Abstract

Although virtually all pediatric patients with acute myeloid leukemia (AML) achieve a complete remission after initial induction therapy, 30%-40% of patients will encounter a relapse and have a dismal prognosis. To prevent relapses, personalized treatment strategies are currently being developed, which target specific molecular aberrations. To determine relevance of established AML type I/II mutations that may serve as therapeutic targets, we assessed frequencies of these mutations and their persistence during disease progression in a large group (n = 69) of paired diagnosis and relapse pediatric AML specimens. In 26 of 42 patients (61%) harboring mutations at either stage of the disease, mutation status changed between diagnosis and relapse, particularly in FLT3, WT1, and RAS genes. Presence or gain of type I/II mutations at relapse was associated with a shorter time to relapse (TTR), whereas absence or loss correlated with longer TTR. Moreover, an adverse outcome was found for patients with activating mutations at relapse, which was statistically significant for FLT3/ITD and WT1 mutations. These findings suggest that mutational shifts affect disease progression. We hence propose that risk stratification, malignant cell detection, and selection of personalized treatment should be based on status of type I/II mutations both at initial diagnosis and during follow-up.

Introduction

Current treatment of acute myeloid leukemia (AML) is successful in almost 70% of pediatric patients. Although virtually all AML patients achieve a complete remission after initial treatment, a large fraction of patients (30%-40%) will have a relapse, and these patients have a dismal prognosis.1,2  Factors that are related to prognosis in de novo pediatric AML are clinical parameters such as age and response to treatment. Moreover, biologic characteristics of AML, including cytogenetics,3,4  minimal residual disease,5,6  and a number of molecular aberrations,7-11  also play an important role. According to the proposed “2 hit” model of leukemogenesis,12  2 types of molecular aberrations are discernible: (1) activating type I mutations, for example, in RAS or FLT3, thus conferring increased proliferative and survival capabilities on leukemic cells; (2) type II abnormalities, for example, chromosomal aberrations such as t(8;21) or mutations in the CEBPA gene, which cause a differentiation arrest and an increase in self-renewal properties. According to this model, both types of aberrations act in concert to initiate and drive AML progression.

Drugs that directly target aberrant proteins according to their corresponding type I/II mutations may therefore greatly improve prognosis. Kinase inhibitors such as lestaurtinib, midostaurin, or the more recently developed FLT3 kinase inhibitor AC220 were shown to be predominantly effective in mutated FLT3/ITD-positive AML samples in vitro.13-15  In clinical trials with kinase inhibitors, for example, lestaurtinib16  or sunitinib,17  responses were observed particularly in patients harboring mutant FLT3. Ultimately, these targeted approaches may allow personalized chemotherapy of AML that could either eliminate subsets of leukemic cells that are resistant to conventional chemotherapy or eradicate minimal residual disease (MRD), thereby preventing the emergence of relapse. Various novel drugs are now being developed to specifically target leukemic cells according to their specific molecular aberrations.18,19 

Apart from the use of type I/II aberrations as putative therapeutic targets, they may also be used as molecular markers for the detection of malignant cells. Type I/II aberrations or expression of the genes involved have been proposed in the literature as markers for the molecular assessment of MRD with the use of different techniques (eg, NPM120,21 , WT122,23 , t(8;21),24  inv(16),25  PML-RAR26,27 ). For the use of the type I/II aberrations for leukemic cell detection and for subsequent targeted therapy, it will be essential to know the robustness of the mutation status during disease progression, that is, at diagnosis and relapse. Following the initial description28  of cytogenetic changes from diagnosis to relapse, it has been shown by others29  as well as by our own group30  that FLT3/ITD aberrations are not persistently present in initial diagnosis and corresponding relapse AML samples. Such changes in molecular aberrations may theoretically result from outgrowth of otherwise undetectable rare clones present at diagnosis or from acquisition of de novo alterations during or after chemotherapy. These phenomena may lead not only to inaccuracies in malignant cell detection but also to suboptimal drug selection. Therefore, further research on the persistence of these aberrations after initial diagnosis is warranted.

In the current study we determined relevant type I/II molecular aberrations in paired diagnosis and relapse samples of patients with pediatric AML to determine their frequency and persistence during disease progression.

Methods

Patient samples

After informed consent was obtained in accordance with the Declaration of Helsinki, bone marrow or peripheral blood samples were collected from 69 paired patients with pediatric AML (n = 138 samples) at initial diagnosis and at relapse. Pediatric samples were obtained from the Dutch Childhood Oncology Group and the AML Berlin-Frankfurt-Münster Study Group. Approval from the Institutional Review Board of VU University Medical Center was obtained. This study is in part an extension of a previous reported study that used paired samples30 : 34 pediatric patients from the previous study were also included in the current study. Mononuclear cells were isolated by standard Ficoll (1.077 g/mL; Amersham Biosciences) gradient centrifugation, and samples were enriched for leukemic blasts by eliminating nonleukemic cells as previously described.31  Central review of the diagnosis, classification by morphology or cytogenetics, and clinical follow-up of the patients were performed by both study groups. Percentages of blast cells were assessed by morphologic analysis of May-Grünwald-Giemsa–stained cytospin slides. Leukemic samples were routinely investigated for cytogenetic abnormalities by standard chromosome-banding analysis and screened for recurrent nonrandom genetic abnormalities, typical for AML. Applied screening methods for cytogenetic abnormalities included reverse transcription–polymerase chain reaction or fluorescent in situ hybridization or both. Cytogenetic data were determined for the initial sample only.

Treatment protocols

Patients were all treated with intensive cytarabine/anthracycline-based protocols in the Netherlands and Germany from 1992 until 2004 (protocols Dutch Childhood Oncology Group AML 87, 94, and 97 and AML–Berlin-Frankfurt-Münster 93, 98, and 04).32-34 

Molecular analysis of paired samples

Genomic DNA was isolated from cytospin slides, frozen cell pellets, or liquid nitrogen cryopreserved cells. Mutation analyses were performed in the VU University Medical Center, Amsterdam, and Erasmus Medical Center/Sophia Children's Hospital, Rotterdam, The Netherlands, with the use of sensitive high-throughput methods, which are summarized in Table 1. Mutations in NPM1 exon 12 were analyzed by PCR on genomic DNA in a multiplex reaction with FLT3/ITD analysis. PCR amplification was performed with the following primers: NPM1 forward, 5′-TTAACTCTCTGGT-GGTAGAATGA-3′, and NPM1 reverse. 5′-CTGACCACCGCTACTACTATGT-3′, located in intron 11 and exon 12, respectively. Subsequent fragment analysis was performed with a tetrachlorofluorescein phosphoramidite–labeled (Biolegio) forward primer. Mutations detected with melting curve analysis were confirmed with the use of 2 methods: (1) reamplification of the exon and repeated melting curve analysis or (2) bidirectional DNA sequencing on an ABI 3100 automated sequencer with the use of the BigDye terminator kit (Applied Biosystems Inc).

Table 1

Summary of mutation analysis assays

Gene Type* Mutation Assay References 
FLT3 Type I ITD Fragment analysis Cloos, 200630  
  D835 Lightcycler/sequencing Cloos, 200630  
N-RAS/K-RAS Type I Codon 12/13 Lightcycler/sequencing Goemans, 200535 ; Kramer, 200936  
  Codon 61 Lightcycler/sequencing Goemans, 200535  
KIT Type I D816V Lightcycler/sequencing Goemans, 200535  
  Exon 11 ITD Fragment analysis Corbacioglu, 200637  
WT1 No consensus Exon 1, 2, 7, 8, 9, 10 dHPLC, sequencing Hollink, 20098  
CEBPA Type II All coding regions dHPLC, sequencing Wouters, 200938 ; Hollink, 200939  
PTPN11 Type I Exon 3/13 Sequencing Goemans, 200540  
NPM1 No consensus Exon 12 Fragment analysis See “Methods” 
Gene Type* Mutation Assay References 
FLT3 Type I ITD Fragment analysis Cloos, 200630  
  D835 Lightcycler/sequencing Cloos, 200630  
N-RAS/K-RAS Type I Codon 12/13 Lightcycler/sequencing Goemans, 200535 ; Kramer, 200936  
  Codon 61 Lightcycler/sequencing Goemans, 200535  
KIT Type I D816V Lightcycler/sequencing Goemans, 200535  
  Exon 11 ITD Fragment analysis Corbacioglu, 200637  
WT1 No consensus Exon 1, 2, 7, 8, 9, 10 dHPLC, sequencing Hollink, 20098  
CEBPA Type II All coding regions dHPLC, sequencing Wouters, 200938 ; Hollink, 200939  
PTPN11 Type I Exon 3/13 Sequencing Goemans, 200540  
NPM1 No consensus Exon 12 Fragment analysis See “Methods” 

ITD indicates internal tandem duplication; dHPLC, denaturing high-performance liquid chromatography.

*

Specifies to which category a mutation for the given gene belongs according to the “2-hit model” by Gilliland et al.12  When consensus is lacking in the literature as to which category a mutation belongs, it is so stated.

Statistical analysis

To compare categorical variables we used χ2 analysis and Fisher exact test in the case of small numbers. Nonparametric Mann-Whitney U test, Spearman rank correlation, and analysis of variance were applied to assess differences in the distribution of continuous variables. To assess outcome, the following parameters were used: complete remission (defined as < 5% blasts in a bone marrow aspirate and no evidence of leukemia at any other site and hematologic recovery according to Cancer and Leukemia Group B criteria, relapse (defined by classical morphologic criteria when containing > 5% blasts), overall survival (OS; defined as the period of time in months between the date of diagnosis and of death from any cause or last follow-up), and the time to relapse (TTR; defined as the number of months between the date of initial diagnosis and of relapse). OS was estimated by the Kaplan-Meier method, and different groups were compared with the log-rank test. Prognostic factors were examined by multivariate Cox regression analysis. P values ≤ .05 were considered statistically significant (2-tailed testing). SPSS statistical software Version 17.0 for Windows (SPSS Inc) was used.

Results

Study population

We successfully obtained genomic DNA from paired initial diagnosis and corresponding relapse samples from 69 patients with pediatric AML. Not all patients were evaluable for all mutations because of limited amounts of genomic DNA. Patient characteristics are depicted in Table 2. The sex of the majority of the study cohort was male (70%). Patients were equally distributed over different treatment protocols, and no significant difference in OS was found between the protocols (data not shown). Because of the selection of patients with a relatively poor outcome (ie, only for those patients who developed a relapse), the results of patient characteristics and type I/II mutation analysis at diagnosis, might be expected to differ from what has been described in literature for newly diagnosed pediatric AML. Indeed, we found a higher frequency of FLT3/ITD (20.8%), RAS (23.8%), and WT1 (17.8%) mutations compared with previous results.7,8,35,41  However, the frequency of NPM1 mutations (7.2%) at initial diagnosis was comparable with the incidence described previously.9  The coincidence of these mutations with each other also differed; for example, NPM1 mutations coincided less frequently (1 of 67 analyzed cases, 1.5%) with FLT3 mutations compared with the 40% coincidence that was previously described.9  Similarly, the numbers of patients with favorable and intermediate cytogenetic aberrations were lower than previously described for unselected patients.42  The selected group is representative for patients with relapsed AML in terms of TTR rates, because approximately one-half of the relapses (57%) were early relapses and occurred within 1 year from diagnosis.1  The 2-year OS rate was 36%, which is lower compared with all patients with newly diagnosed pediatric AML treated with similar protocols and in line with previous reports on outcome of children with relapsed AML.43 

Table 2

Clinical, morphologic, and cytogenetic characteristics of the included patients with AML at diagnosis

Characteristic Value 
No. of patients 69 
Median age at diagnosis, y (range), n = 67 9.1 (8.8; 0.3-16.5) 
PT-BRWBC count, ×109/L, median (25-75 percentiles), n = 56 44.9 (16.6-155.9) 
Sex, n (%)  
    Female 20 (29) 
    Male 49 (71) 
FAB classification, n (%)  
    M0 5 (7) 
    M1 11 (16) 
    M2 14 (20) 
    M3 2 (3) 
    M4 16 (23) 
    M5 12 (17) 
    M6 1 (1) 
    M7 2 (3) 
    Unknown 6 (9) 
Cytogenetic subgroups  
    AML-ETO1 7 (10) 
    MLL 5 (7) 
    PML-RAR 0 (0) 
    CBF-MYH 2 (3) 
    CN-AML 13 (19) 
    Other 22 (32) 
    Unknown 20 (29) 
Characteristic Value 
No. of patients 69 
Median age at diagnosis, y (range), n = 67 9.1 (8.8; 0.3-16.5) 
PT-BRWBC count, ×109/L, median (25-75 percentiles), n = 56 44.9 (16.6-155.9) 
Sex, n (%)  
    Female 20 (29) 
    Male 49 (71) 
FAB classification, n (%)  
    M0 5 (7) 
    M1 11 (16) 
    M2 14 (20) 
    M3 2 (3) 
    M4 16 (23) 
    M5 12 (17) 
    M6 1 (1) 
    M7 2 (3) 
    Unknown 6 (9) 
Cytogenetic subgroups  
    AML-ETO1 7 (10) 
    MLL 5 (7) 
    PML-RAR 0 (0) 
    CBF-MYH 2 (3) 
    CN-AML 13 (19) 
    Other 22 (32) 
    Unknown 20 (29) 

WBC indicates white blood cell; FAB, French-American-British morphology classification; and CN-AML, cytogenetically normal acute myeloid leukemia.

Incidence of type I/II mutations at diagnosis and at relapse

Of the 69 patients studied, 42 patients (61%) had a mutation identified either at diagnosis or at relapse. Table 3 depicts the frequency of mutations measured both at initial diagnosis and at relapse. Overall, the frequency of mutations did not differ significantly between diagnosis and relapse, except for WT1 which had a nearly 2-fold increased incidence of mutations at relapse. Mutations were not mutually exclusive in these patients with relapsed pediatric AML. In particular, FLT3/ITDs coincided with NPM1, CEBPA, RAS, and WT1. Overall, 18 of 42 patients (43%) who had a mutation at any stage of the disease harbored more than one different mutation. An overview of all mutation data for each patient is listed in supplemental Table 1 (available on the Blood Web site; see the Supplemental Materials link at the top of the online article).

Table 3

Overall incidence of type I/II mutations at diagnosis or relapse

Gene Diagnosis
 
Relapse
 
P 
No. of evaluated patients No. of patients with mutation (%) No. of evaluated patients No. of patients with mutation (%) 
FLT3/ITD 67 14 (20.8) 62 10 (16.1) .14 
WT1 45 8 (17.8) 42 14 (33.3) .05 
RAS 63 15 (23.8) 52 10 (19.2) .15 
NPM1 69 5 (7.2) 68 4 (5.9) .22 
CEBPα 31 3 (9.6) 30 4 (13.3) .28 
KIT 33 3 (9.1) 27 2 (7.4) ND 
PTPN11 65 2 (3.5) 58 0 (0.0) ND 
FLT3 D835 57 2 (3.5) 50 1 (2.0) ND 
Gene Diagnosis
 
Relapse
 
P 
No. of evaluated patients No. of patients with mutation (%) No. of evaluated patients No. of patients with mutation (%) 
FLT3/ITD 67 14 (20.8) 62 10 (16.1) .14 
WT1 45 8 (17.8) 42 14 (33.3) .05 
RAS 63 15 (23.8) 52 10 (19.2) .15 
NPM1 69 5 (7.2) 68 4 (5.9) .22 
CEBPα 31 3 (9.6) 30 4 (13.3) .28 
KIT 33 3 (9.1) 27 2 (7.4) ND 
PTPN11 65 2 (3.5) 58 0 (0.0) ND 
FLT3 D835 57 2 (3.5) 50 1 (2.0) ND 

ND indicates not determined.

The observed frequency of mutations was distinct in the cytogenetic subgroups. For instance, FLT3/ITD, WT1, RAS, and NPM1 mutations were predominantly found in the group of cytogenetically normal (CN) patients with AML and the subgroup of “other cytogenetics.” Occasional cases of RAS mutations were observed in mixed-lineage leukemia (MLL), AML-ETO1, and core binding factor AML. Supplemental Figure 1 summarizes the observed incidence of mutations per cytogenetic subgroup at diagnosis.

To assess the retention of mutations during disease progression, we compared the mutation data obtained at diagnosis and their matched relapse pairs. We observed mutational shifts between diagnosis and relapse for the currently analyzed genes in 26 of 69 patients (38%), which constitutes 61% of the 42 samples harboring a mutation at some stage of disease (Table 4; supplemental Table 1). This analysis shows frequent mutational shifts in FLT3/ITD, RAS, and WT1. In FLT3 and RAS, both gains and losses of mutations occurred between diagnosis and relapse. Both types of mutations were more frequently lost than gained. In WT1, however, mutations accumulated at relapse because these mutations were only gained and none were lost. Occasional changes were also found in other targets such as CEBPA and FLT3 D835. The frequencies of mutational shifts per gene of interest are given in Table 4. When stratified according to cytogenetic subgroups, mutational shifts occurred more frequently in patients wit AML with CN-AML or MLL rearrangements (54% and 80% of analyzed cases, respectively), compared with other cytogenetic subgroups (supplemental Table 2).

Table 4

Frequencies of gains and losses of molecular aberrations in pediatric AML (n = 69)

Type of change Gene of interest
 
FLT3/ITD (n = 63) FLT3 D835 (n = 50) RAS (n = 52) KIT (n = 27) WT1 (n = 42) NPM1 (n = 68) CEBPA (n = 30) 
Gain at relapse 
Loss at relapse 
Total no. of changes (%) 6 (9.5) 2 (4) 11 (21) 0 (0) 5 (12) 1 (1.5) 1 (1.5) 
Type of change Gene of interest
 
FLT3/ITD (n = 63) FLT3 D835 (n = 50) RAS (n = 52) KIT (n = 27) WT1 (n = 42) NPM1 (n = 68) CEBPA (n = 30) 
Gain at relapse 
Loss at relapse 
Total no. of changes (%) 6 (9.5) 2 (4) 11 (21) 0 (0) 5 (12) 1 (1.5) 1 (1.5) 

n indicates the number of paired samples that could be analyzed for this mutation.

Association between clinical characteristics and type I/II mutation status at diagnosis versus at relapse

The mutational shifts (Table 4) strongly influenced the associations of mutational status with TTR. The patients who were positive for FLT3/ITD or RAS or WT1 mutations at initial diagnosis showed shorter TTR; however, this did not reach statistical significance (Table 5). The lack of a clear effect of these mutations on TTR could be anticipated because, by definition, our patient subgroup lacks the nonrelapsing good prognosis group. However, when TTR was determined after stratification of groups on the basis of mutation status at relapse, the presence of FLT3/ITD, RAS, or WT1 mutations was associated with a significant or a strong trend (P = .05, P = .12, and P = .06, respectively) for a shorter TTR: 16.5 and 7.7 months for FLT3 wild-type and ITD-positive patients, respectively, as well as 17.4 and 9.4 months for wild-type RAS and patients harboring mutant RAS, respectively, and 17.6 and 8.9 months for wild-type WT1 and patients with WT1 mutations (Table 5). Of this patient group, clinical data were available to perform OS analysis, and we hence stratified the patients according to mutation status either at diagnosis or at relapse. In this patient group, the OS at 2 years was 36%, hence reflecting again the selection of a patient subgroup with poor prognosis. Our results show that the presence of a FLT3 or WT1 mutation at relapse was a more significant predictive factor for an adverse outcome of patients with AML compared with the presence of these mutations at diagnosis (Figure 1). The persistence or shifts of mutational status for individual genes was too low to assess the effect on OS of the mutational shifts in these genes. However, when data were combined for genes with frequent mutational shifts, a significant correlation between shifts or persistence of mutations and OS was found (P = .039; supplemental Figure 2). Multivariate OS analysis, including FLT3, RAS, or WT1 mutations, either at diagnosis or relapse, and age, white blood cell count, French-American-British type and sex showed that only the negative association of FLT3/ITD mutations at relapse with OS remained significant (P = .001).

Table 5

Mean time to relapse for patients with or without FLT3/ITD, RAS, and WT1 molecular aberrancies determined at either diagnosis or relapse

Aberration Disease stage analysis Status Mean TTR (mo) P* 
FLT3/ITD Diagnosis ITD− 14.2 .69 
  ITD15.8  
 Relapse ITD− 16.5 .05 
  ITD7.7  
RAS Diagnosis RAS− 16.0 .22 
  RAS11.0  
 Relapse RAS− 17.4 .12 
  RAS9.4  
WT1 Diagnosis WT1− 15.9 .21 
  WT19.5  
 Relapse WT1− 17.6 .06 
  WT18.9  
Aberration Disease stage analysis Status Mean TTR (mo) P* 
FLT3/ITD Diagnosis ITD− 14.2 .69 
  ITD15.8  
 Relapse ITD− 16.5 .05 
  ITD7.7  
RAS Diagnosis RAS− 16.0 .22 
  RAS11.0  
 Relapse RAS− 17.4 .12 
  RAS9.4  
WT1 Diagnosis WT1− 15.9 .21 
  WT19.5  
 Relapse WT1− 17.6 .06 
  WT18.9  

TTR indicates time (in months) to relapse and was defined as the period of time between diagnosis and relapse; −, wild-type; and +, mutated.

*

Determined by analysis of variance and calculated for the difference between wild-type and mutated patients per aberration and moment of analysis. P < .05 was considered significant.

Figure 1

The effect of mutation analyses at either diagnosis or relapse on overall survival. Kaplan-Meier estimates of overall survival for patients with mutated and wild-type FLT3/ITD, patients with mutated and wild-type RAS, and patients with mutated and wild-type WT1. Lines represent wild-type patients, dashed lines mutated patients. Left plots show estimates with the use of initial diagnosis analyses, right plots are from relapse analyses.

Figure 1

The effect of mutation analyses at either diagnosis or relapse on overall survival. Kaplan-Meier estimates of overall survival for patients with mutated and wild-type FLT3/ITD, patients with mutated and wild-type RAS, and patients with mutated and wild-type WT1. Lines represent wild-type patients, dashed lines mutated patients. Left plots show estimates with the use of initial diagnosis analyses, right plots are from relapse analyses.

Discussion

In the current study on a large cohort of paired diagnosis and relapse pediatric AML samples (n = 69), we examined the incidence and retention of established type I/II molecular aberrations at diagnosis and at relapse. We focused our study to 7 genes and found substantial differences in the mutation status within the predominant leukemic populations at initial diagnosis and at relapse in 38% of the patients. The actual number of patients who experience such differences may even be higher, considering that not all mutations could be determined in all paired samples, because of limited availability of genomic DNA. Moreover, the observed frequency of mutational shifts may be an underestimate, because many other mutated genes that are potentially involved in leukemogenesis or disease progression were not studied here. Therefore, the extent of the alterations between initial diagnosis and relapse remains to be elucidated. The observed mutational shifts occurred most frequently in CN-AML and MLL rearranged AML (54% and 80% of cases, respectively), which suggests that these AML types are genetically less stable at these gene loci. Mutational shifts were more frequently observed for those genes for which mutations at diagnosis are associated with a dismal prognosis (FLT3, WT1) or which are presumed to be involved in leukemogenesis (eg, RAS). The instability of FLT3 and WT1 mutations between diagnosis and relapse is in concordance with previous publications in which FLT3/ITD mutations were shown to be gained or lost during disease progression, whereas WT1 mutations were only gained.8  Given the effects on patient outcome, these changes in FLT3/ITD mutation status and particularly WT1 mutations may resemble the leukemia-driving capacity of these genes (ie, their type I properties). KIT or CEBPA genes that are also implicated in leukemogenesis showed less mutational shifts, which can be explained by the low incidence of mutations in these genes. In the current study we observed a loss of NPM1 mutation in one case; although NPM1 is often regarded as a robust and stable molecular marker,9,21,44  similar losses of NPM1 mutations between diagnosis and relapse were observed in adults by other groups.45,46  To the best of our knowledge, other mutational shifts, including in RAS, have not previously been described.

It is, therefore, tempting to speculate on the origin of these mutational shifts. The latter are consistent with the immunophenotypic shifts47-49  or cytogenetic changes28  that are also observed in AML between diagnosis and relapse. Given the sensitivity of the methods used for the detection of mutations and considering the high blast percentages in the analyzed samples, it is highly unlikely that we have failed to detect mutations that were present in the bulk of leukemic blasts at either stage of the disease. One explanation for the observed shifts may be the presence of small numbers of leukemic clones with a distinct molecular make-up that differs from the bulk of leukemic blasts at initial diagnosis (ie, oligoclonality). These chemotherapy drug–resistant clones appear to initiate the relapse in a posttherapy bone marrow on the basis of their superior tumor-initiating properties. This plausible explanation is substantiated by findings of Pollard et al50  that at diagnosis the immature AML cells are predominantly heterogeneous in their FLT3/ITD mutation status. In particular, the presence of the mutation in this immature subpopulation was associated with a drug-resistant disease. In patients, the selective pressure of induction chemotherapy may hence result in the survival of these leukemia-initiating cells, which expand and give rise to the relapsed blast population. This proposed model is in line with the “leukemic stem cell” model and explains both gains and losses of mutations. The other possible explanation could be that AML cells are genetically unstable and acquire appropriate mutations or chromosomal aberrations necessary for survival during chemotherapy, thereby resulting in clonal expansion and relapse. In this model, complex DNA recombination events, in which loss of heterozygosity occurs, are required to explain the loss of mutations and reversion to the wild-type genotype; clearly, both possibilities remain to be explored experimentally.

In this study we confirm the mutational shifts in FLT3/ITDs in paired samples and provide similar results for other molecular aberrations. In addition, within this extended patient group with relevant clinical data, we could show an effect on outcome for patients with mutational shifts in WT1 or RAS. The clinical relevance of our observations is evident and may further have direct implications for the patients involved, because genetic imbalances may affect the use of molecular markers for MRD assessment as well as the nature and timing of future targeted therapies. Currently, molecular diagnostics are often performed only at the time of definitive diagnosis, and patients are stratified into risk groups accordingly. If subsequent changes in mutational status occur between diagnosis and relapse, the strategy to treat such patients at relapse may not be properly tailored to their actual risks, and patients might be overtreated or undertreated. This will become particularly important when the molecular aberrations will also be used for selecting targeted drugs against the aberrant proteins of these mutated genes. Moreover, other aberrations, apart from mutations, may represent future targets for treatment, and these may also vary between initial diagnosis and relapse. Studies at multiple biologic levels in larger series of consecutive patient samples could elucidate the extent of mutational shifts or other genetic alterations, including aberrant gene expression or DNA methylation status.

The results of this study clearly indicate that mutation analysis at diagnosis alone is not sufficient for proper risk stratification, MRD detection, and optimal selection of personalized treatment. We hence propose that risk stratification, malignant cell detection, and selection of personalized treatment should be based also on type I/II mutation status at both initial diagnosis and relapse.

An Inside Blood analysis of this article appears at the front of this issue.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

Acknowledgments

This work was supported by grants from the Dutch Cancer Society (VU 2005-3666) (J.C.), Children Cancer-free (Y.G.A.), the Netherlands Organization for Scientific Research (Y.G.A.), and The Netherlands Academy of Arts and Sciences (Y.G.A.).

Authorship

Contribution: C.B. performed experiments, analyzed data, and wrote the paper; G.J.S. and J.C. supervised the research and edited the paper; I.H.I.M.H. provided experimental data; Z.J.K. performed experiments and analyzed data; B.F.G. and G.J.L.K. designed the research and edited the paper; C.M.Z. and M.M.v.d.H.-E. provided clinical and experimental data and edited the paper; E.S.J.M.d.B. provided experimental data; V.d.H., D.R., and U.C. provided samples and clinical data; and Y.G.A. discussed the research and edited the paper.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Jacqueline Cloos, Department of Pediatric Oncology/Hematology, VU University Medical Center, PO Box 7057, 1007 MB Amsterdam, The Netherlands; e-mail: j.cloos@vumc.nl.

References

References
1
Webb
DK
Management of relapsed acute myeloid leukaemia.
Br J Haematol
1999
, vol. 
106
 
4
(pg. 
851
-
859
)
2
Kaspers
GJ
Creutzig
U
Pediatric acute myeloid leukemia: international progress and future directions.
Leukemia
2005
, vol. 
19
 
12
(pg. 
2025
-
2029
)
3
Creutzig
U
Zimmermann
M
Reinhardt
D
Dworzak
M
Stary
J
Lehrnbecher
T
Early deaths and treatment-related mortality in children undergoing therapy for acute myeloid leukemia: analysis of the multicenter clinical trials AML-BFM 93 and AML-BFM 98.
J Clin Oncol
2004
, vol. 
22
 
21
(pg. 
4384
-
4393
)
4
Creutzig
U
Buchner
T
Sauerland
MC
, et al. 
Significance of age in acute myeloid leukemia patients younger than 30 years: a common analysis of the pediatric trials AML-BFM 93/98 and the adult trials AMLCG 92/99 and AMLSG HD93/98A.
Cancer
2008
, vol. 
112
 
3
(pg. 
562
-
571
)
5
Sievers
EL
Lange
BJ
Alonzo
TA
, et al. 
Immunophenotypic evidence of leukemia after induction therapy predicts relapse: results from a prospective Children's Cancer Group study of 252 patients with acute myeloid leukemia.
Blood
2003
, vol. 
101
 
9
(pg. 
3398
-
3406
)
6
Campana
D
Status of minimal residual disease testing in childhood haematological malignancies.
Br J Haematol
2008
, vol. 
143
 
4
(pg. 
481
-
489
)
7
Zwaan
CM
Meshinchi
S
Radich
JP
, et al. 
FLT3 internal tandem duplication in 234 children with acute myeloid leukemia: prognostic significance and relation to cellular drug resistance.
Blood
2003
, vol. 
102
 
7
(pg. 
2387
-
2394
)
8
Hollink
IH
van den Heuvel-Eibrink
MM
Zimmermann
M
, et al. 
Clinical relevance of Wilms tumor 1 gene mutations in childhood acute myeloid leukemia.
Blood
2009
, vol. 
113
 
23
(pg. 
5951
-
5960
)
9
Hollink
IH
Zwaan
CM
Zimmermann
M
, et al. 
Favorable prognostic impact of NPM1 gene mutations in childhood acute myeloid leukemia, with emphasis on cytogenetically normal AML.
Leukemia
2009
, vol. 
23
 
2
(pg. 
262
-
270
)
10
Ho
PA
Alonzo
TA
Gerbing
RB
, et al. 
Prevalence and prognostic implications of CEBPA mutations in pediatric acute myeloid leukemia (AML): a report from the Children's Oncology Group.
Blood
2009
, vol. 
113
 
26
(pg. 
6558
-
6566
)
11
Pollard
JA
Alonzo
TA
Gerbing
RB
, et al. 
Prevalence and prognostic significance of KIT mutations in pediatric core binding factor AML patients enrolled on serial pediatric cooperative trials for de novo AML.
Blood
2010
, vol. 
115
 
12
(pg. 
2372
-
2379
)
12
Gilliland
DG
Griffin
JD
The roles of FLT3 in hematopoiesis and leukemia.
Blood
2002
, vol. 
100
 
5
(pg. 
1532
-
1542
)
13
Weisberg
E
Boulton
C
Kelly
LM
, et al. 
Inhibition of mutant FLT3 receptors in leukemia cells by the small molecule tyrosine kinase inhibitor PKC412.
Cancer Cell
2002
, vol. 
1
 
5
(pg. 
433
-
443
)
14
Brown
P
Meshinchi
S
Levis
M
, et al. 
Pediatric AML primary samples with FLT3/ITD mutations are preferentially killed by FLT3 inhibition.
Blood
2004
, vol. 
104
 
6
(pg. 
1841
-
1849
)
15
Zarrinkar
PP
Gunawardane
RN
Cramer
MD
, et al. 
AC220 is a uniquely potent and selective inhibitor of FLT3 for the treatment of acute myeloid leukemia (AML).
Blood
2009
, vol. 
114
 
14
(pg. 
2984
-
2992
)
16
Knapper
S
Burnett
AK
Littlewood
T
, et al. 
A phase 2 trial of the FLT3 inhibitor lestaurtinib (CEP701) as first-line treatment for older patients with acute myeloid leukemia not considered fit for intensive chemotherapy.
Blood
2006
, vol. 
108
 
10
(pg. 
3262
-
3270
)
17
Fiedler
W
Serve
H
Dohner
H
, et al. 
A phase 1 study of SU11248 in the treatment of patients with refractory or resistant acute myeloid leukemia (AML) or not amenable to conventional therapy for the disease.
Blood
2005
, vol. 
105
 
3
(pg. 
986
-
993
)
18
Misaghian
N
Ligresti
G
Steelman
LS
, et al. 
Targeting the leukemic stem cell: the Holy Grail of leukemia therapy.
Leukemia
2009
, vol. 
23
 
1
(pg. 
25
-
42
)
19
Pierotti
MA
Negri
T
Tamborini
E
Perrone
F
Pricl
S
Pilotti
S
Targeted therapies: the rare cancer paradigm.
Mol Oncol
2010
, vol. 
4
 
1
(pg. 
19
-
37
)
20
Gorello
P
Cazzaniga
G
Alberti
F
, et al. 
Quantitative assessment of minimal residual disease in acute myeloid leukemia carrying nucleophosmin (NPM1) gene mutations.
Leukemia
2006
, vol. 
20
 
6
(pg. 
1103
-
1108
)
21
Barragan
E
Pajuelo
JC
Ballester
S
, et al. 
Minimal residual disease detection in acute myeloid leukemia by mutant nucleophosmin (NPM1): comparison with WT1 gene expression.
Clin Chim Acta
2008
, vol. 
395
 
1–2
(pg. 
120
-
123
)
22
Trka
J
Kalinova
M
Hrusak
O
, et al. 
Real-time quantitative PCR detection of WT1 gene expression in children with AML: prognostic significance, correlation with disease status and residual disease detection by flow cytometry.
Leukemia
2002
, vol. 
16
 
7
(pg. 
1381
-
1389
)
23
Willasch
AM
Gruhn
B
Coliva
T
, et al. 
Standardization of WT1 mRNA quantitation for minimal residual disease monitoring in childhood AML and implications of WT1 gene mutations: a European multicenter study.
Leukemia
2009
, vol. 
23
 
8
(pg. 
1472
-
1479
)
24
Viehmann
S
Teigler-Schlegel
A
Bruch
J
Langebrake
C
Reinhardt
D
Harbott
J
Monitoring of minimal residual disease (MRD) by real-time quantitative reverse transcription PCR (RQ-RT-PCR) in childhood acute myeloid leukemia with AML1/ETO rearrangement.
Leukemia
2003
, vol. 
17
 
6
(pg. 
1130
-
1136
)
25
Perea
G
Lasa
A
Aventin
A
, et al. 
Prognostic value of minimal residual disease (MRD) in acute myeloid leukemia (AML) with favorable cytogenetics [t(8;21) and inv(16)].
Leukemia
2006
, vol. 
20
 
1
(pg. 
87
-
94
)
26
Fukutani
H
Naoe
T
Ohno
R
, et al. 
Prognostic significance of the RT-PCR assay of PML-RARA transcripts in acute promyelocytic leukemia. The Leukemia Study Group of the Ministry of Health and Welfare (Kouseisho).
Leukemia
1995
, vol. 
9
 
4
(pg. 
588
-
593
)
27
Krauter
J
Gorlich
K
Ottmann
O
, et al. 
Prognostic value of minimal residual disease quantification by real-time reverse transcriptase polymerase chain reaction in patients with core binding factor leukemias.
J Clin Oncol
2003
, vol. 
21
 
23
(pg. 
4413
-
4422
)
28
Estey
E
Keating
MJ
Pierce
S
Stass
S
Change in karyotype between diagnosis and first relapse in acute myelogenous leukemia.
Leukemia
1995
, vol. 
9
 
6
(pg. 
972
-
976
)
29
Kottaridis
PD
Gale
RE
Langabeer
SE
Frew
ME
Bowen
DT
Linch
DC
Studies of FLT3 mutations in paired presentation and relapse samples from patients with acute myeloid leukemia: implications for the role of FLT3 mutations in leukemogenesis, minimal residual disease detection, and possible therapy with FLT3 inhibitors.
Blood
2002
, vol. 
100
 
7
(pg. 
2393
-
2398
)
30
Cloos
J
Goemans
BF
Hess
CJ
, et al. 
Stability and prognostic influence of FLT3 mutations in paired initial and relapsed AML samples.
Leukemia
2006
, vol. 
20
 
7
(pg. 
1217
-
1220
)
31
Kaspers
GJ
Veerman
AJ
Pieters
R
, et al. 
Mononuclear cells contaminating acute lymphoblastic leukaemic samples tested for cellular drug resistance using the methyl-thiazol-tetrazolium assay.
Br J Cancer
1994
, vol. 
70
 
6
(pg. 
1047
-
1052
)
32
Creutzig
U
Ritter
J
Zimmermann
M
, et al. 
Idarubicin improves blast cell clearance during induction therapy in children with AML: results of study AML-BFM 93. AML-BFM Study Group.
Leukemia
2001
, vol. 
15
 
3
(pg. 
348
-
354
)
33
Creutzig
U
Zimmermann
M
Ritter
J
, et al. 
Treatment strategies and long-term results in paediatric patients treated in four consecutive AML-BFM trials.
Leukemia
2005
, vol. 
19
 
12
(pg. 
2030
-
2042
)
34
Kardos
G
Zwaan
CM
Kaspers
GJ
, et al. 
Treatment strategy and results in children treated on three Dutch Childhood Oncology Group acute myeloid leukemia trials.
Leukemia
2005
, vol. 
19
 
12
(pg. 
2063
-
2071
)
35
Goemans
BF
Zwaan
CM
Miller
M
, et al. 
Mutations in KIT and RAS are frequent events in pediatric core-binding factor acute myeloid leukemia.
Leukemia
2005
, vol. 
19
 
9
(pg. 
1536
-
1542
)
36
Kramer
D
Thunnissen
FB
Gallegos-Ruiz
MI
, et al. 
A fast, sensitive and accurate high resolution melting (HRM) technology-based assay to screen for common K-ras mutations.
Cell Oncol
2009
, vol. 
31
 
3
(pg. 
161
-
167
)
37
Corbacioglu
S
Kilic
M
Westhoff
MA
Reinhardt
D
Fulda
S
Debatin
KM
Newly identified c-KIT receptor tyrosine kinase ITD in childhood AML induces ligand-independent growth and is responsive to a synergistic effect of imatinib and rapamycin.
Blood
2006
, vol. 
108
 
10
(pg. 
3504
-
3513
)
38
Wouters
BJ
Lowenberg
B
Erpelinck-Verschueren
CA
van Putten
WL
Valk
PJ
Delwel
R
Double CEBPA mutations, but not single CEBPA mutations, define a subgroup of acute myeloid leukemia with a distinctive gene expression profile that is uniquely associated with a favorable outcome.
Blood
2009
, vol. 
113
 
13
(pg. 
3088
-
3091
)
39
Hollink
IH
van den Heuvel-Eibrink
MM
Zwaan
CM
CEBPA resembles Roman god Janus.
Blood
2009
, vol. 
113
 
26
(pg. 
6501
-
6502
)
40
Goemans
BF
Zwaan
CM
Martinelli
S
, et al. 
Differences in the prevalence of PTPN11 mutations in FAB M5 paediatric acute myeloid leukaemia.
Br J Haematol
2005
, vol. 
130
 
5
(pg. 
801
-
803
)
41
Meshinchi
S
Alonzo
TA
Stirewalt
DL
, et al. 
Clinical implications of FLT3 mutations in pediatric AML.
Blood
2006
, vol. 
108
 
12
(pg. 
3654
-
3661
)
42
Manola
KN
Cytogenetics of pediatric acute myeloid leukemia.
Eur J Haematol
2009
, vol. 
83
 
5
(pg. 
391
-
405
)
43
Goemans
BF
Tamminga
RY
Corbijn
CM
Hahlen
K
Kaspers
GJ
Outcome for children with relapsed acute myeloid leukemia in the Netherlands following initial treatment between 1980 and 1998: survival after chemotherapy only?
Haematologica
2008
, vol. 
93
 
9
(pg. 
1418
-
1420
)
44
Chou
WC
Tang
JL
Lin
LI
, et al. 
Nucleophosmin mutations in de novo acute myeloid leukemia: the age-dependent incidences and the stability during disease evolution.
Cancer Res
2006
, vol. 
66
 
6
(pg. 
3310
-
3316
)
45
Suzuki
T
Kiyoi
H
Ozeki
K
, et al. 
Clinical characteristics and prognostic implications of NPM1 mutations in acute myeloid leukemia.
Blood
2005
, vol. 
106
 
8
(pg. 
2854
-
2861
)
46
Papadaki
C
Dufour
A
Seibl
M
, et al. 
Monitoring minimal residual disease in acute myeloid leukaemia with NPM1 mutations by quantitative PCR: clonal evolution is a limiting factor.
Br J Haematol
2009
, vol. 
144
 
4
(pg. 
517
-
523
)
47
Hur
M
Chang
YH
Lee
DS
Park
MH
Cho
HI
Immunophenotypic and cytogenetic changes in acute leukaemia at relapse.
Clin Lab Haematol
2001
, vol. 
23
 
3
(pg. 
173
-
179
)
48
Baer
MR
Stewart
CC
Dodge
RK
, et al. 
High frequency of immunophenotype changes in acute myeloid leukemia at relapse: implications for residual disease detection (Cancer and Leukemia Group B Study 8361).
Blood
2001
, vol. 
97
 
11
(pg. 
3574
-
3580
)
49
Langebrake
C
Brinkmann
I
Teigler-Schlegel
A
, et al. 
Immunophenotypic differences between diagnosis and relapse in childhood AML: Implications for MRD monitoring.
Cytometry B Clin Cytom
2005
, vol. 
63
 
1
(pg. 
1
-
9
)
50
Pollard
JA
Alonzo
TA
Gerbing
RB
, et al. 
FLT3 internal tandem duplication in CD34+/CD33- precursors predicts poor outcome in acute myeloid leukemia.
Blood
2006
, vol. 
108
 
8
(pg. 
2764
-
2769
)