Frequency and Prognostic Relevance of FLT3 Mutations in Saudi Acute Myeloid Leukemia Patients

The Fms-like tyrosine kinase-3 (FLT3) is a receptor tyrosine kinase that plays a key role in cell survival, proliferation, and differentiation of hematopoietic stem cells. Mutations of FLT3 were first described in 1997 and account for the most frequent molecular mutations in acute myeloid leukemia (AML). AML patients with FLT3 internal tandem duplication (ITD) mutations have poor cure rates the prognostic significance of point mutations; tyrosine kinase domain (TKD) is still unclear. We analyzed the frequency of FLT3 mutations (ITD and D835) in patients with AML at diagnosis; no sufficient data currently exist regarding FLT3 mutations in Saudi AML patients. This study was aimed at evaluating the frequency of FLT3 mutations in patients with AML and its significance for prognosis. The frequency of FLT3 mutations in our study (18.56%) was lower than many of the reported studies, FLT3-ITD mutations were observed in 14.4%, and FLT3-TKD in 4.1%, of 97 newly diagnosed AML patients (82 adult and 15 pediatric). Our data show significant increase of FLT3 mutations in male more than female (13 male, 5 female). Our results support the view that FLT3-ITD mutation has strong prognostic factor in AML patients and is associated with high rate of relapse, and high leucocytes and blast count at diagnosis and relapse.


Introduction
FLT3 (Fms-like tyrosine kinase-3), also known as FLK2 (fetal liver kinase-2) and STK1 (human stem cell kinase-1), was originally isolated as a hematopoietic progenitor cell-specific kinase and belongs to Class-III receptor tyrosine kinase (RTK) family to which c-Fms, c-Kit, and the PDGFR (platelet derived growth factor receptor) also belong [1]. Normal expression of FLT3 is restricted to hematopoietic progenitor cells in the bone marrow (BM), thymus, and lymph nodes but is also found in other tissues such as placenta, brain, cerebellum, and gonads [2].
FLT3 plays a key role in cell survival, proliferation, and differentiation of hematopoietic stem cells [3]. The human FLT3 gene is located on chromosome 13q12 and encompasses 24 exons. It encodes a membrane-bound glycosylated protein of 993 amino acids with a molecular weight of 158-160 kDa, as well as a nonglycosylated isoform of 130-143 kDa that is not associated with the plasma membrane [4]. The structure of FLT3 is shown in Figure 1. FLT3 is frequently overexpressed in acute leukemia. FLT3 mutations occur in approximately 30% of acute myeloid leukemia (AML) patients and confer a poor prognosis [5].
The two major types of mutation that occur are internal tandem duplication (ITD) mutations of the juxtamembrane region and point mutations in tyrosine kinase domain (TKD), which frequently involve aspartic acid 835 of the kinase domain (D835). Both mutations result in constitutive activation of the receptor's tyrosine kinase activity in the absence of ligand [6].
Many studies have shown that AML patients with FLT3-ITD mutations have poor cure rates due to relapse. This has led to the development of a number of small molecule tyrosine kinase inhibitors (TKI) with activity against FLT3 [7]. Adult patients usually have a higher prevalence of FLT3-ITD (24%) than pediatric AML patients. This observation may partially explain why adult AML has a poorer clinical outcome than pediatric AML [8]. Moreover, the clinical significance of FLT3-TKD mutation which is found in approximately 5-10% of AML patients is not clear yet, but several studies indicate that it is also an adverse prognostic indicator [8]. Clinically AML patients with FLT3-ITD tend to have higher WBC counts and an increased percentage of leukemic blasts [9]. FLT3 mutations have also been seen in myelodysplastic syndrome (MDS) in about 3-5% of newly diagnosed patients and in MDS patients without FLT3 mutations, they sometimes appear when these patients progress to AML [10].
This study which is considered the first study done in Saudi Arabia focuses on frequency and prognosis of the presence of FLT3 mutations in adult and pediatric acute myeloid leukemia patients; only one study was done for detection of FLT3 oncogene mutations in adult acute myeloid leukemia using conformation sensitive gel electrophoresis [11].  [29], and 1 U of gold Taq polymerase, in a volume of 50 L.

Materials and Methods
The PCR consisted of an initial incubation step at 95 ∘ C for 10 minutes followed by 35 cycles at 94 ∘ C for 30 seconds, 57 ∘ C for 60 seconds, and 72 ∘ C for 90 seconds and final extension step at 72 ∘ C for 10 minutes on a GeneAmp PCR system 9700 (Applied Biosystems). PCR products were analyzed on standard 3% agarose gels, and samples showing additional longer PCR products were considered FLT3/ITD+.

Analysis of the D835 Mutation of the FLT3 Gene.
PCR reaction was set up as above using published primers [28]. PCR product was digested with EcoRV (Promega), at 37 ∘ C for 2 h. The digestion products were separated on a 3% agarose gel, and incomplete digestion indicated the presence of a mutant.

Statistical Analysis
The Kaplan-Meier technique was used to analyze the probability of overall survival (OS) and event-free survival (EFS). OS was calculated from time of diagnosis to death and EFS from time of diagnosis to death, evidence of persistent leukemia, or relapse. Continuous variables, such as white blood cell count and hemoglobin, were compared by using the Kruskal-Wallis test. Differences between means were considered as significant at < 0.05.  In FLT3-mutated patients median WBC was 65 × 10 9 /L compared to 12.5 × 10 9 /L in the rest of the patients. Peripheral blood blasts were elevated in FLT3-mutated group compared to FLT3-wild type (WT) patients (40% versus 8%, resp.).
As cytogenetic study was available only in a limited number of cases, correlation for cytogenetic analysis was not possible. In mutated group, cytogenetic and molecular studies revealed that 4 cases (22.2%) are associated with AML specific abnormalities, namely, PML-RARA/t (15; 17)(q22; q21), AML-ETO t(8; 21)(q22; q22), CBFB/inv(16)(p13; q22), and MLL (DC,BAR)/11q23. By conventional karyotype, it was not available for 5 cases because of a low number of analyzed metaphases; 3 cases were trisomy (2 cases with trisomy 8 and one case with trisomy 5) and 6 cases were with normal karyotype. Median overall survival was 10.0 months for FLT3-mutated patients and 20.0 months for WT patients ( = 0.031), and FLT3-positive patients had also a significantly shorter 2-year event-free survival (EFS) than FLT3/WT patients ( = 0.040) because of a higher relapse rate.
The overall frequency of FLT3 mutations in our study (18.55%) is lower than most of the reported studies [3,28,[30][31][32]; however, some reports agree with our report [21]. Also, the only study which was conducted in Saudi Arabia for FLT3 mutations in AML patients showed frequency of 20.15% (26/129), close to our results [11]. The explanations of lower frequency of FLT3 mutations in our study from other several studies may be explained by differences in the sizes of examined groups or might be due to population genetics, environmental factors, selected patient population studies, or because of differences of age as the median age of patients in this study was 36 years in comparison to comparative studies or a combination of the all.
Adult patients usually have a higher prevalence of FLT-3-ITD mutation than pediatric AML patients; this observation may partially explain why adult AML has a poorer clinical outcome than pediatric AML [8]. In our study, the frequency of FLT3-ITD mutation in adult AML was 14.43% which is lower than reported in other published studies [8,19,20,22,28,35], but still in the variation range of frequency of ITD mutations (13-27%) reported by some study group [12,21] ( Table 2). In contrast to published studies the rate of FLT3-ITD mutation in pediatric AML (20%) is more than the adult and higher than most of the reported studies [33,34,36]. This difference in the rate most likely is attributed to small size of samples of pediatric AML (15 cases).
Similarly, the rate of FLT3-TKD mutation (D835) in our study is 4.12% (4/97) which is less frequent than FLT3-ITD mutation (14/97), these results are in accordance with the published studies on the frequency of FLT3-TKD in AML performed by many study groups [20,22,27,28]; the rate of FLT3-TKD mutation (D835) in our study is almost very close to the largest study done by Bacher et al. [23] in which FLT3-TKD mutations were detected in 147 of 3082 (4.8%) patients. However, our results are slightly lower than many studies on the frequency of FLT3-TKD in AML (Table 3) which were performed by Abu-Duhier et al. [27], Yamamoto et al. [28], Thiede et al. [22], Moreno et al. [26], and Fröhling et al. [8]; according to these studies, FLT3-TKD mutation shows an incidence of 5.8% to 7.7% in AML. However, some of the reported studies show incidence higher than these studies ranged from 8 to 12% [22]. Our data are in accordance with those published by other groups [8,13,14,17,22,29,[37][38][39][40][41] and showed that FLT3-ITD mutation has a strong prognostic factor in AML patients and associated disease progression with high rate of relapse and shorter overall survival; median overall survival was 10.0 months for FLT3-mutated patients and 20.0 months for WT patients ( = 0.031), and event-free survival (EFS) was also worse for FLT3-positive patients than FLT3-WT patients ( = 0.040) because of a higher relapse rate. Also high leucocytes count, high blast cells count in peripheral blood, resistant to therapy and confers a poor prognosis, Table 1. This has led to the development of a number of small molecule tyrosine kinase inhibitors (TKI) with activity against FLT3 [7,42]. Moreover, patients with low or absent levels of WT FLT3, consistent with homozygosity for the FLT3-ITD allele, appear to have a particularly dismal outcome [22].
Because of the low frequency, the prognostic significance of FLT3-TKD mutations is still unclear [23]. Many recent studies [21,23,43] showed that FLT3-TKD has little or no prognostic significance in AML patients. In this study, according to our data unlike the FLT3-ITD mutation, which are associated with inferior survival, prognosis was not influenced by mutation of FLT3-TKD. Moreover, Bacher et al. [23] found an additional favorable impact of FLT3-TKD on  EFS in prognostically favorable AML with NPM1 or CEBPA mutations. However, Yamamoto et al. [28] revealed that, in contrast to our study, D835 mutations were not significantly related to the leukocytosis but tended to worsen disease-free survival.
As a surprising finding in our study, the rate of FLT3 mutations in male (13/18; 72.2%) is higher than in female (5/18; 27.8%); this finding may be the first one reported in English literature showing the significant difference between male and female ( = 0.025). One small study was done on 30 AML patients with frequency of FLT3/ITD mutation in AML which was 4/30 (13.3%); three were males and one female [12]. However, these results should be treated with reservation, due to the relatively small sample sizes. These differences in male/female ratio in our study from several reported studies may be due to population genetics, environmental factors, relative small sample sizes, or a combination of all and further research and investigation are required.
As one interesting rare finding, one adult AML patient in our cohort study who did not have FLT3 mutations at diagnosis has been found to acquire FLT3/ITD at the time of relapse; this finding was reported by another study group [10] and raises the importance of assessing FLT3 gene on all relapses. This finding also suggests that FLT3 mutations are unstable and that there is potential clinical value in continuously monitoring FLT3 mutation status [44].

Conclusion
Currently no sufficient data exist regarding FLT3 mutations in Saudi patients. This study was aimed at evaluating the prevalence of FLT3 mutations in patients with AML and its significance for prognosis. The frequency of FLT3 mutations in our study was lower than (18.56%) other reported studies; however, some reports agree with our report or are very close. Our data as other several reports support the view that FLT3/ITD has strong prognostic factor in AML patients and is associated with high rate of relapse, is resistant to therapy, and confers a poor prognosis. Our data show increase of FLT3 mutations in male more than female which need more research studies using large size samples. This study also raises the importance of assessing FLT3 gene on all relapses as one case which did not have FLT3 mutations at diagnosis has been found to acquire FLT3/ITD at the time of relapse. Early detection of FLT3 mutations and an intensification of induction therapy might thus be useful for this group of patients to overcome the poor prognosis.

FLT3:
Fms-like tyrosine kinase-3 FLK2: Fetal liver kinase-2 STK1: Human stem cell kinase-1 BM: Bone marrow AML: Acute myeloid leukemia ITD: Internal tandem duplication TKD: Tyrosine kinase domain RTK: Receptor tyrosine kinase PDGFR: Platelet derived growth factor receptor MDS: Myelodysplastic syndrome WT: Wild type OS: Overall survival FISH: Fluorescence in situ hybridization EFS: Event-free survival TKI: Tyrosine kinase inhibitors. thank all the Hematology, Cytogenetic and Molecular Laboratories staffs and data managers, especially Mr. Mohamed Asiri and Dalal Alkhammash from Hematology Laboratory for data collection and management and Ms. Nadia Halawani from Molecular Laboratory for great help in molecular work of this study. Finally, they thank Ms. Khowla Al-Fayez and Inesse Abdullah from Cytogenetic Laboratory for excellent help and data collection in cytogenetic aspect of this study.