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The FMS-like tyrosine kinase 3 (FLT3) gene is located on chromosome 13q12 and encodes a membrane-bound receptor tyrosine kinase that has an important role in hematopoiesis.1, 2, 3 FLT3 is one of the most frequently mutated genes in hematological malignancies, present in over 30% of adults with acute myeloid leukemia.4, 5, 6, 7, 8 The most common type of FLT3 mutation is internal tandem duplications (FLT3-ITDs) in the juxtamembrane domain of the receptor, which have been found in 15–35% of adult acute myeloid leukemia patients.4, 5, 9, 10 Point mutations in the heavily converted areas of the intracellular tyrosine kinase domain (TKD), most commonly the nucleotide substitution of aspirate 835 (FLT3-D835), occur in 5–10% of adult acute myeloid leukemia patients.3, 7, 8, 11 Although most patients have only one type of the FLT3 mutation, 1–3% of acute myeloid leukemia patients have both FLT3-ITD and FLT-D835.8, 11, 12 FLT3-ITD and FLT-D835 cause constitutive activation of FLT3, leading to aberrant activation of multiple downstream pathways, such as phosphatidyl-inositol 3–kinase, mitogen-activated protein kinase, and signal transducer and activator of transcription 5.13

Patients with acute myeloid leukemia associated with FLT3 mutation usually present as de novo disease with high peripheral leukocyte count, high bone marrow blast count, and normal cytogenetics.14 FLT3-ITD is an independent predictor of poor prognosis and is associated with increased relapse risk after chemotherapy, and decreased disease-free survival and overall survival.6, 8, 10, 15, 16 The clinical significance of FLT3-D835 is still unclear. Some studies have shown that FLT3-D835 is associated with shorter disease-free and overall survival.7, 14, 17, 18 Other studies did not identify differences in these parameters.8, 14 Conflicting data may be due to small patient numbers, different treatment regimens, and patient selection.

The high frequency of FLT3 mutations in acute myeloid leukemia has made the FLT3 gene a promising target for developing therapeutic agents. There are several classes of FLT3 inhibitors currently in development or in clinical trials with varying degrees of potency and selectivity for the target.19 FLT3 inhibitors offer a potential paradigm shift in the standard therapy of acute myeloid leukemia patients. Some studies have even reported potential benefits of administering FLT3 inhibitors to acute myeloid leukemia patients without detectable FLT3 mutations.20 One potential explanation is that FLT3 mutations are unstable, and that patients may acquire or lose mutations along the disease course.

Change of FLT3 status (gain or loss) at time of initial diagnosis and subsequently, usually at time of relapse, has been observed repeatedly in a small fraction of the patients in earlier studies.10, 16, 21, 22, 23, 24, 25, 26, 27, 28, 29 However, owing to a lack of a sufficient number of patients, no study has been able to provide statistical significant correlations between FLT3 status changes and disease progression. The goal of this study is to examine the clinical features and outcome of a large cohort of acute myeloid leukemia patients in whom FLT3 mutation status changed. In a retrospective review of 3555 acute myeloid leukemia patients, we identified 42 (6.2%) with a change in FLT3 status. Our findings suggest that acquiring a FLT3 mutation during the course of disease has a negative impact on patient survival and may justify changes in clinical management.

Materials and methods

Case Selection

We retrospectively reviewed all clinical and laboratory data of all adult acute myeloid leukemia patients assessed for FLT3 mutation at the University of Texas M. D. Anderson Cancer Center between May 2002 and January 2011. All cases were diagnosed and classified as acute myeloid leukemia according to World Health Organization criteria. In all patients, FLT3 mutation testing was performed on bone marrow aspirate specimens obtained at initial diagnosis and at time of multiple follow-up visits. To assess the impact of a change in FLT3 mutation status on patient outcome, the study cohort was divided into the following four groups based on the mutation-testing results at the time of initial diagnosis and at follow-up: 1) a Negative/Negative group of patients who were negative for FLT3 mutations at the time of initial diagnosis and remained negative at time of all follow-up visits, including during clearly documented hematological relapses; 2) a Positive/Positive group of patients who were positive for FLT3 mutations at the time of initial diagnosis and remained positive at time of all follow-up visits when their bone marrow blast count was at least 5% (ie, above the sensitivity of our FLT3 assay); 3) a Negative/Positive group of patients who were negative for FLT3 mutation at the time of initial diagnosis but acquired mutation in at least one of their follow-up tests (ie, gained FLT3 mutation during persistent disease after treatment or at relapse); and 4) a Positive/Negative group of patients who were positive for FLT3 mutations at the time of initial diagnosis but became wild type in all follow-up test, including during documented relapse of acute myeloid leukemia (ie, lost FLT3 mutation). Patients who had received stem cell transplantation or FLT3 inhibitor treatments were excluded. The patients' clinical information, complete blood count, morphological findings in bone marrow, and cytogenetics results were obtained from the medical records. This study was approved by the institutional review board and conducted in accordance with the Declaration of Helsinki.

Cytogenetic and Molecular Analyses

Conventional cytogenetic analysis was performed on bone marrow aspirate samples in all cases, as described previously.30 Karyotypes were reported using the International System for Human Cytogenetic Nomenclature.

Genomic DNA was extracted from fresh bone marrow aspirate samples for FLT3 mutation analysis using the Autopure extractor (QIAGEN/Gentra, Valencia, CA, USA). FLT3-ITD and FLT3-D835 mutations were screened using polymerase chain reaction (PCR) followed by capillary electrophoresis on an Applied Biosystems Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA), as previously described.31 For FLT3-D835 point mutation analysis, PCR products were digested with EcoRV before capillary electrophoresis. Sequences were analyzed and mutant allelic ratios were calculated using GeneScan software (Applied Biosystems). The specimens were also analyzed for other genes commonly mutated in acute myeloid leukemia, including NPM1, KRAS, and NRAS. These genes were assessed by PCR followed by capillary electrophoresis or direct sequencing (Sanger sequencing or pyrosequencing), according to previously described protocols.31, 32 The sensitivity of the NPM1 and FLT3 assays is approximately 2.5%. The sensitivity of Sanger sequencing is approximately 20% and for pyrosequencing, 5–10%.

Statistical Analysis

Categorical variables were compared using the Chi-square test. Patient survivals were estimated by the Kaplan–Meier method, and differences among groups were determined by the log-rank test.

Results

FLT3 Mutation Status Changes in a Subset of Acute Myeloid Leukemia Patients

During our review period of May 2002 to January 2011, a total of 3555 patients were diagnosed with acute myeloid leukemia at our institution and had multiple bone marrow samples tested for FLT3 mutation. Among these patients, 2875 (80.9%) never tested positive for FLT3 mutation throughout their clinical course, whereas 680 (19.1%) tested positive for FLT3 mutation at least once. These mutated cases included 541 (79.6%) with FLT3-ITD and 139 (20.4%) with FLT3-D835.

In most patients with FLT3 mutation (n=638; 93.8%), mutation was detected at time of initial diagnosis and the mutation persisted in all follow-up specimens with 5% or more blasts. In 42 (6.2%) patients who changed their mutation status, 36 (5.3%) were negative for FLT3 mutation at initial diagnosis but mutations were later detectable in follow-up samples, including 18 patients with FLT3-ITD alone, 11 patients with FLT3- D835 alone, and 7 patients with both types of mutations. These patients were designated as the Negative/Positive group. Another 6 (0.9%) patients were positive for FLT3 mutation at the time of initial diagnosis but subsequently became wild type for FLT3 in all follow-up, including 5 patients with FLT3-ITD alone, and 1 patient with both FLT3-ITD and FLT3-D835. These patients were designated as the Positive/Negative group. Representative results demonstrating the change of mutation status are shown in Figure 1.

Figure 1
figure 1

Representative examples of GeneScan results of FLT3 mutation status changes in acute myeloid leukemia patients. A patient diagnosed acute myeloid leukemia with 30% bone marrow blasts and wild-type FLT3 (a) went into complete remission after treatment, but relapsed 2 months later with 7% blasts and a detectable FLT3-ITD mutation (b). The patient became resistant to therapy and a bone marrow biopsy 3 months later showed 78% blasts and more prominent FLT3-ITD mutation (c). Another patient diagnosed acute myeloid leukemia with 46% bone marrow blasts and FLT3-ITD mutation (d) went into complete remission after treatment. This patient relapsed 5 months later with 23% blasts and no detectable FLT3 mutation (e).

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FLT3 Status Changes and Clinical Characteristics

We compared the clinical characteristics and molecular profiles of the 42 patients with a change in FLT3 status to a random selection of 57 patients with persistently negative FLT3 mutation test results (Negative/Negative) as well as 49 randomly selected patients who persistently retained FLT3 mutation in follow-up sample (Positive/Positive). The results are summarized in Table 1.

Table 1 Clinopathological characteristics of acute myeloid leukemia patients according to FLT3 mutation statuses at diagnosis and follow-ups
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No statistically significant differences were found with respect to age and gender among these groups. Significantly higher bone marrow blast counts were observed in the FLT3-mutated groups (Positive/Positive, Negative/Positive, and Positive/Negative), compared with the Negative/Negative group (P=0.001), consistent with earlier studies.6, 8 The frequency of FLT3-ITD versus FLT3-D835 was similar among the FLT3-mutated groups (P=0.135). The mutant allelic ratio was also similar among these groups (P=0.676 and 0.450 for FLT3-ITD and FLT3-D835, respectively).

As expected, NPM1 mutations were more frequently seen in FLT3-mutated groups compared with the Negative/Negative group (P=0.005), due to the strong association between these two mutations. RAS mutation rates were similar among all groups (7–14%) except the Positive/Negative group, in which a higher rate (33%) was observed. However, due to the small sample size, the difference was not statistically significant (P=0.339). Additionally, a higher frequency of cytogenetic clonal evolutions was found in patients with FLT3 mutations (P=0.006), especially in patients in the Negative/Positive group.

Patients in the Positive/Positive group had a significant lower complete remission rate than those of the Negative/Negative group (P=0.003). The other two groups of patients with changed FLT3 mutation status showed complete remission rates closer to that of the Negative/Negative group. However, the higher complete remission rates in these two groups may be biased due to the fact that our patient selection criteria excluded patients without follow-up testing, and most of the patients who did not reach a complete remission died before they were re-tested for FLT3. As a result, the majority of these two groups were patients who went into complete remission and then relapsed.

FLT3 Status Changes Impact Patient Survival

The Kaplan–Meier curves plotting the overall survival among the four study groups are shown in Figure 2a. Patients who gained mutations (Negative/Positive) had an overall survival similar to that of the Negative/Negative group (P=0.464), and was significant better than that of the Positive/Positive group (P<0.001). The best survival appeared to be the six patients in the Positive/Negative group who lost FLT3 mutation after first complete remission, although admittedly the sample size is too small to accurately predict their behavior. However, when examining survival time of the Negative/Positive patients after they turned positive for FLT3 mutations, their survival curve tracked that of the Positive/Positive group after their first relapse (Figure 2b; P=0.761), and was significantly worse than that of the Negative/Negative group (P<0.001). In the Negative/Positive group, the median lag time from initial diagnosis without FLT3 mutation to testing positive for mutation was 15 months, ranging from 2 to 45 months. The survival, therefore, became dramatically worse for these patients after gaining FLT3 mutation and all but one patient died shortly after FLT3 mutation was acquired.

Figure 2
figure 2

Survival of acute myeloid leukemia patients according to FLT3 mutation statuses at diagnosis and follow-ups. (a) Overall survival of all patients in the four groups (P<0.001). (b). Survival after first relapse or after FLT3 mutation detected in the Negative/Positive group (P<0.001).

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We further compared the survival of patients with FLT3-ITD and those with FLT3-D835 mutation in the Positive/Positive and Negative/Positive groups (Figure 3). No significant difference was found between acute myeloid leukemia patients with these two types of mutations in either overall survival or survival time after first relapse in Positive/Positive group or after FLT3 mutation becoming positive in Negative/Positive group.

Figure 3
figure 3

Comparaison of survival between acute myeloid leukemia patients with FLT3-ITD mutation and those with FLT3-D835 mutation. (a) Overall survival in the Positive/Positive groups (P=0.169). (b) Survival after first relapse in the Positive/Positive groups (P=0.780). (c) Overall survival in the Negative/Positive groups (P=0.086). (d) Survival after FLT3 mutations detected in the Negative/Positive groups (P=0.368).

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Discussion

It is well established that FLT3 mutation is an important prognostic factor in patients with acute myeloid leukemia. Therefore, accurate assessment of FLT3 mutation status is crucial to risk stratification, clinical management, and treatment selection for acute myeloid leukemia patients. Several studies have observed FLT3 mutation status changes in small subsets of acute myeloid leukemia patients and have implied the prognostic importance.10, 16, 21, 22, 23, 24, 25, 26, 27, 28, 29In aggregate, these data suggest that gain of FLT3 mutations may be associated with worse prognosis.24, 27, 29, 33 These patients also seem not to benefit from intensifying chemotherapy.34, 35, 36 The number of patients in these studies, however, are truly small. We present the first study focused on a large group of such patients, thereby making it possible to statistically analyze clinical characteristics and the prognostic impact of a change in FLT3 status.

In this study, approximately 6% of acute myeloid leukemia patients had a demonstrated change in FLT3 status during their disease course, similar to reports in earlier studies.10, 16, 21, 22, 23, 24, 25, 26, 27, 28, 29 One previous study reported that FLT3-ITD is more often acquired than FLT3-TKD at time of relapse, 8% and 2%, respectively, whereas FLT3-TKD is more often lost than FLT3-ITD, 7% and 4%, respectively.29 In our study, FLT3 mutations were acquired far more often than lost, in a ratio of 6 to 1, during the disease course. In addition, FLT3-ITD was more often involved than FLT3-TKD in both patients with gain or loss of FLT3 mutation.

We compared the clinical presentation of patients with a change in FLT3 mutation status to randomly chosen groups of patients without change in FLT3 status. There was no significant difference in age and gender distribution between these groups. However, patients who gained the mutations during follow-up (Negative/Positive group) presented with a higher average bone marrow blast count. The Negative/Positive group also had a higher frequency of NPM1 mutations compared with the Negative/Negative group. Although the predictive power of these two findings is low, given the high frequency of a high blast count and NPM1 mutation, and the relative low frequency of a change in FLT3 status, these features in a patient presented with wild-type FLT3 may serve as an early indicator for possible emergence of FLT3 mutations.

Importantly, our results show that a gain of FLT3-ITD mutation strongly correlates with disease progressions and worse prognosis. Patients in the Negative/Positive group initially enjoyed similar survival rate as patients in the Negative/Negative group, even after relapse, but this was no longer the case once a FLT3 mutation became detectable. Affected patients showed dramatic exacerbations of their disease after relapse with FLT3 mutation and commonly died shortly thereafter. The median survival of these patients after acquiring a FLT3 mutation was very similar to that of patients in the Positive/Positive group after first relapse, often less than 5 months post first relapse.34, 36 Based on these results, we suggest that all acute myeloid leukemia patients be tested repeatedly for FLT3 mutation over their disease course to monitor the mutation status.

The exact mechanism of the change in FLT3 mutation status is not clear. One possibility is that FLT3 mutations may emerge owing to the instability of the tumor genome.24, 25 The higher frequency of cytogenetic clonal evolution known to be associated with FLT3 mutations found in this study potentially supports this possibility. A second possibility is that, leukemia cells with FLT3 mutations may be present in bone marrow at time of initial diagnosis in a very small number that is below the level of detection of the assays to assess FLT3. These FLT3-mutated leukemia cells have a survival advantage over their wild-type counterparts and eventually become a dominant clone. There is evidence that conventional therapies for acute myeloid leukemia appear to give a selection advantage to leukemia cells depending on FLT3 signaling, and resulting in increased expression of the mutant allele or loss of the wild-type allele at relapse.37 This phenomenon may be related to clonal expansion after therapy. Findings in this study also show that emergence of FLT3-mutated leukemia cells in the Negative/Positive patient group correlated with a poor response to conventional chemotherapy, significantly impacting patient survival in a negative manner. Therefore, change in FLT3 mutation status has practical implications for patient management, and is not simply an academic issue.

Conventional chemotherapy is not effective in eradicating FLT3-mutated leukemia cells. In our study, only 6 of 644 patients who were initially positive for FLT3 mutations successfully eliminated these mutations during their disease course, presumably as a result of conventional therapy alone. Other studies also suggest that FLT3 signaling may be a key survival mechanism in leukemia cells.38 Overexpression of FLT3 also has been detected in acute myeloid leukemia patients without FLT3 mutations and it is associated with a poor overall survival.39 These data argue that patients with FLT3 mutations may require their own therapeutic regimens, including FLT3 inhibitors, agents that target the aberrantly activated FLT3 kinase. In fact, FLT3 inhibitors have attracted broad attention as new therapeutic agents for acute myeloid leukemia, and there are multiple clinical trials that are currently in progress. Both small molecules and FLT3-directed antibodies are currently in trials.19 It has been suggested that all acute myeloid leukemia patients might benefit from receiving FLT3 inhibitor regimens starting from their initial diagnosis regardless of their mutation status.20, 37 Patients in the Negative/Positive group in our study would support this view.

In summary, this study demonstrates that a change of FLT3 status occurs in a small subset of acute myeloid leukemia patients, approximately 6%. As a result of the large cohort of AML patients at our institution, we have reported on 42 patients who showed a change in FLT3 status. Importantly, acquisition of FLT3 mutation has a negative clinical impact on survival. The findings suggest that FLT3 mutations are unstable and that acute myeloid leukemia cells are constantly evolving. Our data suggest that continuous testing for FLT3 mutation throughout the disease course is important, and may be most important for patients that initially tested negative for FLT3 mutation. Adding FLT3 inhibitors to standard therapeutic regimens may be beneficial for all patients who suffer from acute myeloid leukemia.