High FLT3-ITD/wildtype (wt) load in FLT3-ITD-mutated AML has been associated with adverse impact on outcome in several studies. To clarify whether FLT3-ITD load as expressed as FLT3-ITD/wt ratio is also relevant in patients with NPM1 mutated AML, we assessed the FLT3-ITD mutation status and FLT3-ITD/wt ratio by fragment analysis in 638 NPM1mut AML (339 females; 299 males; 17.8–88.0 years), and analyzed its prognostic relevance in 355 patients. FLT3-ITD of various length and load were detected in 243/638 cases (38.1%). Median EFS (19.3 vs 9.7 months, P<0.001) and median 2-year survival rate (72.0 vs 52.7%, P=0.006) was better in FLT3wt (n=212 with available follow-up data) than FLT3-ITD (n=143). A higher FLT3-ITD/wt ratio as continuous variable was correlated with a shorter EFS (P=0.028). When patients were separated into subgroups according to the FLT3-ITD mutation load, only a FLT3-ITD/wt ratio ⩾0.5 conferred an independent adverse impact on EFS and OS, and retained its prognostic significance also in multivariate analysis (P=0.009 for EFS, P=0.008 for OS). In conclusion, for risk estimation in NPM1 mutated AML not only the FLT3-ITD status, but also the FLT3-ITD load has to be taken into account. These data might contribute to clinical decision making in AML.
Mutations of the nucleophosmin (NPM1) gene were identified in 35% of patients with acute myeloid leukemia (AML), and in 55% of those with a normal karyotype (NK-AML).1 The NPM1 mutations mediate aberrant localization of the nucleophosmin protein to the cytoplasm. Therefore, the nucleophosmin protein is unable to maintain its shuttle function between the nucleus and the cytoplasm, which alters the ARF–P53 tumor suppressor pathway.2 Prognosis of patients with isolated NPM1 mutations in normal karyotype has been found to be more favorable when compared to many other subgroups of AML.3, 4, 5 According to the new WHO classification,6 NPM1 mutated AML is defined as a separate so called provisional subentity of AML.7
The second most frequent molecular mutations, with a frequency of 35–45% in NK-AML, are the prognostically unfavorable internal tandem duplications within the FLT3 gene (FLT3-ITD).8, 9, 10 The FLT3 gene encodes for a class III receptor tyrosine kinase. Mutations result in constitutive activation of the receptor and downstream activation of various signaling pathways.11, 12 When the NPM1 mutations are found in coincidence with the FLT3-ITD, outcomes are inferior when compared to occurrence of the NPM1 mutations only.3, 4, 5 The FLT3-ITDs are heterogeneous, first, according to length, and second, according to the mutation load compared to the wildtype (wt) alleles. Mutation length varies between three nucleotides and several hundreds of nucleotides.10, 13
Several studies described that a high mutation load as determined by calculation of FLT3-ITD/FLT3wt ratio indicates a worse prognosis in mutation carriers.14, 15, 16 Therefore, it was suggested that not the FLT3-ITD per se, but more likely loss of heterozygosity is associated with the unfavorable outcome in FLT3-ITD mutated AML. However, most studies that analyzed the prognostic impact of the FLT3-ITD/wt ratio were carried out before the detection of the NPM1 mutation and did not focus specifically on the FLT3-ITD/wt ratio in NPM1 mutated AML. We thus performed characterization of the FLT3-ITD mutation status and the FLT3-ITD/wt ratio in 638 patients with NPM1 mutated AML, and characterized its prognostic relevance in 355 patients within this specific setting.
Materials and methods
The study cohort consisted of 638 consecutive patients with NPM1 mutated AML at diagnosis (339 females and 299 males) (Table 1) who were selected according to the positive NPM1 status at diagnosis of AML. Median age was 65.2 years (range 17.8–88.0 years). A total of 596 patients had de novo AML, 26 had secondary AML following MDS, and 16 developed the AML in association with previous chemo- or radiotherapy (t-AML). Treatment was carried out according to standard chemotherapy protocols including one or two courses of induction therapy, at least one course of consolidation therapy and, in patients with HLA-identical siblings, allogeneic hematopoietic stem cell transplantation. Follow-up data were available in 355 patients: 143 cases with FLT3-ITD and 212 cases with FLT3wt (Table 1). Samples were referred to our laboratory for diagnosis in the period of August 2005–June 2009. Patients gave consent to genetic analysis and the use of laboratory results for research purposes. The study was approved by the Bavarian Medical Association (Bayerische Landesärztekammer) and adhered to the Declaration of Helsinki.
Isolation of mononucleated cells, mRNA extraction and random primed complementary DNA (cDNA) synthesis was previously described.10 Analysis for NPM1 mutations was carried out by LightCycler (Roche, Mannheim, Germany)-based melting curve analysis (Figure 1a).4 Quantification of the NPM1 mutation at diagnosis and during follow up was carried out by quantitative real-time PCR (RQ-PCR).17 Determination of the length of the FLT3-ITD and quantification of the FLT3-ITD mutation load were realized by fragment analysis (GeneScan, 3130 sequence detection system, ABI, Darmstadt, Germany) (Figure 1b).15 The FLT3-ITD load was quantified as the ratio of the mutation compared to the wt allele. In case of 2–3 different FLT3-ITD mutations, FLT3-ITD/wt ratios were summed up. Ratios ⩾1 were indicative for complete or partial loss of the wt allele (FLT3wt).
Cytomorphology, cytogenetics and immunophenotyping
Cytomorphological classification of AML was based on May-Gruenwald-Giemsa stains and cytochemistry with myeloperoxidase (MPO) and non-specific esterase (NSE) using alpha-naphtyl-acetate.18 Cases were diagnosed according to the FAB19 and the WHO classification.6 Chromosome banding combined with interphase fluorescence in situ hybridization (FISH)20 and immunophenotyping followed previous descriptions.21
Overall survival (OS) and event-free survival (EFS) were calculated according to Kaplan–Meier. Comparison of survival was based on the two-sided log-rank test. OS was the time from diagnosis to death or last follow up, EFS the time from diagnosis to treatment failure, relapse or death, or last follow up. Relapse of AML was defined according to International Working Group criteria published by Cheson et al.22 Cox regression was performed for OS and EFS with different parameters as covariates. Parameters that were significant in univariate analysis (P<0.05) were included into multivariate analysis. Dichotomous variables were compared between different groups using the χ2-test and continuous variables by Student's t-test. Spearman's rank correlation was used to analyze correlations between continuous parameters. Results were significant at a level of P<0.05 at both sides. SPSS (version 14.0.1) software (SPSS, Chicago, IL, USA) was used for statistical analysis.
Characterization of the NPM1 mutations
In the total cohort (n=638), the NPM1 mutations were distributed as follows: Most frequent was the A subtype (n=488/638; 76.5%), being followed by B (n=54; 8.5%) and D (n=33; 5.2%) subtypes. In all, 63 patients (9.9%) had rare NPM1 mutation subtypes.
Frequency and characterization of FLT3 mutations in NPM1 mutated AML
FLT3-ITD mutations were detected in 243/638 (38.1%) of the NPM1 mutated AML cases. The median length of the mutation was 51 nucleotides with a wide range from 9 to 579 nucleotides. The FLT3-ITD/wt ratio of the mutated cases was ranging from 0.016 to 44.85 (median: 0.565). Three cases had two and one case three different FLT3-ITD mutations.
Karyotypes were available in 603/638 cases (94.5% of all patients). The majority (513/603; 85.1%) of cases with available cytogenetics had a normal karyotype. In 90/603 cases (14.9%) karyotype aberrations were detected, which are summarized in Table 1. The most frequent cytogenetic alteration was trisomy 8 (n=25; 27.8% of all aberrant cases).
Influence of the FLT3-ITD mutation status on prognosis
Follow-up data were available in 355 patients: 143 cases with FLT3-ITD and 212 cases with FLT3wt. Median follow-up was 12.8 months (12.5 months in the FLT3-ITD and 13.0 months in the FLT3wt patients). The FLT3wt cohort had significantly better median EFS compared to the FLT3-ITD cohort (19.3 vs 9.7 months, P<0.001; Figure 2a). Median OS was not reached in both groups, but the 2-year survival rate was also superior in the FLT3wt cohort compared to the FLT3-ITD cohort (72.0 vs 52.7%, P=0.006) (Table 2a). A significant influence of the length of the FLT3-ITD on prognosis could not be shown.
Influence of the FLT3-ITD mutation load on prognosis
To analyze the prognostic impact of FLT3-ITD load, Cox regression analysis was carried out, which showed that an increasing FLT3-ITD/wt ratio as a continuous variable was correlated with decreasing EFS (P=0.028).
Subsequently, patients were subdivided in three subgroups separating the FLT3-ITD-positive cases according to the FLT3-ITD/wt ratio: FLT3wt (group 1, n=212) vs FLT3-ITD with a mutation ratio <1 (group 2, n=114) vs FLT3-ITD with a mutation ratio ⩾1 (group 3, n=29) (Table 2a; Figure 2b). The FLT3wt group had the longest median EFS of 19.3 months compared to 14.5 months in group 2 (ratio <1), and 4.4 months in group 3 with ratio ⩾1 (P=0.025 for comparisons between groups 1 and 2; P<0.001 groups 1 and 3; and P<0.001 groups 2 and 3).
For further clarification, we separated patients in four subgroups, introducing thresholds of 0.5 and 1.0 for the FLT3-ITD/wt ratio. Doing so, median EFS showed no significant differences between group 1 (FLT3wt) and group 2 (FLT3-ITD/wt ratio <0.5), but median EFS was significantly superior in group 1 compared to group 3 (FLT3-ITD/wt ratio ⩾0.5 but <1; P=0.014) and group 4 (FLT3-ITD/wt ratio ⩾1; P<0.001) and significantly superior in group 2 when compared to group 4 (P<0.001). Group 3 showed superior EFS compared to group 4; P=0.009 (Table 2a; Figure 2c).
In a next step, further subdivision of the patients with a FLT3-ITD/wt ratio <0.5 was performed thus leading to five different subgroups (Figure 2d): (1) FLT3wt (n=212), (2) FLT3-ITD/wt ratio <0.25 (n=39), (3) ratio ⩾0.25 but <0.5 (n=27), (4) ratio ⩾0.5 but <1 (n=48), and (5) ratio ⩾1 (n=29). Median EFS showed a decrease from the subgroups 1 to 5 (group 1: 19.3 months; 2: 20.2 months; 3: 13.8 months; 4: 7.7 months; 5: 4.4 months). Differences were statistically significant only when the FLT3wt cohort (group 1) was compared to groups 4 (P=0.014) and 5 (P<0.001). As well significance was reached between groups 2 vs 5 (P<0.001), 3 vs 5 (P=0.020) and 4 vs 5 (P=0.009). Therefore, only FLT3-ITD/wt ratios ⩾0.5 were associated with significantly worse EFS in NPM1 mutated AML compared to the FLT3wt patients (Figure 2e). Similar relations were observed regarding OS when patients were separated in 2, 3, 4 or 5 groups (Table 2a; Figures 2a–d).
When patients were separated according to a threshold of FLT3-ITD/wt ratio of 0.5, those with a ratio <0.5 (n=278) had better median OS (544 vs 216 days; P<0.001) and median EFS (not reached vs 502 days; P<0.001) when compared to those with a ratio ⩾0.5 (n=77) (Figure 3a).
Influence of the FLT3-ITD mutation load on prognosis according to age
As this study includes a significant number of older patients we applied the threshold of 0.5 FLT3-ITD/wt ratio separately on patients <60 years and ⩾60 years of age. In patients <60 years median EFS and OS were longer in those with FLT3-ITD/wt ratio <0.5 (1.017 days vs 204 days; P<0.001 and not reached vs 625 days; P=0.001, respectively; Figure 3b). For patients ⩾60 years this effect was less strong, but still significant (EFS: 441 vs 216 days; P=0.015, and OS: 812 days vs 342 days; P=0.046; Figure 3c). Thus the 0.5 FLT3-ITD/wt ratio threshold was shown to be relevant in both age groups.
Uni and multivariate analysis
In univariate analysis, the following parameters were associated with worse EFS: male sex (P=0.042), higher age (P<0.001), higher WBC count (P<0.001) and FLT3-ITD/wt ratio higher than 0.5 (P<0.001). An aberrant karyotype, thrombocyte count and hemoglobin level had no significant influence on EFS. In multivariate analysis, only age (P<0.001), WBC count (P=0.002) and FLT3-ITD/wt ratio ⩾0.5 (P=0.009) maintained their relevance for EFS in NPM1 mutated AML. Investigating OS, age (P<0.001) and WBC (P<0.001) and the FLT3-ITD ratio ⩾0.5 (P<0.001) were prognostically relevant in univariate analysis. An aberrant karyotype, thrombocyte count and hemoglobin level had no significant impact on OS in univariate analysis. In multivariate analysis, age (P<0.001), WBC count (P=0.001) and FLT3-ITD/wt ratio ⩾0.5 (P=0.008) all retained their prognostic impact (Table 2b).
Numerous large studies univocally demonstrated that prognosis of patients with a coincidence of NPM1 mutations and FLT3-ITD was inferior when compared to carriers of an isolated occurrence of the NPM1 mutation.3, 4, 23, 24 On the other hand, several studies have analyzed the specific prognostic influence of the FLT3-ITD/wt ratio in the past irrespective of NPM1 status, which all demonstrated that only a high mutant allele/wt ratio was prognostically unfavorable.14, 15, 16
In this study, we characterized the FLT3-ITD status in 638 patients with NPM1 mutations and analyzed the clinical impact of the FLT3-ITD mutation status and ITD/wt ratio in 355 patients with NPM1 mutated AML in whom follow-up data were available. First, we were able to confirm an adverse influence of a mutated FLT3-ITD status on OS (P=0.006) and EFS (P=0.001) in this NPM1 mutated cohort. However, when we subdivided patients into smaller subgroups in accordance with the FLT3-ITD/wt ratio, only FLT3-ITD/wt ratios ⩾0.5 had a significantly adverse impact on EFS or OS. In contrast, survival outcomes of patients with FLT3-ITD/wt ratios <0.5 did not differ significantly from FLT3-ITD negative patients. Aiming to consider the parameter with the strongest impact, in univariate analysis, FLT3-ITD/wt ratio ⩾0.5 (but not FLT3-ITD +/− mutation status) was included. This parameter maintained its prognostic impact also in multivariate analysis (EFS: P=0.009, RR 1.71; OS: P=0.008, RR 2.02) when other parameters (age, WBC count and gender) were considered.
These results were corresponding to previous studies that had been carried out before the detection of the NPM1 mutations: When Thiede et al.15 correlated the FLT3-ITD/wt ratio with the clinical outcomes in 200 mutation carriers, they described a 1.6-fold increased relative relapse risk in patients with a FLT3-ITD/wt ratio ⩾0.78 when compared to patients without FLT3 aberrations (n=797). Patients with a high FLT3-ITD/wt ratio (⩾0.78) had significantly shorter OS and DFS, whereas survival in patients with ratios <0.78 did not differ from those without FLT3 aberrations. Finally, FLT3-ITD/wt ratio >2 was associated with the worst outcomes, as 93% of affected patients died by 12 months.
Whitman et al.14 evaluated the clinical impact of the FLT3-ITD/wt ratio in 23 adult mutation carriers <60 years of age who received uniform high-dose AML therapy. Three genotypic subgroups were identified: FLT3wt, heterozygous and hemizygous FLT3-ITD (who had no wt allele). Disease-free survival was significantly inferior for patients with hemizygous FLT3-ITD (P=0.0017). Although OS and disease-free survival did not differ significantly for FLT3wt and -heterozygous patients, OS of the hemizygous FLT3-ITD group was worse when compared to both the wildtype (P=0.0005) and the heterozygous groups (P=0.008). The authors thus proposed that cells with FLT3-ITD confer a greater growth advantage when compared to cells having FLT3 wt or a heterozygous genotype.
In a further study by Gale et al.16 in young adult AML patients, relapse risk and overall survival (OS) were significantly worsened with increasing FLT3-/ITD mutant levels (P<0.001 for both). The threshold of 0.5 for FLT3-ITD/wt ratio was also validated by separate analysis of patients <60 and ⩾60 years of age. The relative FLT3-ITD mutant level remained a significant adverse prognostic factor for relapse risk and OS in multivariate analysis, when parameters such as age, cytogenetics, history of the AML, WBC and NPM1 mutation status were taken into account. In contrast, we did not find a significant impact of the FLT3-ITD size on clinical outcomes. Thus, our results were similar to the findings from the Medical Research Council (MRC) cohort.16
Although the follow-up of the present series is relatively short, our findings on FLT3-ITD load and outcome in NPM1 mutated AML are overall in good accordance with the data published by Whiteman et al., Thiede et al. and Gale et al. in FLT3-ITD sole mutated AML before the detection of the NPM1 mutations or evaluating the FLT3-ITD mutation load irrespective of the NPM1 mutant status. However, thresholds used for the calculation of the impact on survival are different in all studies. Whiteman defined those with complete or nearly complete loss of the FLT3wt allele as the unfavorable group, whereas Thiede et al. put the threshold as a FLT3-ITD/wt ratio of 0.78, which was the median of all patients analyzed, and Gale defined a 50% threshold. In addition all previous studies used genomic DNA in most of the cases, whereas we did the whole study on cDNA. Previously, we have shown that FLT3-ITD/FLT3wt ratios as assessed on the cDNA level are in good correlation to those assessed at the genomic level.25 However, large series showing comparisons of parallel assessments of DNA and cDNA are missing, and it remains unclear whether these values always go in parallel. Thus, the definition of the most valid threshold and the comparison of genomic DNA vs cDNA as template should be a major point for future studies.
In conclusion, whereas the FLT3-ITD status per se is predictive for OS and EFS in NPM1 mutated AML, it has no independent prognostic impact when other prognostically relevant parameters are taken into account, whereas a FLT3-ITD/wt ratio ⩾0.5 retained its independent prognostic impact on OS and EFS in multivariate analysis in this study. In a retrospective study by Schlenk et al.24, it has been suggested that NPM1mut/FLT3wt AML do not benefit from early allogeneic hematopoietic stem cell transplantation in first remission. Prospective studies with respect to this issue are anticipated. Our results suggest that not only the NPM1mut/FLT3wt, but also NPM1mut/FLT3-ITD-positive cases with low FLT3-ITD load (FLT3-ITD/wt ratio <0.5) are prognostically favorable AML. Therefore, the FLT3-ITD/wt ratio should be considered for therapeutic decisions in NPM1 mutated AML. For further validation, multicentre studies should focus on the definition of the best cutoff for a clinically relevant FLT3-ITD load in NPM1 mutated AML.
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SS, WK, CH and TH declare part ownership of the MLL Munich Leukemia Laboratory. TA is employed by the MLL Munich Leukemia Laboratory. UB has nothing to disclose.
SS did molecular analysis, performed data analysis and contributed to writing of manuscript. UB performed data analysis and wrote manuscript. WK was responsible for immunophenotyping and, together with TA, performed statistics. CH was responsible for cytogenetics. TH performed cytomorphology and was responsible for study design. All authors contributed to writing of the manuscript and approved the final version.
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Schnittger, S., Bacher, U., Kern, W. et al. Prognostic impact of FLT3-ITD load in NPM1 mutated acute myeloid leukemia. Leukemia 25, 1297–1304 (2011). https://doi.org/10.1038/leu.2011.97
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