Acute myeloid leukemia (AML) is a clinically and genetically heterogeneous but aggressive disease in which cytogenetic findings are considered to be the most powerful single prognostic factor.1 Most patients belong to the category with standard-risk disease and the stratification of this heterogeneous group seems to be insufficient. Along with cytogenetics, mutational analysis is one of the most important approaches to gain understanding of the molecular bases of leukemogenesis. Dash and Gilliland2 suggested that AML blasts develop from normal blasts affected by two types of genetic damage: class I and class II mutations. Class I mutations result in constitutive activation of cell surface receptors and confer a proliferative and survival advantage on the blast cells. This group includes activating mutations in receptor tyrosine kinases such as FLT3 (fms-like tyrosine kinase-3) and c-KIT and in the GTPase RAS family. In contrast, class II mutations seem to impair differentiation, but are not sufficient to cause leukemia when expressed alone. This second group includes chromosomal rearrangements leading to AML1-ETO, CBFβ-MYH11 or MLL fusion genes, deregulation of HOX genes expression and mutations in CEBPα (CCAAT/enhancer-binding protein α) gene. More recently, gene mutations in two new molecular markers, NPM1 (nucleophosmin) and WT1 (Wilms'mor 1), were reported to have a critical function in leukemia. The two-hit model of leukemogenesis, combining an activating lesion of tyrosine kinase pathways with event-blocking myeloid differentiation, seems very interesting to screen molecular events in AML patients, in view of the frequent association of a class I and a class II mutation.
There exist many studies of the frequency and prognostic significance of gene mutations in adult AML. Very few studies have been performed in pediatric populations. We studied a well-characterized cohort of 76 children with de novo AML and evaluated at diagnosis the frequency and the impact on clinical outcome of FLT3 internal tandem duplication (FLT3-ITD) and tyrosine kinase domain (FLT3-TKD), c-KIT, N-RAS, K-RAS, CEBPα, NPM1 and WT1 gene mutations in pediatric AML with a long clinical follow-up (median, 72 months). The main aim of the study was to determine whether these molecular lesions could be used to improve the molecular-risk stratification to better predict the response to therapy.
In 76 children diagnosed with de novo AML, we found FLT3-ITD, FLT3-TKD, c-KIT, N-RAS, K-RAS, CEBPα, NPM1 and WT1 gene mutations in 8, 6.5, 4, 10.5, 1.3, 9, 5 and 8% of cases, respectively (Table 1). The most frequent mutations in this study occurred in the FLT3 gene, with 11 (14.5%) of the 76 patients displaying an activating mutation of FLT3. FLT3-ITD and FLT3-TKD were found in 6 and 5 cases, respectively. Mutation of the other receptor tyrosine kinases, c-KIT was detected in three AML patients. In all, 11% (n=9) showed an activating mutation of N-RAS (n=8) or K-RAS (n=1). N-RAS mutation involved either exon 1 (n=6) or exon 2 (n=2), whereas K-RAS mutation was found only in exon 1. Among the seven patients (9%) with CEBPα mutations, three had mutations in both the N-terminal and bZIP domains, one had only an N-terminal mutation and three had only a mutation in the bZIP domain. Four of the 76 AML patients displayed a mutation of NPM1, whereas a WT1 mutation was detected in six (8%) cases, all in exon 7.
Overall, 40 mutations were detected in 34 patients, 28 patients having one mutation and 6 two mutations (17.5%). The coexistence of WT1 and FLT3-ITD mutations was detected in four patients and was the most frequent association. Mutations in NPM1 were associated with an FLT3-TKD mutation in one case and an N-RAS mutation in another (Figure 1a).
Among the 76 children diagnosed with de novo AML, 72 (95%) had a chromosome analysis, which could be interpreted by standard or FISH techniques. Patients belonged to the MRC1, MRC2 and MRC3 groups in 15% (n=11), 68% (n=49) and 17% (n=12) of cases, respectively. Normal karyotype and 11q23/MLL rearrangement were observed in 30% (n=22) and 19.5% (n=14) of cases. As expected, NPM1, CEBPα and FLT3 mutations were mainly associated with a normal karyotype or the intermediate MRC2 prognostic subgroup. In our study, the repartition of c-KIT and N-RAS mutations was unusual with no association between AML1-ETO rearrangement and c-KIT mutation, an over representation of N-RAS mutation in t(6;9) with 3/4 cases and only one N-RAS mutation in the core-binding factor (CBF) AML subgroup. The only K-RAS mutation was associated with an 11q23/MLL rearrangement. WT1 mutations were detected in all MRC groups and represented 2/22 of normal karyotypes, 2/15 of other MRC2 karyotypes, 1/11 of CBF AML and 1/10 of MRC3 patients (Figure 1b). In general, gene mutations were more frequently found in AML patients with t(6;9), inv(16)/t(16;16) or normal karyotype. On the contrary, few mutations were identified in AML with 11q23/MLL rearrangement, involving a different MLL partner in each case. All gene mutations and their prevalence according to cytogenetic findings are summarized in Figure 1a and b.
A total of 71 patients (93%) achieved complete remission and with a median follow-up of 72 months (range, 44–127), the 5-year event-free survival (EFS) and overall survival (OS) rates were 59±6% and 67±5%, respectively.
Prognostic factors for adverse events and death in the cohort (n=76) are reported in Table 2. In univariate analysis, MRC3 classification, M7 FAB subgroup and FLT3-ITD, N-RAS and WT1 mutations were associated with an adverse outcome. At 5 years, the estimated OS was 25±15% in the presence of an N-RAS mutation and 72±5% in its absence (P=0.009). This adverse prognosis associated with N-RAS mutations was also observed in terms of EFS (P=0.02). At 5 years, the estimated EFS was 17±15% in the presence of either an FLT3-ITD (P=0.004) or a WT1 mutation (P=0.01) and 63±6% in the absence of these mutations. However, the presence of FLT3-ITD or WT1 mutations was not associated with an inferior response to induction chemotherapy or poorer OS as compared with wild-type cases. In our study, there were no statistical differences in 5-year EFS or OS between patients with and without CEBPα mutations. Although the number of cases of pediatric AML with c-KIT, NPM1, K-RAS or FLT3-TKD gene mutations were too small to reliably evaluate OS and EFS, all these patients were alive at 5 years (range, 45–127 months). Overall, 14 patients received a matched-sibling allograft in first complete remission without any significant impact on the survival (P=0.84, Cox with a time-dependent variable).
At diagnosis, six patients (8%) had one WT1 mutation in exon 7 (Table 3), whereas no patient had a WT1 mutation in exon 9. Patients with WT1 mutations tended to be older (P=0.1), to have higher WBC counts (P=0.1) and to harbor more often FLT3-ITD mutations (P<0.01), but displayed no specific cytogenetic profile. Interestingly, all these patients expressed very high levels of WT1 transcripts (data not shown). Although all of them achieved complete remission, they relapsed more frequently than patients with a wild-type karyotype (83 versus 39%, P=0.01) (Figure 2). The estimated 5-year EFS was only 17% for WT1 mutated patients as compared with 63% for wild-type AML cases.
In multivariate analysis, there were too many WT1mut7/FLT3-ITD double positive patients to reliably examine the independent impact of these two markers. We chose to focus on WT1 mutations, which had not yet been evaluated in pediatric AML. In a multivariate analysis (Table 4) considering age, sex, FAB subtype and cytogenetic data, N-RAS and WT1 mutations independently predicted a poorer EFS (P=0.02 and 0.004, respectively), the estimated risk of relapse being 4.5-fold higher for children with WT1 mutations as compared with children without WT1 mutations.
The mutation frequencies we observed in FLT3, RAS, c-KIT, CEBPα and NPM1 are consistent with those reported in earlier pediatric studies.3, 4, 5, 6 WT1 mutations were described in up to 15% of adult AML patients 10 years ago and more recently were implicated in 10% of cases of CN-AML, mainly mutations in exon 7.7, 8 One recent pediatric study showed WT1 mutations in 12% of AML patients.9 Consistent with these results, we found WT1 mutations in 8% of our pediatric series, exclusively in exon 7.
The prognostic implications of these mutations were analyzed and substantially extend the findings of earlier studies: (1) an adverse prognosis for 5-year EFS and OS was associated with N-RAS mutations, (2) no statistical differences were found in 5-year EFS or OS between patients with and without CEBPα mutations, (3) patients with NPM1 or FLT3-TKD mutations had an excellent outcome and (4) FLT3-ITD or WT1 mutations were associated with inferior 5-year EFS.
Contrary to earlier studies, our results do not confirm that CEBPα mutations predict favorable outcome in pediatric6 and adult10 AML. As c-KIT mutations were only found in inv(16)(p13q22) AML, these patients had an excellent outcome. In contrast, some recent studies showed an adverse prognostic impact of c-KIT mutation in CBF AML, especially with t(8;21)(q22;q22).11 The prognostic impact of N-RAS mutations is still very controversial. In our study, N-RAS mutations were identified only in one case of CBF AML and in 4/8 cases of AML with unfavorable cytogenetics, especially t(6;9), but nevertheless remained an independent adverse prognosis factor.
In two large-scale adult and one recent pediatric studies, patients with WT1 mutations displayed an inferior response to induction chemotherapy in adult and an increased cumulative incidence of relapse with a reduction in both relapse free and OS in both children and adults. On the whole, the presence of a WT1 mutation was an independent adverse prognostic factor.7, 8, 9 In our study, all children with a WT1 mutation reached complete remission, but the estimated risk of relapse was 4.5 times higher than in children without WT1 mutations and half of them died. Our results do not confirm a higher rate of resistant disease but do confirm the adverse prognostic impact of WT1 mutations on EFS.
A concomitant FLT3-ITD was found in 4/6 patients carrying WT1 mutations, in agreement with adult studies.7, 8, 9 On the basis of the two-hit model of AML pathogenesis, this frequent association between FLT3-ITD and WT1 mutations argues for a function of the latter in hematopoietic differentiation rather than proliferation. The WT1 gene is located on chromosome 11p13, consists of 10 exons and encodes a transcription factor with two major functional domains, an N-terminal transcriptional regulatory domain (exons 1–6) and a C-terminal DNA-binding domain composed of four zinc fingers (exons 7–10). The precise function of WT1 in normal and malignant hematopoiesis is still controversial. Whereas low levels of WT1 expression have been shown in normal bone marrow and CD34+ progenitor cells,12 WT1 gene is overexpressed at diagnosis in 70–80% of AML cases in adults12 and children.13 WT1 exon 7 mutations give rise to a truncated protein lacking the four zinc fingers, which disrupts the binding domains for interacting proteins and the nuclear localization signal. Concomitantly, WT1 gene was overexpressed in all our WT1 mutants, suggesting that WT1 exon 7 mutations might cause failure of negative feedback. It is also possible that WT1 exon 9 mutations, which lead to the loss of two zinc fingers, may have different effects on cellular regulation and prognostic impact in AML.
In conclusion, we showed that the presence of WT1 mutations at diagnosis is an important factor predicting relapse in pediatric AML. Further pediatric studies will be necessary to extend our knowledge and more precisely define the prognostic significance of WT1 mutations. In view of our results and those of others, NPM1 and WT1 mutations should be prospectively investigated at diagnosis in pediatric AML to improve the current risk and treatment stratification.
Conflict of interest
The authors declare no conflict of interest.
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This study was supported by the Association pour la recherche dans les maladies hématologiques de l'ant (ARMHE), the Association Laurette Fugain, fondation de France (comité leucémie) and northwest canceropole (axis 2). We thank all the technicians of the laboratories involved for their excellent contribution to this work.
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Lapillonne, H., Llopis, L., Auvrignon, A. et al. Extensive mutational status of genes and clinical outcome in pediatric acute myeloid leukemia. Leukemia 24, 205–209 (2010) doi:10.1038/leu.2009.172
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