Prognostic significance of FLT3 internal tandem duplication and tyrosine kinase domain mutations for acute myeloid leukemia: a meta-analysis

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1 Prognostic significance of FLT3 internal tandem duplication and tyrosine kinase domain mutations for acute myeloid leukemia: a meta-analysis M Yanada 1,2, K Matsuo 3, T Suzuki 3, H Kiyoi 2 and T Naoe 1 (2005) 19, & 2005 Nature Publishing Group All rights reserved /05 $ Department of Hematology, Nagoya University Graduate School of Medicine, Nagoya, Japan; 2 Department of Infectious Diseases, Nagoya University Graduate School of Medicine, Nagoya, Japan; and 3 Division of Epidemiology and Prevention, Aichi Cancer Center Research Institute, Nagoya, Japan Two distinct forms of fms-like tyrosine kinase (FLT3) gene aberrations, internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations, have been recognized in a substantial proportion of patients with acute myeloid leukemia (AML). To investigate their prognostic significance, we performed a meta-analysis of the four published studies that provided survival information according to the FLT3 status: ITD, TKD mutation, and wild type. The summary hazard ratios for disease-free survival () were 1.88 (95% confidence interval (CI) ; Po0.001) for FLT3 mutations, 1.86 (95% CI: ; Po0.001) for ITD, and 1.90 (95% CI: ; Po0.001) for TKD mutation. The corresponding ratios for overall survival were 1.61 (95% CI: ; Po0.001), 1.68 (95% CI: ; Po0.001), and 1.37 (95% CI: ; P ¼ 0.104). Neither white blood cell count at diagnosis nor cytogenetic risk category was a significant source of heterogeneity. These findings indicate that FLT3 mutations have an adverse effect on the outcome for AML, and that the negative impact of TKD mutation seems comparable to that of ITD with regard to. Although it should be borne in mind that this meta-analysis was based on data abstracted from observational studies, these results may justify the risk-adapted therapeutic strategies for AML according to the FLT3 status. (2005) 19, doi: /sj.leu ; published online 16 June 2005 Keywords: acute myeloid leukemia; FLT3; ITD; TKD mutation; prognosis; meta-analysis Introduction Prognostic assessment is important for the management of acute myeloid leukemia (AML) since treatments can be optimized on the basis of accurate estimation of outcome. Although the use of karyotype analysis for risk-stratification is widely accepted, 1 5 prognosis of AML is not sufficiently predictable so that additional prognostic markers are required for more accurate estimation. A potential prognostic genetic marker is the fms-like tyrosine kinase 3 (FLT3) gene, which encodes a class 3 receptor tyrosine kinase and plays an important role in hematopoiesis. 6 8 Two distinct forms of FLT3 mutations have been identified, internal tandem duplication (ITD) in the juxtamembrane domain and point mutation within the activation loop of the tyrosine kinase domain (TKD), which mostly affects asparate 835 (D835). Both mutations are thought to be involved in leukemogenesis by constitutively activating the receptor. 6 8 FLT3-ITD is found in 20 30% of patients with AML, and many studies have shown that its presence correlates with poor outcome On the other hand, the prognostic relevance of TKD mutation is less Correspondence: Dr M Yanada, Department of Hematology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi , Japan; Fax: þ ; myanada@med.nagoya-u.ac.jp Received 16 April 2005; accepted 13 May 2005; published online 16 June 2005 clear. 9 14,24 Because of the relatively infrequent occurrence of TKD mutation, it can be assumed that a single study might be inadequate to accurately determine the effect of this mutation. For this reason, we performed a meta-analysis of the published studies to investigate the prognostic significance of FLT3 mutations for AML. Materials and methods Selection of studies Studies were eligible for inclusion in the meta-analysis if they met all the following criteria. (1) They were published up to December 2004 as original articles written in English. (2) They dealt only with untreated AML patients. (3) They offered survival information based on the FLT3 status: ITD, TKD mutation, and wild type. (4) They provided data on either or both of overall survival (OS) and disease-free survival (). Studies were excluded if they focused exclusively on children or on acute promyelocytic leukemia. Multiple reports of a single study were considered as one publication, and only the most recent article was examined. A computerized literature search of the MEDLINE database was conducted by using the free text search term AML AND FLT3 AND survival, with the publication period limited to before December 2004, and the language to English. The primary search yielded a total of 80 publications, 49 of which were excluded by title screening. Abstracts of the remaining 31 papers were reviewed, resulting in eight of them being excluded, and leaving 23 as candidate articles. To reach a final decision on which articles to include in the meta-analysis, we examined all the 23 papers in detail, which resulted in further exclusion of 19 papers, because survival information was not available for either or both of the FLT3 mutations. 12,13,15,16,18,20 23,25 34 Eventually, four studies 9 11,24 were considered to meet all the criteria specified above (Table 1). Citations in the four papers were also followed up. All potentially relevant articles were reviewed by two independent investigators (MY and KM). Data abstraction To avoid bias in the data abstraction process, the two reviewers (MY and KM) independently abstracted the data from the articles and subsequently compared the results. All data were checked for internal consistency, and disagreements were resolved by discussion. Characteristics abstracted from the articles included the name of the first author, year of publication, location of the study, number of subjects, mean or median values of age and initial white blood cell (WBC) counts, percentage of cases with favorable cytogenetics and hazard ratios (HRs), and 95% confidence intervals (CIs) for and OS according to the

2 1346 Table 1 List of studies included in the meta-analysis Authors, country, publication year FLT categories No. of subjects Age (years) Initial WBC count (x10 9 /ml) %of favorable cytogenetics HR for 95% CI for HR for OS 95% CI for OS Yamamoto et al Wild type (15 85) 20.8 ( ) Reference 1.00 Reference Japan ITD (15 77) 54.6 ( ) TKD mutation (17 70) 25.7 ( ) Frohling et al Wild type (16 60) 12.5 ( ) Reference 1.00 Reference Germany ITD (19 60) 44.8 ( ) TKD mutation (16 60) 24.8 ( ) Thiede et al Wild type 480 NR 10.9 ( ) Reference 1.00 Reference Germany ITD 111 NR 52.0 ( ) TKD mutation 37 NR 40.1 ( ) Moreno et al Wild-type 81 NR NR Reference NA NA Spain ITD 19 NR NR NA NA 2003 TKD mutation 11 NR NR NA NA FLT3 ¼ fms-like tyrosine kinase 3; WBC ¼ white blood cell; HR ¼ hazard ratio; 95% CI ¼ 95% confidence interval; ¼ disease-free survival; OS ¼ overall survival; ITD ¼ internal tandem duplication; TKD ¼ tyrosine kinase domain; NR ¼ not reported; NA ¼ not assessed. The HRs for the Thiede and Moreno studies were estimated based on Kaplan Meier survival curves by using the method by Parmar et al. HR higher than unity indicates that the presence of FLT3 mutation is associated with worse prognosis. For WBC and cytogenetic data in the Thiede study, values for all patients (n ¼ 979) were used because data for those subjected to survival analysis were not available. FLT3 status. When the data required for the analysis could not be abstracted, attempts were made to contact the investigators who conducted the studies. Quantitative data synthesis HRs were used to assess the survival effect of FLT3 mutations compared with wild type. The natural logarithm of a crude HR and its variance for each of the subcategories within the study was calculated by using the abstracted survival probabilities at each time point with the methods proposed by Parmar et al 35 and described elsewhere. 36 HRs were calculated to show how many times higher the probability of failure was for patients with FLT3 mutations than for those with wild type, as an HR higher than unity indicates that FLT3 mutations yield a worse survival rate than wild type. Because no patient carried ITD-TKD dual mutations in one study, 24 and survival data for dual mutants were not available in two studies, 10,11 those with dual mutations were excluded from the analysis. A general variance-based method was used to estimate the summary HRs and their 95% CIs. We also calculated the between-study variation (t 2 ) from the Q statistic with the method described by DerSimonian and Laird. 37 At first, both the fixedeffect model and the random-effect model were used to calculate summary HRs, but eventually the random-effect model was finally chosen. Begg s funnel plots 38 and Egger s test 39 were used to detect possible publication bias, and meta-regression analysis was employed to detect the source of heterogeneity in the survival analysis. The factors examined in the metaregression analysis were WBC counts at diagnosis, percentage of cases with favorable cytogenetic risk, and type of FLT3 mutations. All statistical analyses were conducted with Stata ver. 8 software (College Station, TX, USA). We defined a P-value of less than 0.05 as a statistically significant test result for a summary HR. To avoid false negative results due to the small number of studies entered in the regression analysis, a P-value of less than 0.15 was defined as significant for a meta-regression test. No adjustment of multiple comparisons was performed because of the lack of statistical power of the study and the existence of an a priori hypothesis. Results As shown in Table 1, four studies covering a total of 1160 subjects (833 with FLT3 wild type, 243 with ITD, and 84 with TKD mutation) were finally included in the meta-analysis. 9 11,24 Three of them were from Europe 9 11 and one from Japan. 24 One study 11 reported, but not OS. WBC counts at diagnosis were reported in three studies, 9,10,24 and indicated that FLT3- mutant cases seemed to have higher WBC counts than subjects with wild type. No common trend was observed for percentages of patients with favorable cytogenetics. We did not find any graphical or statistical evidence of publication bias for either or OS. The summary HRs for were 1.88 (95% CI: ; Po0.001) for FLT3 mutations, 1.86 (95% CI: ; Po0.001) for ITD, and 1.90 (95% CI: ; Po0.001) for TKD mutation by random-effect models, suggesting that the presence of either mutation is a deleterious prognostic factor for (Figure 1). The test for heterogeneity in the overall analysis showed no significant heterogeneity (Q ¼ 2.03, df ¼ 7, P ¼ 0.958, t 2 ¼ 0). Figure 2 shows the results of a similar analysis for OS. The summary HRs for OS were 1.61 (95% CI: ; Po0.001), 1.68 (95% CI: ; Po0.001), and 1.37 (95% CI: ; P ¼ 0.104), respectively. The test for heterogeneity in the overall analysis showed no significant heterogeneity (Q ¼ 5.692, df ¼ 5, P ¼ 0.337, t 2 ¼ 0.005) for OS either. Since WBC counts at diagnosis and cytogenetic risk category are the currently generally accepted risk factors for survival, 3 we used meta-regression analysis to further evaluate the effect of these two factors, with the results shown in Table 2, which demonstrates that neither was a significant source of heterogeneity.

3 1347 Figure 1 Forrest plots of the hazard ratios (HRs) and 95% confidence intervals for disease-free survival. The size of the diamonds represents the weight for the random-effect model in the meta-analysis. A HR higher than unity indicates that the presence of FLT3 mutation is associated with a worse prognosis. ITD: internal tandem duplication; TKD: tyrosine kinase domain mutation. Figure 2 Forrest plots of the hazard ratios (HRs) and 95% confidence intervals for overall survival. The size of the diamonds represents the weight for the random-effect model in the meta-analysis. A HR higher than unity indicates that the presence of FLT3 mutation is associated with a worse prognosis. ITD: internal tandem duplication; TKD: tyrosine kinase domain mutation. Discussion It has been suggested that FLT3-ITD has a significant prognostic impact on AML, 9 23 but whether another type of FLT3 mutation affecting TKD provides relevant prognostic information remains controversial. 9 14,24 Since TKD mutation enhances the proliferative activity of AML cells in vitro to the same level as ITD does, 24 TKD mutation may also have a prognostic value. In contrast with ITD, however, which is found in 20 30% of AML cases, the frequency of TKD mutation ranges from 5 to 10%,

4 1348 Table 2 Results of meta-regression analysis for heterogeneity among and OS analyses OS Coefficient SE P-value Coefficient SE P-value Initial WBC count /ml increase Favorable cytogenetics 1 percent increase ¼ disease-free survival; OS ¼ overall survival; SE ¼ standard error; WBC ¼ white blood cell; HR ¼ hazard ratio. Coefficients show how much actual summary HR by random effect model is changed by the factor of interest. Change is reflected in a natural logarithm. making it difficult to detect its actual effect on outcome. Metaanalysis is a useful statistical method for integrating results from independent studies for a specified outcome. Combining the relevant studies increases statistical power, and makes it possible to detect effects that may be missed by individual studies. The meta-analysis reported here demonstrated that the effects of FLT3 mutations were quite robust with the summary HRs of 1.88 (95% CI: ) for, and 1.61 (95% CI: ) for OS. In line with the results of the individual studies, ITD was found to be associated with poor prognosis. In addition, our study indicated that TKD mutation has a negative effect on outcome, and its significance was, unexpectedly, comparable to that of ITD with regard to. These findings support the notion that FLT3 plays a determinative role in configurating the clinical characteristics of AML, and suggest that its status may contribute to a more effective stratification of treatment. Our study has several limitations. The first and major problem is that analyses were based on observational studies rather than prospective controlled studies. Secondly, we used abstracted data, while an individual patient data-based meta-analysis would have provided a more robust estimate of the association. The results reported here should therefore be interpreted carefully by clinical physicians. Thirdly, as is often the case with meta-analysis, a substantial effect of heterogeneity needs to be taken into account. Although WBC counts and ratios of favorable cytogenetics were not identified as sources of heterogeneity, we still cannot rule out the potential effect of other factors, such as differences in treatment, distribution of the intermediate and adverse cytogenetics, which were not examined in our analysis. Finally, publication bias, although not directly detected by us, may also have had a negative effect on the accuracy of our study. Although these limitations need to be borne in mind, our meta-analysis showed that FLT3 mutations do have an adverse effect on outcome for AML, and this negative prognostic impact was found to apply not only to ITD but also to TKD mutation. These findings may make it advisable to distinguish AML with FLT3 mutations from AML without mutations and justify the risk-adapted therapeutic strategy for AML based on the FLT3 status. However, a large number of patients should be prospectively studied to make it possible to reach any definitive conclusions, especially with regard to TKD mutation. In addition to the presence or absence of FLT3 mutations, several factors relevant to FLT3 have been put forward as having prognostic value, including expression levels of FLT3 transcripts, 34 mutant/wild type allelic ratios for ITD, 10,19 ITD-TKD dual mutations, 40 and types of TKD mutation. These factors should also be further investigated in order to arrive at a more accurate estimation of the prognosis for AML. Acknowledgements We are indebted to Drs Richard Schlenk and Konstanze Döhner (the AML Study Group Ulm) for providing additional data. We also thank Ms Mariko Nakano (Aichi Cancer Center Research Institute) for her technical support. References 1 Grimwade D, Walker H, Oliver F, Wheatley K, Harrison C, Harrison G et al. 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