BFM, acute myeloid leukemia-Berlin-Frankfurt-Muenster; n, number;WT1,Wilms Tumor 1;FLT3-ITD, fms-related tyrosine kinase 3-internal tandem duplication;NPM1,nucleophosmin 1;NRAS,neuroblastoma RAS viral oncogene homolog;c-KIT,KIT proto-oncogene; CBF, core binding factor; MLL, rearrangements ofMLL gene;NUP98-NSD1,Nucleoporin-Nuclear Receptor Binding SET Domain Protein 1fusion gene; CN, cytogenetic-normal AML; AR, allelic ratio; CCR, continued complete remission; LFU, lost to followup; NR, non-response; PR, partial remission. ​aCBF aberrations include translocation of chromosomes 8 and 21 and inversion or translocation of chromosome 16. ​bOther cytogenetic aberrations such as trisomy 8, various chromosomal translocations, and complex karyotype alterations.Clinical end-points were defined as previously described [28, 29] and survival rates were calculated via Kaplan-Meier analysis and compared by log-rank test. Multivariate analysis was performed using Cox regression model evaluating the hazard ratio (HR) of each covariate with 95% confidence interval (CI). Stem cell transplantation was included in the Cox regression model as a time-dependent variable. Differences with a p value less than 0.05 were considered as significant. Data were analyzed using the Statistical Analysis System software version 9.4 (SAS Institute, Cary, NC). Data acquisition was stopped at June 30, 2018, with a median follow-up of 3.6 years.


## 3. Results

### 3.1. Study Cohort and Patient Characteristics

In this study, we included 353 patients treated on either the AML-BFM 2004 or AML-BFM 2012 protocol for whom sufficient material and information were available (Figure1(a)). As shown in Table 1, 48 (14%) patients hadWT1and 52 (15%)FLT3-ITDmutations in their leukemic blasts at diagnosis. Mutations inNPM1,NRAS, andc-KIT were present in the blasts of 9%, 17%, and 12% of patients, respectively. Most patients with mutatedWT1 (n=35, 73%) harbored at least one co-occurring mutation in the AML blasts, with the most common beingFLT3-ITD (n=19, 40%) followed byNRAS mutations (n=11, 23%, Table 1 and Figure 1(b)). Comparably, the majority of patients withFLT3-ITD had additional mutations in other genes (n=32, 62%), most commonly inWT1 (n=19, 37%) andNPM1 (n=11, 21%). Patients with mutatedWT1orFLT3-ITD were older compared to the rest of the study cohort, and AML FAB M1/M2 was the most common morphologic subtype in both groups (Table 1). In addition, the AML blasts of more than half of patients withWT1(n=25/48, 52%) andFLT3-ITD(n=28/52, 54%) mutations had a normal karyotype at diagnosis; these percentages were significantly higher than those in patients without mutations in each of the two genes (p<0.0001, Table 1).Table 1
Patient Characteristics.
FEATURES All patients WT1wild-type WT1mutated  FLT3-ITDneg. FLT3-ITDpos. n % n % n % p∗ n % n % p∗ Study Population  Number ( % ) 353 100 305 100 48 100  301 100 52 100  Age (years), median (range) 9.09 7.97 10.68 0.03 7.8 12.95 0.0001 (0 -18) (0 - 18) (0.8 - 17.8) (0 -18) (2.7 - 17.9)  Gender male 183 52% 159 52% 24 50% 0.783 153 51% 30 58% 0.36 female 170 48% 146 48% 24 50% 148 49% 22 42%  WBC count at diagnosis  median x 109 cells/L (range) 24.5 24 42.85 0.1 20.1 73.5 0.0001 (0.019 - 475) (0.23 - 475) (0.019 - 324) (0.019-475) (1.8 - 324)  Morphological Classification  FAB M0 9 3% 7 2% 2 4%  5 2% 4 8% M1/M2 134 38% 107 35% 27 56%  100 33% 34 65% M4Eo+ 31 9% 27 9% 4 8%  29 10% 2 4% M4Eo-/M5 132 37% 125 41% 7 15% 0.008 121 40% 11 21% 0.0001 M6 3 1% 2 1% 1 2%  3 1% 0 0% M7 22 6% 21 7% 1 2%  22 7% 0 0% AUL/other 22 6% 16 5% 6 13%  21 7% 1 2%  Cytogenetics  t(8;21) 38 11% 38 12% 4 8%  36 12% 2 4% inv(16) 32 9% 28 9% 0 0%  31 10% 1 2% MLL rearr. 61 17% 60 20% 1 2% <0.0001 60 20% 1 2% 0.0001 others 117 33% 99 32% 18 38% 97 32% 20 38% normal 97 27% 72 24% 25 52%  69 23% 28 54% no data 8 2% 8 3% 0 0%  8 3% 0 0%  NUP98-NSD1  positive 15 4% 7 2% 8 17% <0.0001 6 2% 9 17% <0.0001 negative 231 65% 198 65% 33 69% 197 65% 34 65% no data 107 30% 100 33% 7 15% 98 33% 9 17%  Co-mutations  FLT3-ITD negative 301 85% 272 89% 29 60% <0.0001 positive 52 15% 33 11% 19 40%  WT1 wildtype 305 86%      272 90% 33 63% 0.0001 mutated 48 14%  29 10% 19 37%  NPM1 wildtype 316 90% 273 90% 43 90%  277 92% 39 75% mutated 31 9% 26 9% 5 10% 0.69 20 7% 11 21% 0.0004 no data 6 2% 6 2% 0 0%  4 1% 2 4%  nRAS wildtype 282 80% 245 80% 37 77%  235 78% 47 90% mutated 59 17% 48 16% 11 23% 0.26 57 19% 2 4% 0.008 no data 12 3% 12 4% 0 0%  9 3% 3 6%  KIT wildtype 308 87% 267 88% 41 85% 0.51 266 88% 42 81% 0.31 mutated 41 12% 34 11% 7 15% 33 11% 8 15% no data 4 1% 4 1% 0 0% 2 1% 2 4%  CEBPA wildtype 163 46% 121 40% 42 88% <0.0001 122 41% 41 79% <0.0001 single 6 2% 4 1% 2 4% 6 2% 0 0% double 10 3% 9 3% 1 2% 8 3% 2 4% no data 174 49% 174 57% 0 0% 165 55% 9 17%  HSCT  HSCT in 1ts CR 64 18% 56 18% 8 17% 0.78 53 18% 11 21% 0.54 Chemotherapy only 289 82% 249 82% 40 83% 248 82% 41 79%  Patient Status  alive 251 71% 221 72% 30 63%  225 75% 26 50% deceased 73 21% 58 19% 15 31% 0.14 51 17% 22 42% 0.0001 LFU 29 8% 26 9% 3 6%  25 8% 4 8% n, number;WT1, Wilms tumor 1; FLT3-ITD, fms related tyrosine kinase 3-internal tandem duplication; WBC, white blood cell; FAB, French-American-British; M4Eo+, AML M4 subtype with the presence of atypical eosinophils; M4Eo-, AML M4 subtype without the presence of atypical eosinophils; AUL, acute undifferentiated leukemia; t, translocation; inv, inversion; MLL rear., rearrangement of MLL gene; NUP98-NSD1, Nucleoporin-Nuclear Receptor Binding SET Domain Protein 1 fusion gene; NPM1, nucleophosmin 1; NRAS, neuroblastoma RAS viral oncogene homolog; c-KIT, KIT proto-oncogene; CEBPA, CCAAT/enhancer binding protein (C/EBP) alpha; HSCT, hematopoietic stem cell transplantation; CR, complete remission; HSCT, hematopoietic stem cell transplantation; LFU, lost to follow-up. ∗p-values derived from Pearson’s Chi-squares test.


### 3.2. Characteristics of WT1 Mutations

We identified 64 differentWT1 sequence alterations in 48 patients (Table 2). These alterations were frequently located in exon 7 (n=55, 86%) and predominantly resulted in frameshifts producing premature termination codons (PTCs). In total, nine single nucleotide variants (SNVs) were found, mostly in exon 9 (n=7, 78%). Only three of the nine SNVs were not previously reported as pathogenic (Table 2). Using NGS, we characterized multiple distinctWT1 mutations with highly diverse variant allele frequencies in 13 patients (11 patients had two and 2 patients, three distinct mutations). We then analyzed the heterozygosity of these mutations via the integrative genomic viewer (Broad Institute, MA, USA) and determined that they were all located on individual/different alleles/reads (Table 2).Table 2
Characteristics ofWT1 Variants.
UPN exon seq. read mutation sequence​a amino acid alteration VF (%) dbSNP or COSMIC ID published previously reported sample outcome missense substitutions  8 9  c.1333C>T p.Arg445Trp 19.1 rs121907900, COSM21417 Yes WT CCR  15 9  c.1345C>A p.Leu449Met 5.49  No  CCR  20 9  c.1385G>A p.Arg462Gln 47.21 rs121907903, COSM4191067 Yes AML, colon cancer, adenocarcinoma CCR  21 9  c.1343A>G p.His448Arg 33.12 COSM7335365 Yes AML, mesothelioma CCR  23 9  c.1333C>T p.Arg445Trp 72.42 rs121907900 Yes WT, DDS CCR  26 7  c.1097C>G p.Ser366Cys 2.57  No  CCR  35 9 different c.1334G>A p.Arg445Gln 3.12 rs121907903, COSM4191067 Yes AML, colon cancer, adenocarcinoma Relapse 9 different c.1307G>A p.Cys436Tyr 44.21 COSM21438 Yes AML  nonsense substitutions/insertions, deletions or duplications  1 7  c.1090_1093dupTC p.Ala365Valfs∗4 43 COSM5487332 Yes AML CCR  2 7  c.1048-4_1056dupGCAGGATGTGCGA p.Arg353Alafs∗19 30.25  No  LFU in CCR  3 7  c.1087_1161dup74 p.Lys387Asnfs∗44 n.d.  No  Relapse  4 7 different c.1087_1091dupCGGTC p.Ala365Glyfs∗69 5.08 COSM28954 Yes AML, T-ALL Relapse 4 7 different c.1091C>A p.Ser364∗ 28.38 COSM27307 Yes AML, WT  5 7  c.1083_1098delTGTACGGTCGGCATCT p.Val362Argfs∗65 46.82  No  NR/PR  6 7  c.1059dupT p.Val354Cysfs∗14 35.9 COSM1317324 Yes AML Relapse  7 7  c.1179dupG p.His394Alafs∗8 25  No  CCR  9 7 different c.1078_1079insGCCGA p.Thr360Serfs∗74 38.7  No  NR/PR 7 different c.1084_1085insGC p.Val362Glyfs∗71 52.9  No  10 7  c.1074_1077dupCCCG p.Thr360Profs∗9 9.9  No  CCR  11 7  c.1079_1090delCTCTTGTACGGTinsTGGG p.Thr360Metfs∗5 55.23  No  CCR  12 7  c.1058_1059insGA p.Val354Metfs∗5 31.6  No  CCR  13 7 different c.1058_1059insGGTG p.Pro355Cysfs∗14 5.6  No  Relapse 7 different c.1078_1084dupACTCTTG p.Val362Aspfs∗8 8.3 COSM5879281 Yes AML  14 7  c.1090_1093dupTCGG p.Ala365Valfs∗4 22.81 COSM21392 Yes AML CCR  16 7  c.1054_1084dup p.Val362Alafs∗16 7.3  No  CCR  17 7  c.1087delCinsGGG p.Arg363Glyfs∗70 24.3  No  CCR  18 7  c.1054_1055insT p.Arg352Leufs∗16 67.2 COSM5751511 Yes T-ALL CCR  19 7  c.1077_1078insTGTTTCTTCCGCCCAG p.Thr360Cysfs∗13 36.95  No  Relapse  22 7  c.1087delCinsGG p.Arg363Glyfs∗5 41.88  Yes AML CCR  24 7  c.1083_1090dupTGTACGGT p.Ser364Leufs∗71 3.8 COSM27309 Yes AML CCR  25 9 different c.1323_1338dupAAAGTTCTCCCGGTCC p.Asp447Lysfs∗18 40.1  No  CCR 9 different c.1322_1332dupGAAAGTTCTCC p.Arg445Glufs∗9 40.5  No  27 7 different c.1077_1078insGTTG p.Thr360Valfs∗9 43.71  No  CCR 7 different c.1089dupG p.Ser364Valfs∗4 49.24 COSM28966 Yes AML  28 7 different c.1058delGinsCCA p.Arg353Profs∗6 19.72  No  NR/PR 7 different c.1054_1055insAAAAAGATT p.Arg352delins4 19.55  No  29 7  c.1179dupG p.His394Alafs∗8 25  No  CCR  30 7  c.1048_1057delGATGTGCGACinsAAGG p.Asp350_Arg353 46.34  No  CCR  31 7  c.1093dupG p.Ala365Glyfs∗3 44.49  Yes AML NR/PR  32 7  c.1048-8_1055dupGCCTGCAGGATGTGCG p.Arg353Profs∗20 2.5  No  NR/PR  33 7  c.1090_1091dupTC p.Ala365Argfs∗68 44.25 COSM28955 Yes AML Relapse  34 7 different c.1087delCinsGA p.Arg363Glufs∗5 4.1  No  NR/PR 7 different c.1086dupA p.Arg363Thrfs∗5 5.33 COSM1166631 Yes AML 7 different c.1090_1093dupTC p.Ala365Valfs∗4 36.29 COSM5487332 Yes AML  36 7 different c.1090_1093dupTCGG p.Ala365Valfs∗4 6.94 COSM5487332 Yes AML NR/PR 7 different c.1091dupC p.Ala365Glyfs∗3 39.42 COSM27304 Yes AML  37 7 different c.1057delCinsGG p.Arg353Glyfs∗15 42.78  Yes AML CCR 7 different c.1087delCinsGGG p.Arg363Glyfs∗70 52.11  No  38 7  c.1068_1076delAGTAGCCCCinsGACGGTCGTTATTA p.Val357Thrfs∗77 42.14  No  CCR  39 7  c.1087delCinsGG p.Arg363Glyfs∗5 47.54  Yes AML CCR  40 7 different c.1058_1059insGGTGCCGCTCG p.Gly356Leufs∗6 48.49  No  Early Death 7 different c.1082_1091dupTTGTACGGTC p.Ala365Cysfs∗6 41.83 COSM27303 Yes AML  41 7 different c.1123dupA p.Met375Asnfs∗9 44.5  Yes AML CCR 7 different c.1057_1058insTA p.Arg353Leufs∗6 45.8  No  42 7  c.1051_1055dupGTGCG p.Arg353Cysfs∗7 34.73  No  Relapse  43 7  c.1058delGinsCC p.Arg353Profs∗15 44.78 COSM28946 Yes AML Relapse  44 7 different c.1079_1101delinsGAA p.Thr360Argfs∗4 20.37  No  Relapse 7 different c.1088_1089insCTCGG p.Ala365Glyfs∗69 10.69  No  45 7  c.1090_1091insAGGT p.Ser364∗fs∗1 42.97  No  Relapse  46 7  c.1058delGinsCC p.Arg353Profs∗15 51.08 COSM28946 Yes AML, T-ALL NR/PR  47 7 different c.1048-3_1055dupCAGGATGTGCG p.Val354Metfs∗8 2.49  No  CCR 7 different c.1053dupG p.Arg352Alafs∗16 3.83 COSM28980 Yes AML 7 different c.1054delCinsGG p.Arg352Glyfs∗16 35.86 COSM28970 Yes AML, T-ALL  48 7  c.1089_1090insGGCCTCTTGTACGG p.Ser364Glyfs∗73 40.49  No  Relapse UPN, unique patient number; Seq. read, sequence read; VF, variant allele frequency; dup, duplication; ins, insertion; indel, Insertion-deletion; fs, frame-shift;∗termination codon; WT, Wilms tumor; DDS, Denis-Drash syndrome; T-ALL, T-cell acute lymphoblastic leukemia; CCR, continued complete remission, NR, non-response; PR, partial response; LFU, lost to follow-up. aTranscript ID: NM_000378 was used to describe all alterations.


### 3.3. Survival Significance of the Genomic Aberrations

Next, we analyzed the impact of each mutation on the clinical outcomes. Our analysis identifiedWT1andFLT3-ITD,but notNRAS,NPM1, orc-KIT mutations as single factors that significantly increased the chance of relapse or treatment failure and reduced the probability of 3-year overall survival (OS) in our patient cohort (Figures 2(a), 2(b), and 3). In addition,FLT3-ITDbut notWT1mutations significantly decreased the 3-year probability of event-free survival (EFS, Figure 2(b)). When we grouped the two mutations together, the survival analysis revealed a 3-year EFS of 29±11% for patients with bothWT1andFLT3-ITDmutations compared to 63±3% for patients with none of these mutations (p=0.0004) and 61±11% or 45±9% for patients with onlyWT1mutation (p=0.016) orFLT3-ITD (p=0.16), respectively (Figure 2(c)). Corresponding to this low EFS, co-occurrence of these two mutations was associated with an increased cumulative incidence of relapse (CIR) of 65±12% compared to 32±12% for patients with none of these mutations (p=0.002) and 39±11% or 46±9% for patients with onlyWT1mutation (p=0.05) orFLT3-ITD(p=0.08), respectively (Figure 2(c)). Furthermore, we identified a low 3-year OS probability of 33±12% in patients with co-occurrence ofWT1andFLT3-ITD, which was significantly lower than those of patients without these mutations (81±3%, p<0.0001), patients with only mutatedWT1(87±7%, p=0.0007), and patients with onlyFLT3-ITD(67±9%, p=0.017, Figure 2(c)). Comparing the curves for EFS and OS clearly demonstrated that our second line treatment was not able to rescue any patient with co-occurrence ofWT1andFLT3-ITD mutations, while the OS rates increased by more than 20% for the other three subgroups (Figure 2(c)).Figure 2
Co-occurrence of WT1 and FLT3-ITD mutations at initial diagnosis of pediatric AML predicts poor survival outcomes.(a)WT1 mutation as single factor increased the incidence of relapse, reducing the probability of survival. (b) The presence ofFLT3-ITD, individually, leads to an increased chance of relapse and decreased patient survival. (c) Clinical consequences ofWT1 mutations andFLT3-ITD were dependent on each other.WT1,Wilms Tumor 1;FLT3-ITD, fms related tyrosine kinase 3-internal tandem duplication; pEFS, probability of event-free survival; pOS, probability of overall survival; CIR, cumulative incidence of relapse; SE, standard error; n, number. ​aNo response to treatment was considered as the occurrence of an event at time zero.Figure 3
Mutations in NPM1, NRAS, and c-KIT had no impact on survival.(a) Prognostic impact of mutatedNPM1 on EFS, OS, and CIR. (b) Prognostic impact of mutatedNRAS on EFS, OS, and CIR. (c) Prognostic impact ofc-KIT mutation on EFS, OS, and CIR.NPM1,nucleophosmin 1;NRAS,neuroblastoma RAS viral oncogene homolog;c-KIT,KIT protooncogene; pEFS, probability of event-free survival; pOS, probability of overall survival; CIR, cumulative incidence of relapse; SE, standard error; n, number. ​aNo response to treatment was considered as the occurrence of an event at time zero.


### 3.4. Impact of NUP98-NSD1 Fusion

To further characterize the prognostic significance ofWT1andFLT3-ITDmutations, we analyzed the expression ofNUP98-NSD1fusion in our patient cohort (Figure 1(a)). From 246 patients with available material for this retrospective real-time quantitative PCR analysis, 15 (6%) of them were identified to have theNUP98-NSD1translocation. Most of these patients (12/15, 80%) harbored additionalWT1 orFLT3-ITDmutations: 3 patients carried bothWT1 andNUP98-NSD1, 4 had a co-occurrence ofFLT3-ITDandNUP98-NSD1, and 5 patients carried all three genetic alterations (Figure 1(b)). Only 1 of these 15 patients had a previous known status ofNUP98-NSD1 by conventional karyotyping: 2 others were previously diagnosed with deletion of chromosome 5, 1 carried an inversion of chromosome 16 (no other mutations and still in continuous complete remission), 4 carried complex karyotypes or rare aberrations, and 7 had no other cytogenetic abnormalities (data not shown).We then analyzed the prognostic significance ofNUP98-NSD1 in the cohort of 246 patients with the known status of this fusion gene (Figure 1(a)). As a single factor, the presence ofNUP98-NSD1in AML blasts of patients at diagnosis was associated with a significant increase in CIR (81%) in addition to decreased probabilities of 3-year EFS and OS (Figure 4(a)). CombiningNUP98-NSD1withWT1 andFLT3-ITDmutations in our multifactor survival analysis revealed that patients with all three or either two of these mutations had worse survival outcomes. These patients had a higher CIR of 73±11% compared to the CIR of 30±4% for patients with none of these aberrations orNUP98-NSD1alone (p<0.0001) and the CIR of 37±13% or 38±10% for patients with only mutatedWT1(p=0.0078) orFLT3-ITD(p=0.013), respectively (Figures 4(a) and 4(b)). The increased CIR translated into a lower 3-year EFS probability of 23±10% for patients with triple or double mutations compared to the EFS of 62±4% for patients with none of these mutations or onlyNUP98-NSD1 (p<0.0001) and the EFS of 63±13% or 54±10% for patients with onlyWT1 (p=0.003) orFLT3-ITD (p=0.036) mutations, respectively (Figure 4(b)). Moreover, co-occurrence of all three or any double mutations resulted in a significantly lower 3-year OS probability of 42±12% compared to 80±8% for patients with none of the mutations or onlyNUP98-NSD1(p=0.0003) and 88±8% or 73±10% for patients with onlyWT1 (p=0.0007) orFLT3-ITD(p=0.049) mutations, respectively (Figure 4(b)).Figure 4
Prognostic significance of NUP98-NSD1 fusion. (a)NUP98-NSD1 as single factor predicted poor outcomes. (b) Inclusion ofNUP98-NSD1 as poor prognostic factor withWT1 mutation andFLT3-ITD, predicted poor outcomes for patients harboring all three factors in addition to patients withNUP98-NSD1 andWT1 mutation orFLT3-ITD. Patients with unknown status ofNUP98-NSD1 fusion were excluded from this analysis. WT1,Wilms Tumor 1;FLT3-ITD, fms related tyrosine kinase 3-internal tandem duplication;NUP98-NSD1,Nucleoporin-Nuclear Receptor Binding SET Domain Protein 1 fusion gene; pEFS, probability of event-free survival; pOS, probability of overall survival; CIR, cumulative incidence of relapse; SE, standard error; mut, mutated; pos, positive; neg, negative. ​aNo response to treatment was considered as the occurrence of an event at time zero. ​bThree patients withNUP98-NSD1are included in this group.


### 3.5. Survival Significance of the FLT3-ITD Allelic Ratio

We have previously established the prognostic significance of anFLT3-ITD allelic ratio of ≥0.4 in pediatric AML [12]. Therefore, to determine the impact of the mutational burden ofFLT3-ITD on treatment outcomes in the present cohort, we calculated theFLT3-ITD AR in patients with available data/material. As indicated in Figure 1(b), 27 patients had an AR ≥0.4 at diagnosis. Analyzing the survival impact of theFLT3-ITD AR ≥0.4 revealed that as a single factor, it was associated with an EFS of only 25±8% and an OS of only 47±10%, respectively (Figure 5(a)). Remarkably, the co-occurrence ofFLT3-ITDAR ≥0.4,WT1, andNUP98-NSD1 as triple or double mutations significantly increased the CIR to 93±15% compared to the CIR of 31±4% for patients with no mutations or onlyNUP98-NSD1orFLT3-ITDAR <0.4 (p<0.0001) and to the CIR of 31±11% or 36±15% in patients with onlyWT1 (p<0.0001) orFLT3-ITDAR ≥0.4 (p=0.001) mutations, respectively (Figure 5(b)). The probability of 3-year EFS was zero in patients with double or tripleWT1,FLT3-ITDAR ≥0.4, andNUP98-NSD1 mutations as opposed to 61±4% in patients with no mutations or onlyNUP98-NSD1orFLT3-ITDAR <0.4 (p<0.0001) and 69±11% or 45±15% for patients with only mutatedWT1 (p<0.0001) orFLT3-ITDAR ≥0.4 (p=0.019), respectively (Figure 5(b)). Finally, the co-occurrence of double or triple mutations resulted in a 3-year OS probability of 27±13%, which was significantly lower than the 3-year OS of 79±3% in patients with no mutations or onlyNUP98-NSD1orFLT3-ITDAR <0.4 (p<0.0001) and 90±7% or 73±13% in patients with onlyWT1(p=0.0003) orFLT3-ITDAR ≥0.4 (p=0.06) mutations, respectively (Figure 5(b)). By multivariate analysis includingWT1mutation,FLT3-ITDAR ≥0.4, core-binding factor aberrations, early bone marrow response to treatment, and stem cell transplantation as covariables, we confirmed that the interaction of these three factors, and not each of the aberrations individually, was a significant predictor of poor prognosis for EFS (p=0.008, HR: 3.88, 95% CI: 1.42 – 10.6) and OS (p=0.042, HR: 3.42, 95% CI: 1.04 – 11.21, Table 3). Importantly, none of the patients with triple mutations survived and the only patients who could be rescued harbored doubleNUP98-NSD1 andWT1 orNUP98-NSD1 andFLT3-ITD mutations (Figure 1(b)), thus resulting in an OS of 27±13% (Figure 5(b)).Table 3
Multivariate analysis.
Cox regression analysis - Event-free survival  Parameters Hazard ratio 95% confidence interval p value Lower limit Upper limit  WT1 mutation 0.79 0.41 1.53 0.479  FLT3-ITD AR ≥ 0.4 1.55 0.69 3.51 0.288  WT1mutation, FLT3-ITD ≥ 0.4 and 3.88 1.42 10.66 0.008 NUP98-NSD1 interaction  t(8;21) and/or inv(16) 0.51 0.27 0.96 0.037  Unsatisfactory early response to treatment​a 1.31 0.79 2.18 0.294  HSCT​b 0.25 0.1 0.64 0.004  Cox regression analysis - Overall survival  WT1 mutation 0.84 0.35 2.06 0.710  FLT3-ITD ≥ 0.4 1.51 0.57 4.02 0.404  WT1mutation, FLT3-ITD ≥ 0.4and 3.42 1.04 11.21 0.042 NUP98-NSD1 interaction  t(8;21) and/or inv(16) 0.45 0.16 1.31 0.143  Unsatisfactory early response to treatment​a 1.21 0.61 2.42 0.589  HSCT​b 1.18 0.51 2.73 0.700 WT1, Wilms tumor 1; FLT3-ITD, fms related tyrosine kinase 3-internal tandem duplication; NUP98-NSD1, Nucleoporin-Nuclear Receptor Binding SET Domain Protein 1 fusion gen; t, translocation; inv, inversion; HSCT, hematopoietic stem cell transplantation. ​ aUnsatisfactory early response to treatment was defined as persistence of >5% blasts in bone marrow at day 15 and/or 28 after treatment. ​bhematopoietic stem cell transplantation events at first complete remission or after no-response to other treatments were included in the multivariate analysis as a time-dependent variable.Figure 5
Prognostic significance of mutational burden of FLT3-ITD. (a)FLT3-ITD with an allelic ratio ≥0.4 as a single factor predicted poor outcomes. (b) High mutational burden ofFLT3-ITD was another predictor of poor prognosis when it occurred withWT1 and/orNUP98-NSD1. Patients with an unknownFLT3-ITD AR were excluded from this analysis.NUP98-NSD1,Nucleoporin-Nuclear Receptor Binding SET Domain Protein 1 fusion gene;FLT3-ITD, fms related tyrosine kinase 3-internal tandem duplication; pEFS, probability of event-free survival; pOS, probability of overall survival; CIR, cumulative incidence of relapse; AR, allelic ratio; SE, standard error; n, number. ​aNo response to treatment was considered as the occurrence of an event at time zero. ​bThree patients withNUP98-NSD1 are included in this group.


## 3.1. Study Cohort and Patient Characteristics

In this study, we included 353 patients treated on either the AML-BFM 2004 or AML-BFM 2012 protocol for whom sufficient material and information were available (Figure1(a)). As shown in Table 1, 48 (14%) patients hadWT1and 52 (15%)FLT3-ITDmutations in their leukemic blasts at diagnosis. Mutations inNPM1,NRAS, andc-KIT were present in the blasts of 9%, 17%, and 12% of patients, respectively. Most patients with mutatedWT1 (n=35, 73%) harbored at least one co-occurring mutation in the AML blasts, with the most common beingFLT3-ITD (n=19, 40%) followed byNRAS mutations (n=11, 23%, Table 1 and Figure 1(b)). Comparably, the majority of patients withFLT3-ITD had additional mutations in other genes (n=32, 62%), most commonly inWT1 (n=19, 37%) andNPM1 (n=11, 21%). Patients with mutatedWT1orFLT3-ITD were older compared to the rest of the study cohort, and AML FAB M1/M2 was the most common morphologic subtype in both groups (Table 1). In addition, the AML blasts of more than half of patients withWT1(n=25/48, 52%) andFLT3-ITD(n=28/52, 54%) mutations had a normal karyotype at diagnosis; these percentages were significantly higher than those in patients without mutations in each of the two genes (p<0.0001, Table 1).Table 1
Patient Characteristics.
FEATURES All patients WT1wild-type WT1mutated  FLT3-ITDneg. FLT3-ITDpos. n % n % n % p∗ n % n % p∗ Study Population  Number ( % ) 353 100 305 100 48 100  301 100 52 100  Age (years), median (range) 9.09 7.97 10.68 0.03 7.8 12.95 0.0001 (0 -18) (0 - 18) (0.8 - 17.8) (0 -18) (2.7 - 17.9)  Gender male 183 52% 159 52% 24 50% 0.783 153 51% 30 58% 0.36 female 170 48% 146 48% 24 50% 148 49% 22 42%  WBC count at diagnosis  median x 109 cells/L (range) 24.5 24 42.85 0.1 20.1 73.5 0.0001 (0.019 - 475) (0.23 - 475) (0.019 - 324) (0.019-475) (1.8 - 324)  Morphological Classification  FAB M0 9 3% 7 2% 2 4%  5 2% 4 8% M1/M2 134 38% 107 35% 27 56%  100 33% 34 65% M4Eo+ 31 9% 27 9% 4 8%  29 10% 2 4% M4Eo-/M5 132 37% 125 41% 7 15% 0.008 121 40% 11 21% 0.0001 M6 3 1% 2 1% 1 2%  3 1% 0 0% M7 22 6% 21 7% 1 2%  22 7% 0 0% AUL/other 22 6% 16 5% 6 13%  21 7% 1 2%  Cytogenetics  t(8;21) 38 11% 38 12% 4 8%  36 12% 2 4% inv(16) 32 9% 28 9% 0 0%  31 10% 1 2% MLL rearr. 61 17% 60 20% 1 2% <0.0001 60 20% 1 2% 0.0001 others 117 33% 99 32% 18 38% 97 32% 20 38% normal 97 27% 72 24% 25 52%  69 23% 28 54% no data 8 2% 8 3% 0 0%  8 3% 0 0%  NUP98-NSD1  positive 15 4% 7 2% 8 17% <0.0001 6 2% 9 17% <0.0001 negative 231 65% 198 65% 33 69% 197 65% 34 65% no data 107 30% 100 33% 7 15% 98 33% 9 17%  Co-mutations  FLT3-ITD negative 301 85% 272 89% 29 60% <0.0001 positive 52 15% 33 11% 19 40%  WT1 wildtype 305 86%      272 90% 33 63% 0.0001 mutated 48 14%  29 10% 19 37%  NPM1 wildtype 316 90% 273 90% 43 90%  277 92% 39 75% mutated 31 9% 26 9% 5 10% 0.69 20 7% 11 21% 0.0004 no data 6 2% 6 2% 0 0%  4 1% 2 4%  nRAS wildtype 282 80% 245 80% 37 77%  235 78% 47 90% mutated 59 17% 48 16% 11 23% 0.26 57 19% 2 4% 0.008 no data 12 3% 12 4% 0 0%  9 3% 3 6%  KIT wildtype 308 87% 267 88% 41 85% 0.51 266 88% 42 81% 0.31 mutated 41 12% 34 11% 7 15% 33 11% 8 15% no data 4 1% 4 1% 0 0% 2 1% 2 4%  CEBPA wildtype 163 46% 121 40% 42 88% <0.0001 122 41% 41 79% <0.0001 single 6 2% 4 1% 2 4% 6 2% 0 0% double 10 3% 9 3% 1 2% 8 3% 2 4% no data 174 49% 174 57% 0 0% 165 55% 9 17%  HSCT  HSCT in 1ts CR 64 18% 56 18% 8 17% 0.78 53 18% 11 21% 0.54 Chemotherapy only 289 82% 249 82% 40 83% 248 82% 41 79%  Patient Status  alive 251 71% 221 72% 30 63%  225 75% 26 50% deceased 73 21% 58 19% 15 31% 0.14 51 17% 22 42% 0.0001 LFU 29 8% 26 9% 3 6%  25 8% 4 8% n, number;WT1, Wilms tumor 1; FLT3-ITD, fms related tyrosine kinase 3-internal tandem duplication; WBC, white blood cell; FAB, French-American-British; M4Eo+, AML M4 subtype with the presence of atypical eosinophils; M4Eo-, AML M4 subtype without the presence of atypical eosinophils; AUL, acute undifferentiated leukemia; t, translocation; inv, inversion; MLL rear., rearrangement of MLL gene; NUP98-NSD1, Nucleoporin-Nuclear Receptor Binding SET Domain Protein 1 fusion gene; NPM1, nucleophosmin 1; NRAS, neuroblastoma RAS viral oncogene homolog; c-KIT, KIT proto-oncogene; CEBPA, CCAAT/enhancer binding protein (C/EBP) alpha; HSCT, hematopoietic stem cell transplantation; CR, complete remission; HSCT, hematopoietic stem cell transplantation; LFU, lost to follow-up. ∗p-values derived from Pearson’s Chi-squares test.


## 3.2. Characteristics of WT1 Mutations

We identified 64 differentWT1 sequence alterations in 48 patients (Table 2). These alterations were frequently located in exon 7 (n=55, 86%) and predominantly resulted in frameshifts producing premature termination codons (PTCs). In total, nine single nucleotide variants (SNVs) were found, mostly in exon 9 (n=7, 78%). Only three of the nine SNVs were not previously reported as pathogenic (Table 2). Using NGS, we characterized multiple distinctWT1 mutations with highly diverse variant allele frequencies in 13 patients (11 patients had two and 2 patients, three distinct mutations). We then analyzed the heterozygosity of these mutations via the integrative genomic viewer (Broad Institute, MA, USA) and determined that they were all located on individual/different alleles/reads (Table 2).Table 2
Characteristics ofWT1 Variants.
UPN exon seq. read mutation sequence​a amino acid alteration VF (%) dbSNP or COSMIC ID published previously reported sample outcome missense substitutions  8 9  c.1333C>T p.Arg445Trp 19.1 rs121907900, COSM21417 Yes WT CCR  15 9  c.1345C>A p.Leu449Met 5.49  No  CCR  20 9  c.1385G>A p.Arg462Gln 47.21 rs121907903, COSM4191067 Yes AML, colon cancer, adenocarcinoma CCR  21 9  c.1343A>G p.His448Arg 33.12 COSM7335365 Yes AML, mesothelioma CCR  23 9  c.1333C>T p.Arg445Trp 72.42 rs121907900 Yes WT, DDS CCR  26 7  c.1097C>G p.Ser366Cys 2.57  No  CCR  35 9 different c.1334G>A p.Arg445Gln 3.12 rs121907903, COSM4191067 Yes AML, colon cancer, adenocarcinoma Relapse 9 different c.1307G>A p.Cys436Tyr 44.21 COSM21438 Yes AML  nonsense substitutions/insertions, deletions or duplications  1 7  c.1090_1093dupTC p.Ala365Valfs∗4 43 COSM5487332 Yes AML CCR  2 7  c.1048-4_1056dupGCAGGATGTGCGA p.Arg353Alafs∗19 30.25  No  LFU in CCR  3 7  c.1087_1161dup74 p.Lys387Asnfs∗44 n.d.  No  Relapse  4 7 different c.1087_1091dupCGGTC p.Ala365Glyfs∗69 5.08 COSM28954 Yes AML, T-ALL Relapse 4 7 different c.1091C>A p.Ser364∗ 28.38 COSM27307 Yes AML, WT  5 7  c.1083_1098delTGTACGGTCGGCATCT p.Val362Argfs∗65 46.82  No  NR/PR  6 7  c.1059dupT p.Val354Cysfs∗14 35.9 COSM1317324 Yes AML Relapse  7 7  c.1179dupG p.His394Alafs∗8 25  No  CCR  9 7 different c.1078_1079insGCCGA p.Thr360Serfs∗74 38.7  No  NR/PR 7 different c.1084_1085insGC p.Val362Glyfs∗71 52.9  No  10 7  c.1074_1077dupCCCG p.Thr360Profs∗9 9.9  No  CCR  11 7  c.1079_1090delCTCTTGTACGGTinsTGGG p.Thr360Metfs∗5 55.23  No  CCR  12 7  c.1058_1059insGA p.Val354Metfs∗5 31.6  No  CCR  13 7 different c.1058_1059insGGTG p.Pro355Cysfs∗14 5.6  No  Relapse 7 different c.1078_1084dupACTCTTG p.Val362Aspfs∗8 8.3 COSM5879281 Yes AML  14 7  c.1090_1093dupTCGG p.Ala365Valfs∗4 22.81 COSM21392 Yes AML CCR  16 7  c.1054_1084dup p.Val362Alafs∗16 7.3  No  CCR  17 7  c.1087delCinsGGG p.Arg363Glyfs∗70 24.3  No  CCR  18 7  c.1054_1055insT p.Arg352Leufs∗16 67.2 COSM5751511 Yes T-ALL CCR  19 7  c.1077_1078insTGTTTCTTCCGCCCAG p.Thr360Cysfs∗13 36.95  No  Relapse  22 7  c.1087delCinsGG p.Arg363Glyfs∗5 41.88  Yes AML CCR  24 7  c.1083_1090dupTGTACGGT p.Ser364Leufs∗71 3.8 COSM27309 Yes AML CCR  25 9 different c.1323_1338dupAAAGTTCTCCCGGTCC p.Asp447Lysfs∗18 40.1  No  CCR 9 different c.1322_1332dupGAAAGTTCTCC p.Arg445Glufs∗9 40.5  No  27 7 different c.1077_1078insGTTG p.Thr360Valfs∗9 43.71  No  CCR 7 different c.1089dupG p.Ser364Valfs∗4 49.24 COSM28966 Yes AML  28 7 different c.1058delGinsCCA p.Arg353Profs∗6 19.72  No  NR/PR 7 different c.1054_1055insAAAAAGATT p.Arg352delins4 19.55  No  29 7  c.1179dupG p.His394Alafs∗8 25  No  CCR  30 7  c.1048_1057delGATGTGCGACinsAAGG p.Asp350_Arg353 46.34  No  CCR  31 7  c.1093dupG p.Ala365Glyfs∗3 44.49  Yes AML NR/PR  32 7  c.1048-8_1055dupGCCTGCAGGATGTGCG p.Arg353Profs∗20 2.5  No  NR/PR  33 7  c.1090_1091dupTC p.Ala365Argfs∗68 44.25 COSM28955 Yes AML Relapse  34 7 different c.1087delCinsGA p.Arg363Glufs∗5 4.1  No  NR/PR 7 different c.1086dupA p.Arg363Thrfs∗5 5.33 COSM1166631 Yes AML 7 different c.1090_1093dupTC p.Ala365Valfs∗4 36.29 COSM5487332 Yes AML  36 7 different c.1090_1093dupTCGG p.Ala365Valfs∗4 6.94 COSM5487332 Yes AML NR/PR 7 different c.1091dupC p.Ala365Glyfs∗3 39.42 COSM27304 Yes AML  37 7 different c.1057delCinsGG p.Arg353Glyfs∗15 42.78  Yes AML CCR 7 different c.1087delCinsGGG p.Arg363Glyfs∗70 52.11  No  38 7  c.1068_1076delAGTAGCCCCinsGACGGTCGTTATTA p.Val357Thrfs∗77 42.14  No  CCR  39 7  c.1087delCinsGG p.Arg363Glyfs∗5 47.54  Yes AML CCR  40 7 different c.1058_1059insGGTGCCGCTCG p.Gly356Leufs∗6 48.49  No  Early Death 7 different c.1082_1091dupTTGTACGGTC p.Ala365Cysfs∗6 41.83 COSM27303 Yes AML  41 7 different c.1123dupA p.Met375Asnfs∗9 44.5  Yes AML CCR 7 different c.1057_1058insTA p.Arg353Leufs∗6 45.8  No  42 7  c.1051_1055dupGTGCG p.Arg353Cysfs∗7 34.73  No  Relapse  43 7  c.1058delGinsCC p.Arg353Profs∗15 44.78 COSM28946 Yes AML Relapse  44 7 different c.1079_1101delinsGAA p.Thr360Argfs∗4 20.37  No  Relapse 7 different c.1088_1089insCTCGG p.Ala365Glyfs∗69 10.69  No  45 7  c.1090_1091insAGGT p.Ser364∗fs∗1 42.97  No  Relapse  46 7  c.1058delGinsCC p.Arg353Profs∗15 51.08 COSM28946 Yes AML, T-ALL NR/PR  47 7 different c.1048-3_1055dupCAGGATGTGCG p.Val354Metfs∗8 2.49  No  CCR 7 different c.1053dupG p.Arg352Alafs∗16 3.83 COSM28980 Yes AML 7 different c.1054delCinsGG p.Arg352Glyfs∗16 35.86 COSM28970 Yes AML, T-ALL  48 7  c.1089_1090insGGCCTCTTGTACGG p.Ser364Glyfs∗73 40.49  No  Relapse UPN, unique patient number; Seq. read, sequence read; VF, variant allele frequency; dup, duplication; ins, insertion; indel, Insertion-deletion; fs, frame-shift;∗termination codon; WT, Wilms tumor; DDS, Denis-Drash syndrome; T-ALL, T-cell acute lymphoblastic leukemia; CCR, continued complete remission, NR, non-response; PR, partial response; LFU, lost to follow-up. aTranscript ID: NM_000378 was used to describe all alterations.


## 3.3. Survival Significance of the Genomic Aberrations

Next, we analyzed the impact of each mutation on the clinical outcomes. Our analysis identifiedWT1andFLT3-ITD,but notNRAS,NPM1, orc-KIT mutations as single factors that significantly increased the chance of relapse or treatment failure and reduced the probability of 3-year overall survival (OS) in our patient cohort (Figures 2(a), 2(b), and 3). In addition,FLT3-ITDbut notWT1mutations significantly decreased the 3-year probability of event-free survival (EFS, Figure 2(b)). When we grouped the two mutations together, the survival analysis revealed a 3-year EFS of 29±11% for patients with bothWT1andFLT3-ITDmutations compared to 63±3% for patients with none of these mutations (p=0.0004) and 61±11% or 45±9% for patients with onlyWT1mutation (p=0.016) orFLT3-ITD (p=0.16), respectively (Figure 2(c)). Corresponding to this low EFS, co-occurrence of these two mutations was associated with an increased cumulative incidence of relapse (CIR) of 65±12% compared to 32±12% for patients with none of these mutations (p=0.002) and 39±11% or 46±9% for patients with onlyWT1mutation (p=0.05) orFLT3-ITD(p=0.08), respectively (Figure 2(c)). Furthermore, we identified a low 3-year OS probability of 33±12% in patients with co-occurrence ofWT1andFLT3-ITD, which was significantly lower than those of patients without these mutations (81±3%, p<0.0001), patients with only mutatedWT1(87±7%, p=0.0007), and patients with onlyFLT3-ITD(67±9%, p=0.017, Figure 2(c)). Comparing the curves for EFS and OS clearly demonstrated that our second line treatment was not able to rescue any patient with co-occurrence ofWT1andFLT3-ITD mutations, while the OS rates increased by more than 20% for the other three subgroups (Figure 2(c)).Figure 2
Co-occurrence of WT1 and FLT3-ITD mutations at initial diagnosis of pediatric AML predicts poor survival outcomes.(a)WT1 mutation as single factor increased the incidence of relapse, reducing the probability of survival. (b) The presence ofFLT3-ITD, individually, leads to an increased chance of relapse and decreased patient survival. (c) Clinical consequences ofWT1 mutations andFLT3-ITD were dependent on each other.WT1,Wilms Tumor 1;FLT3-ITD, fms related tyrosine kinase 3-internal tandem duplication; pEFS, probability of event-free survival; pOS, probability of overall survival; CIR, cumulative incidence of relapse; SE, standard error; n, number. ​aNo response to treatment was considered as the occurrence of an event at time zero.Figure 3
Mutations in NPM1, NRAS, and c-KIT had no impact on survival.(a) Prognostic impact of mutatedNPM1 on EFS, OS, and CIR. (b) Prognostic impact of mutatedNRAS on EFS, OS, and CIR. (c) Prognostic impact ofc-KIT mutation on EFS, OS, and CIR.NPM1,nucleophosmin 1;NRAS,neuroblastoma RAS viral oncogene homolog;c-KIT,KIT protooncogene; pEFS, probability of event-free survival; pOS, probability of overall survival; CIR, cumulative incidence of relapse; SE, standard error; n, number. ​aNo response to treatment was considered as the occurrence of an event at time zero.


## 3.4. Impact of NUP98-NSD1 Fusion

To further characterize the prognostic significance ofWT1andFLT3-ITDmutations, we analyzed the expression ofNUP98-NSD1fusion in our patient cohort (Figure 1(a)). From 246 patients with available material for this retrospective real-time quantitative PCR analysis, 15 (6%) of them were identified to have theNUP98-NSD1translocation. Most of these patients (12/15, 80%) harbored additionalWT1 orFLT3-ITDmutations: 3 patients carried bothWT1 andNUP98-NSD1, 4 had a co-occurrence ofFLT3-ITDandNUP98-NSD1, and 5 patients carried all three genetic alterations (Figure 1(b)). Only 1 of these 15 patients had a previous known status ofNUP98-NSD1 by conventional karyotyping: 2 others were previously diagnosed with deletion of chromosome 5, 1 carried an inversion of chromosome 16 (no other mutations and still in continuous complete remission), 4 carried complex karyotypes or rare aberrations, and 7 had no other cytogenetic abnormalities (data not shown).We then analyzed the prognostic significance ofNUP98-NSD1 in the cohort of 246 patients with the known status of this fusion gene (Figure 1(a)). As a single factor, the presence ofNUP98-NSD1in AML blasts of patients at diagnosis was associated with a significant increase in CIR (81%) in addition to decreased probabilities of 3-year EFS and OS (Figure 4(a)). CombiningNUP98-NSD1withWT1 andFLT3-ITDmutations in our multifactor survival analysis revealed that patients with all three or either two of these mutations had worse survival outcomes. These patients had a higher CIR of 73±11% compared to the CIR of 30±4% for patients with none of these aberrations orNUP98-NSD1alone (p<0.0001) and the CIR of 37±13% or 38±10% for patients with only mutatedWT1(p=0.0078) orFLT3-ITD(p=0.013), respectively (Figures 4(a) and 4(b)). The increased CIR translated into a lower 3-year EFS probability of 23±10% for patients with triple or double mutations compared to the EFS of 62±4% for patients with none of these mutations or onlyNUP98-NSD1 (p<0.0001) and the EFS of 63±13% or 54±10% for patients with onlyWT1 (p=0.003) orFLT3-ITD (p=0.036) mutations, respectively (Figure 4(b)). Moreover, co-occurrence of all three or any double mutations resulted in a significantly lower 3-year OS probability of 42±12% compared to 80±8% for patients with none of the mutations or onlyNUP98-NSD1(p=0.0003) and 88±8% or 73±10% for patients with onlyWT1 (p=0.0007) orFLT3-ITD(p=0.049) mutations, respectively (Figure 4(b)).Figure 4
Prognostic significance of NUP98-NSD1 fusion. (a)NUP98-NSD1 as single factor predicted poor outcomes. (b) Inclusion ofNUP98-NSD1 as poor prognostic factor withWT1 mutation andFLT3-ITD, predicted poor outcomes for patients harboring all three factors in addition to patients withNUP98-NSD1 andWT1 mutation orFLT3-ITD. Patients with unknown status ofNUP98-NSD1 fusion were excluded from this analysis. WT1,Wilms Tumor 1;FLT3-ITD, fms related tyrosine kinase 3-internal tandem duplication;NUP98-NSD1,Nucleoporin-Nuclear Receptor Binding SET Domain Protein 1 fusion gene; pEFS, probability of event-free survival; pOS, probability of overall survival; CIR, cumulative incidence of relapse; SE, standard error; mut, mutated; pos, positive; neg, negative. ​aNo response to treatment was considered as the occurrence of an event at time zero. ​bThree patients withNUP98-NSD1are included in this group.


## 3.5. Survival Significance of the FLT3-ITD Allelic Ratio

We have previously established the prognostic significance of anFLT3-ITD allelic ratio of ≥0.4 in pediatric AML [12]. Therefore, to determine the impact of the mutational burden ofFLT3-ITD on treatment outcomes in the present cohort, we calculated theFLT3-ITD AR in patients with available data/material. As indicated in Figure 1(b), 27 patients had an AR ≥0.4 at diagnosis. Analyzing the survival impact of theFLT3-ITD AR ≥0.4 revealed that as a single factor, it was associated with an EFS of only 25±8% and an OS of only 47±10%, respectively (Figure 5(a)). Remarkably, the co-occurrence ofFLT3-ITDAR ≥0.4,WT1, andNUP98-NSD1 as triple or double mutations significantly increased the CIR to 93±15% compared to the CIR of 31±4% for patients with no mutations or onlyNUP98-NSD1orFLT3-ITDAR <0.4 (p<0.0001) and to the CIR of 31±11% or 36±15% in patients with onlyWT1 (p<0.0001) orFLT3-ITDAR ≥0.4 (p=0.001) mutations, respectively (Figure 5(b)). The probability of 3-year EFS was zero in patients with double or tripleWT1,FLT3-ITDAR ≥0.4, andNUP98-NSD1 mutations as opposed to 61±4% in patients with no mutations or onlyNUP98-NSD1orFLT3-ITDAR <0.4 (p<0.0001) and 69±11% or 45±15% for patients with only mutatedWT1 (p<0.0001) orFLT3-ITDAR ≥0.4 (p=0.019), respectively (Figure 5(b)). Finally, the co-occurrence of double or triple mutations resulted in a 3-year OS probability of 27±13%, which was significantly lower than the 3-year OS of 79±3% in patients with no mutations or onlyNUP98-NSD1orFLT3-ITDAR <0.4 (p<0.0001) and 90±7% or 73±13% in patients with onlyWT1(p=0.0003) orFLT3-ITDAR ≥0.4 (p=0.06) mutations, respectively (Figure 5(b)). By multivariate analysis includingWT1mutation,FLT3-ITDAR ≥0.4, core-binding factor aberrations, early bone marrow response to treatment, and stem cell transplantation as covariables, we confirmed that the interaction of these three factors, and not each of the aberrations individually, was a significant predictor of poor prognosis for EFS (p=0.008, HR: 3.88, 95% CI: 1.42 – 10.6) and OS (p=0.042, HR: 3.42, 95% CI: 1.04 – 11.21, Table 3). Importantly, none of the patients with triple mutations survived and the only patients who could be rescued harbored doubleNUP98-NSD1 andWT1 orNUP98-NSD1 andFLT3-ITD mutations (Figure 1(b)), thus resulting in an OS of 27±13% (Figure 5(b)).Table 3
Multivariate analysis.
Cox regression analysis - Event-free survival  Parameters Hazard ratio 95% confidence interval p value Lower limit Upper limit  WT1 mutation 0.79 0.41 1.53 0.479  FLT3-ITD AR ≥ 0.4 1.55 0.69 3.51 0.288  WT1mutation, FLT3-ITD ≥ 0.4 and 3.88 1.42 10.66 0.008 NUP98-NSD1 interaction  t(8;21) and/or inv(16) 0.51 0.27 0.96 0.037  Unsatisfactory early response to treatment​a 1.31 0.79 2.18 0.294  HSCT​b 0.25 0.1 0.64 0.004  Cox regression analysis - Overall survival  WT1 mutation 0.84 0.35 2.06 0.710  FLT3-ITD ≥ 0.4 1.51 0.57 4.02 0.404  WT1mutation, FLT3-ITD ≥ 0.4and 3.42 1.04 11.21 0.042 NUP98-NSD1 interaction  t(8;21) and/or inv(16) 0.45 0.16 1.31 0.143  Unsatisfactory early response to treatment​a 1.21 0.61 2.42 0.589  HSCT​b 1.18 0.51 2.73 0.700 WT1, Wilms tumor 1; FLT3-ITD, fms related tyrosine kinase 3-internal tandem duplication; NUP98-NSD1, Nucleoporin-Nuclear Receptor Binding SET Domain Protein 1 fusion gen; t, translocation; inv, inversion; HSCT, hematopoietic stem cell transplantation. ​ aUnsatisfactory early response to treatment was defined as persistence of >5% blasts in bone marrow at day 15 and/or 28 after treatment. ​bhematopoietic stem cell transplantation events at first complete remission or after no-response to other treatments were included in the multivariate analysis as a time-dependent variable.Figure 5
Prognostic significance of mutational burden of FLT3-ITD. (a)FLT3-ITD with an allelic ratio ≥0.4 as a single factor predicted poor outcomes. (b) High mutational burden ofFLT3-ITD was another predictor of poor prognosis when it occurred withWT1 and/orNUP98-NSD1. Patients with an unknownFLT3-ITD AR were excluded from this analysis.NUP98-NSD1,Nucleoporin-Nuclear Receptor Binding SET Domain Protein 1 fusion gene;FLT3-ITD, fms related tyrosine kinase 3-internal tandem duplication; pEFS, probability of event-free survival; pOS, probability of overall survival; CIR, cumulative incidence of relapse; AR, allelic ratio; SE, standard error; n, number. ​aNo response to treatment was considered as the occurrence of an event at time zero. ​bThree patients withNUP98-NSD1 are included in this group.


## 4. Discussion

Treatment of pediatric AML has significantly improved over the past three decades due to the development of intensified first-line treatments, efficient second-line therapies, and optimized supportive care [2, 30]. The success is, at least partly, achieved by more efficient risk group stratification using factors such as somatic mutations and cytogenetic aberrations of AML blasts at diagnosis as well as considering the primary response to treatment to optimize the allocation of patients to standard or enhanced treatment options [1]. In the present study, we analyzed the influence of three parameters, mutations inWT1andFLT3and the translocation ofNUP98-NSD1, on the outcome of pediatric patients in the German AML-BFM 2004 and 2012 protocols. Although all three parameters have been established by us and others as important prognostic factors in both pediatric and adult patients [8–14, 20–22], their combined utility to identify high-risk patients likely to experience dismal treatment results has not yet been reported in a contemporary pediatric AML trial.In a cohort of 237 patients treated within the AML-BFM 2004 and 2012 protocols and with sufficient material for re-analysis, we observed favorable outcomes for 3-year EFS of 61% and 69% and OS of 79% and 90% in patients withoutWT1 mutations orNUP98-NSD1fusion or with only one of these factors. Patients with leukemic blasts that wereFLT3-ITD positive but negative forWT1 andNUP98-NSD1 mutations and that had anFLT3-ITD AR ≥0.4 still achieved an EFS of 45% and an OS of 73%. Surprisingly, our data therefore suggests that withoutWT1 andNUP98-NSD1mutations, the negative impact ofFLT3-ITD even with an AR≥0.4 might not be as severe as previously published [12, 17]. However, all patients positive for at least two of the three risk factors and with anFLT3-ITD AR ≥0.4 had events within the first three years and only 27% could be rescued by our salvage therapies. These unfavorable results in our double or triple mutated group unequivocally demonstrate that our current first-line treatment strategies for these patients are still insufficient/inadequate and urgently need improvement.Of the three risk factors, currently only theFLT3-ITD mutation can be specifically targeted with inhibitors [31]. Although the first generations of these drugs only achieved limited and often transient efficacy due to intrinsic and extrinsic adaptations in the AML blasts and/or the environment [31], combination therapies of newer tyrosine kinase inhibitors such as Quizartinib with standard chemotherapy seem to be relatively well tolerated and in initial studies have demonstrated survival improvement in relapsed or refractory AML patients [32–34]. Due to the important role of FLT3 pathway activation in AML, numerous combinations of FLT3 inhibitors with other drugs are currently being tested. Whether these results will also be helpful for the treatment of pediatric AML will need to be carefully determined in future studies, especially considering the clonal heterogeneity ofFLT3-ITD and the additional survival burden that it causes by increasing drug resistance through clonal evolution or selection and further expansion of resistant AML clones [35, 36]. Nevertheless, it is tempting to speculate that the simple addition of a newer FLT3 inhibitor to our standard therapy might be a feasible, well-tolerated, and effective approach for all patients with blasts that are positive for theFLT3-ITD mutation, regardless of the status of alterations inWT1 orNUP98.The role of WT1 in patients with AML is still controversial [4]. AlthoughWT1 is overexpressed in the majority of leukemias and can be used as a marker for minimal residual disease and maybe even vaccination attempts, the prognostic and therapeutic relevance of high or absent WT1 expression levels is not unequivocally accepted [37–39]. In contrast, mutations inWT1 are clearly identified as determinants of poor prognosis and, as we showed here, confer a dismal prognosis especially in combination withFLT3-ITD orNUP98-NSD1 fusion. In the present study, we identified 64 monoallelicWT1sequence alterations in exon 7 or exon 9 in the leukemic blasts of 48 patients. The majority of these alterations leads to frameshifts and/or premature terminations codons and thus shortened proteins. These mutant proteins can act in a dominant negative manner [40], which may contribute to a myeloid differentiation block present in AML blasts [41]. However, similar mutations have also been described in the context of Wilms tumors as gain-of-function mutations promoting proliferation [42]. Here, we show a favorable prognosis for patients with singleWT1mutations, with 26 out of 29 cases reaching continued complete remission (CCR) (Figure 1(b)). Therefore, based on a 3-year EFS of 69% and an OS of 90%, the development of new treatment approaches is not as urgently needed for these patients withWT1 mutated blasts that do not harborFLT3-ITD orNUP98-NSD1 mutations.Among the 31 different fusion gene partners ofNUP98 identified so far, theNUP98-NSD1 t(5:11) translocation is the most frequent and present in 4-7% of patients in pediatric AML patients [20–22]. Importantly, theNUP98 translocations that occur in AML all share the N-terminus of the protein and are thought to initially lead to epigenetic dysregulation of different leukemia-associated genes includingHOXA7,HOXA9, andHOXA10 in myeloid precursor cells [20]. Additional somatic mutations in other genes occur as secondary events and promote malignant transformation and uncontrolled cell growth [20]. As also shown in our patient data set, these secondary alterations often include activating mutations inFLT3 (FLT3-ITD)or truncating mutationsin WT1 [21]. Strikingly, only three patients in our study had aNUP98-NSD1 translocation without mutations inFLT3 orWT1; two of these patients achieved and remained in first CCR at the end of data acquisition. The third patient had no other genetic risk factors but a very high initial white blood cell count of almost 400,000 cells/μl. Complete remission induction was delayed, and the patient relapsed a year later but was successfully treated by allogeneic stem cell transplantation with a follow-up of 10 years. Therefore, as also described previously [21], our patients withNUP98-rearranged blasts withWT1 and/orFLT3-ITD mutations had a poor prognosis, especially in contrast to patients with onlyWT1 andFLT3-ITD mutations, who could at least partially be rescued by allogeneic transplantation. However, due to the high risk of failure of the first-line treatment, stem cell transplantation already in first CCR seems to be an attractive option for cases ofNUP98-rearranged AML [21, 22]. Nevertheless, it should be noted that even allogeneic stem cell transplantation is not always effective in improving the treatment outcome in patients with a high probability of treatment failure based on risk stratification. Thus, introducing novel treatment approaches such as the use of small inhibitors, e.g., venetoclax and isadanutlin [43] or cellular therapies with allogeneic NK-cells or engineered T-cells with chimeric antigen receptors (CARs) [44] targeting leukemic blasts harboringNUP98 rearrangement orWT1mutations should be taken into consideration in future clinical studies.Recent analysis from a collaborative study between the American and Dutch children oncology groups (COG and DCOG) included patients from three clinical COG/DCOG trials and also young adults less than 39 years of age in the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) AML initiative [45]. Analysis of the different cohorts revealed similarly unfavorable outcomes with an EFS of 14-25% and an OS of 15-40% for patients withFLT3-ITDandWT1mutations and/or theNUP98-NSD1translocation [45]. In contrast to our findings however, the authors reported an EFS range of 15-35% in patients withFLT3-ITDonly, which is lower than that achieved with current protocols, for which an EFS of 45% and an OS of 73% were found for patients withFLT3-ITD only. Notably, in the American-Dutch study, patients with co-occurrence ofNPM1mutations andFLT3-ITD(and withoutWT1 andNUP98-NSD1) were separated from patients withFLT3-ITDonly and had a slightly increased, albeit probably not statistically significant, survival. Similarly, we have previously observed favorable outcomes for patients withNPM1mutations in their AML blasts with normal karyotype and proved this impact was not affected by the presence ofFLT3-ITD [46]. In the current cohort, five patients were positive for mutations inFLT3-ITD andNPM1 and negative forWT1 andNUP98 alterations. At present, four patients with a normal karyotype are still in first CCR, and the fifth patient with a complex karyotype and anFLT3-ITD AR >11 experienced early death. In summary, the principle findings of this American-Dutch study and the present study are very similar. However, the treatment outcomes for our patient groups are superior, most likely due to the fact that we included only patients between 0 and 18 years of age treated in Germany according to two contemporary protocols from the AML BFM study group.


## 5. Conclusion

Despite the fact that our study was partly based on data collected prospectively since 2004 and partly on data assessedde novo on stored material by either NGS or PCR, we can safely conclude that co-occurrence of the three factors, mutatedWT1andFLT3-ITD and/or NUP98-NSD1translocation,still defines a subgroup of AML patients with devastating EFS and OS outcome, even with our current treatment protocols. Although the number of pediatric AML patients available for analysis of these three risk factors was limited and therefore not all interesting factors could be assessed in multivariate analysis, it is obvious that patients with double or triple mutations benefitted very little from the improved EFS and OS in our AML-BFM studies in recent years. Thus, for these pediatric patients, new and more targeted approaches are urgently needed for both first- and second-line treatments.



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