G.B. collected and analyzed the data, drafted the brief report, and provided clinical care to patients. H.K., F.R., S.F., T.K., and J.E.C. coauthored the article and provided clinical care to patients. S.P., K.P.P., R.L., and W.Q. collected data, analyzed results. and coauthored the article. W.Q. performed statistical analyses and coauthored the article.
The impact of single versus multiple fms-like tyrosine kinase receptor 3 internal tandem duplication (FLT3-ITD) mutations on the clinical outcome of patients with acute myelogenous leukemia has not been well studied, and particularly has not been investigated while simultaneously accounting for the quantitative mutation burden.
The authors conducted a multivariate analysis of overall survival, event-free survival, and complete remission duration, including numeric variation (single vs multiple) and quantitative mutant burden of FLT3-ITD as variables among other clinically relevant factors.
An analysis of a cohort of 1043 patients with AML demonstrated that, among patients with normal-karyotype acute myelogenous leukemia and FLT3-ITD mutation, overall survival and event-free survival were not affected by the number of FLT3-ITD mutations, but complete remission duration was significantly longer in patients who had multiple FLT3-ITD mutations (median, 86 weeks vs 34 weeks; P = .03).
Multiple reports have confirmed the adverse impact of fms-like tyrosine kinase receptor 3 internal tandem duplication (FLT3-ITD) mutation on clinical outcomes among patients with acute myelogenous leukemia (AML) and normal-karyotype AML (AML-NK).1-6 A high FLT3-ITD mutant level is associated with a poor outcome, whereas the clinical outcomes of patients with low mutant level may be similar to the outcomes of patients without FLT3-ITD mutation.2, 4
Data on the number (1 vs multiple) of FLT3-ITD mutants are conflicting. In a retrospective analysis involving 854 patients with AML (mostly aged ≥60 years) who were treated on United Kingdom Medical Research Council (MRC) trials, the number of ITD mutations (0 vs 1 vs >2) replaced the presence or absence of FLT3-ITD mutation as the most important independent variable predicting for the risk of relapse and for disease-free survival in a multivariate model, and a higher mutant number was associated with a worse outcome.3 The number of ITD mutations was identified as the most important factor after cytogenetic risk group was predictive of event-free and overall survival. Among the patients with AML who had FLT3-ITD mutation, the risk of relapse was similar for patients with 1 mutation and patients with ≥2 mutations. A subsequent analysis that involved a larger cohort of patients from the MRC group revealed that quantitative mutation burden was more prognostic than the number or size of ITD mutations.2 Among pediatric patients, the presence of more than 1 ITD mutation was not clinically relevant.7
MATERIALS AND METHODS
We retrospectively analyzed clinical outcome data on patients with newly diagnosed AML who received treatment at The University of Texas MD Anderson Cancer Center from January 2003 to December 2009. Event-free survival was calculated from the beginning of treatment until an event; which was defined as either relapse, resistance to induction therapy. or death. Overall survival was calculated from the time of diagnosis until death. Complete remission (CR) duration (CRD) was calculated from the date of CR until the date of either relapse or death in CR. Patients were censored at their last follow-up. Categorical and continuous variables were compared using the Fisher exact test and the Wilcoxon test, respectively. Kaplan-Meier product-limit survival probability estimates of overall survival, event-free survival, and CRD were calculated, and the log-rank test was used to compare the times to events. FLT3 mutation analysis and quantification of mutant burden were done on genomic DNA extracted from bone marrow aspirate samples according to published methods.8, 9 The detection of ITD mutation was achieved by fluorescent-based polymerase chain reactions and amplification of the juxtamembrane domain using fluorescent dye-labeled primers that flanked the ITD region. Polymerase chain reaction product was subjected to fluorescent-based capillary electrophoresis using a 3100 Genetic Analyzer (Applied Biosystems, Foster City, Calif).
The allelic ratio for FLT3-ITD was calculated as the ratio of an area under an ITD/mutant FLT3 peak to the area under total (mutant + wild-type) FLT3. The ratios were adjusted for the number of blasts in the aspirate as judged by a 500-cell manual differential count performed on the bone marrow aspirate smears to obtain the blast-normalized FLT3 mutation burden (FLT3 normalized).
A Cox proportional hazards regression model was used to evaluate the ability of prognostic variables at presentation, including the FLT3-ITD normalized mutation burden and the number of FLT3-ITD (multiple vs single) mutations, to predict for overall survival, event-free survival, and CRD.
RESULTS AND DISCUSSION
Data from 1043 patients with newly diagnosed AML were analyzed. Five hundred thirty patients (51%) had AML-NK, and 93 patients (18%; 59 with a single ITD mutation and 34 with ≥2 ITD mutations [21 with 2 ITDs and 13 with 3 ITDs]) had FLT3-ITD mutations. The median FLT3-ITD normalized value was 0.41 (range, 0.01-1.09) for a single ITD mutation and 0.52 (range, 0.02-2.35) for multiple ITD mutations. The characteristics of patients with single versus multiple FLT3-ITD mutations are summarized in Table 1. Patients with single and multiple (≥2) ITD mutations were comparable in terms of age, performance status, white blood cell and platelet counts, and percentage of bone marrow blasts. Overall survival (P = .59) (Fig. 1A) and event-free survival (P = .53) were similar among patients with single or multiple ITD mutations.
Table 1. Characteristics of Patients With Single Versus Multiple fms-Like Tyrosine Kinase Receptor 3 Internal Tandem Duplication Mutations
FLT3-ITD Group: Median [Range]
Single Mutation, N = 59 (63.44%)
Multiple Mutations, N = 34 (36.52%)
Abbreviations: FLT3-ITD, fms-like tyrosine kinase receptor 3 internal tandem duplication; WBC, white blood cells.
This ratio was adjusted for the number of blasts in the aspirate as determined by a 500-cell manual differential count performed on bone marrow aspirate smears to obtain the blast-normalized FLT3 mutation burden (“FLT3 normalized”).
Of the 93 patients with AML and FLT3-ITD mutation, 65 patients (70%) achieved CR or CR with incomplete platelet recovery. The CR rate was 66% in the single ITD group and 76% in multiple ITD group. Sixty-eight patients (83%) in the entire group and 56 patients (86%) in the group that achieved CR had received cytarabine-based induction chemotherapy. The time to CR did not differ between the single ITD group and the multiple ITD group. CRD was significantly longer in patients who had multiple FLT3-ITD mutations compared with patients who had a single FLT3-ITD mutation (median, 86 weeks vs 34 weeks; P = .03) (Fig. 1B). Seven patients (12%) who had a single FLT3-ITD mutation and 2 patients (6%) who had multiple FLT3-ITD mutations underwent stem cell transplantation in first remission. For the current analysis, these patients were not censored at the time of stem cell transplantation. On univariate and multivariate analyses that included age, performance status, and white blood cell and platelet counts, among other variables, the number of FLT3-ITD mutations (single vs multiple) and FLT3 normalized were the only predictors of CRD (P = .004 and P = .02, respectively). Because it was reported previously that high FLT3-ITD mutant burden was predictive of outcome, we wanted to rule out the possibility that the patients with multiple ITDs may have had a lower mutant burden (thus explaining the longer CRD). Our analysis, on the contrary, revealed that patients with multiple ITDs had a higher mutant burden than patients with a single ITD (P = .02). Although older age and higher white blood cell counts were predictors for shorter overall survival (P = .008 and P = .006, respectively), older age was the only predictor for event-free survival (P = .04).
Although the presence of single versus multiple ITDs does not appear to impact overall or event-free survival, a biologic explanation for longer CRD in patients with multiple FLT3 ITDs in our cohort is unclear. Multiple ITDs can develop if cells carrying an FLT3-ITD mutation in 1 allele develop a different sized ITD mutation in the wild-type allele. Alternately, different leukemic blast populations may carry different sized ITD mutations. Single-cell polymerase chain reaction analysis may help to unravel these possibilities. Subclones of AML cells with multiple ITDs have demonstrated different engraftment potential in nonobese-diabetic/severe combined immunodeficiency (NOD/SCID) mice, with some clones engrafting and others failing to engraft and disappearing, suggesting that FLT3-ITD mutations can occur at different stages of leukemogenesis and may have differential sensitivity to chemotherapy.10 Similarly, observations of loss of FLT3 mutation at relapse of AML may indicate the presence of some mutations in more mature leukemic cells rather than leukemic “stem” cell.11 Although it is not believed that the remission rate is influenced by the presence of FLT3-ITD mutation, to our knowledge, the impact of dose intensity on clinical outcomes among patients with FLT3-ITD mutation has not been analyzed in large data sets.
In conclusion, clinical outcome analysis in patients with AML-NK and FLT3-ITD mutation should take in to account the mutant allele burden, size, and ITD number in addition to the categorical variable of presence or absence of mutation. If all of these parameters turn out to be important in an analysis of data pooled from large groups, then they should be incorporated into the stratification and randomization of patients who participate in clinical trials that involve FLT3-directed therapy.