Molecular Study of FLT3 Gene Mutations in Acute Myeloid Leukemia from Pakistan: Correlation with Clinicopathological Parameters

In acute myeloid leukaemia (AML), mutations for cell differentiation and proliferation are considered to be effective factors amongst various factors for its development. Fms-like tyrosine kinase3 (FLT3) genes belong to the family of tyrosine kinase class III receptors that induce signals for cell proliferation. Mutations of the genes in the form of internal tandem duplication in the juxtamembrane region (FLT3/ITD) has been described as single most common molecular genetic abnormality in acute myeloid leukemia with direct clinical impact on the disease outcome [1-2]. Another mutation called D835 activation loop domain mutation has also been reported. In literature, an average frequency of approximately 20% for FLT3/ITD and 7% for D835 has been reported [3]. The FLT3/ITD has been reported to occur in 16.5% of paediatric AML patients [3]. The occurrence increases with Abstract


Introduction
In acute myeloid leukaemia (AML), mutations for cell differentiation and proliferation are considered to be effective factors amongst various factors for its development. Fms-like tyrosine kinase3 (FLT3) genes belong to the family of tyrosine kinase class III receptors that induce signals for cell proliferation. Mutations of the genes in the form of internal tandem duplication in the juxtamembrane region (FLT3/ITD) has been described as single most common molecular genetic abnormality in acute myeloid leukemia with direct clinical impact on the disease outcome [1][2]. Another mutation called D835 activation loop domain mutation has also been reported. In literature, an average frequency of approximately 20% for FLT3/ITD and 7% for D835 has been reported [3]. The FLT3/ITD has been reported to occur in 16.5% of paediatric AML patients [3]. The occurrence increases with

Molecular Study of FLT3 Gene Mutations in Acute Myeloid Leukemia from Pakistan: Correlation with Clinicopathological Parameters
Mariam Faiz 1 , Farzana Rashid 2 age, i.e 20% of adult AML patients [3] and 34% of elderly AML patients [4]. These mutations are associated more frequently with standard risk cytogenetics, PML/RARα rearrangement [4][5]. However, they are less frequent with core binding factor leukemia, secondary or pediatric AML [6]. This mutation is thought to be associated with leukaemia progression and a poorer clinical outcome in both paediatric and adult patients. These mutations are recommended in international clinical guidelines as for estimating prognosis and deciding treatment after complete remission (CR), particularly in cytogeneticallynormal patients with acute leukaemia [7]. In Pakistan, cases of Acute leukaemia have been defined solely by the French-American-British (FAB) classification system [8]. As such, little is known about the prevalence of mutations and their prognostic importance in terms of 1 Acute leukaemia classified according to the World Health Organization (WHO) classification system in Pakistan.
In the present study, we have examined the cohort of 63 AML patients for mutation and determined its correlation to basic hematological data in them. Due to limited data on these mutations in Pakistan, the diagnosis and frequency of these mutations with different FAB subtypes in Pakistani AML patients is an important concern.

Patient samples
Blood samples from 63 adult AML patients with various French-American-British (FAB) classifications were collected from different Haematology departments of Lahore, Pakistan. The diagnosis of AML was based on morphology and FAB classification. The cytogenetic and immunophenotypic data for these patients were not available due to lack of such facilities in these departments. Informed consent was obtained from the patients before start of therapy.

Molecular studies
DNA extraction from blood samples of 63 AML patients was performed by the proteinase K and Phenol methods [9]. The patients DNA was isolated and stored at -20 o C for further analysis.

Analysis of ITD/FLT3 and D835 Mutations
PCR amplification of FLT3/ITDs exons 14 and 15 were amplified using primers as described elsewhere [4].
D835 and I836 amino acids are encoded by GATATC, which is the recognition sequence for EcoRV. PCR product was digested in a reaction volume of 15 ml, with 5U of EcoRV (New England BioLabs), at 37 o C for 3 hours. The digestion products were separated on a 3.5% agarose gel, and mutants were detected by the loss of GATATC site.

Statistical analysis
Chi square χ 2 and Fisher's exact tests were used to analyze differences in the distribution of variables among subsets of patients using SPSS 16.0.

Clinical Characteristics of the AML patients harboring the FLT3/ITD + and D835mutations
Of the 63 AML patients studied for mutations, 14 (22%) had FLT3/ITD, indicated by presence of longer PCR fragments. While 4 AML patients (6.3%) were found to contain the D835 mutation. None of the patients  reported [14]. This study is the first attempt to determine the incidence of these mutations in our patients in the absence of any cytogenetic data. It is important to note that incidence of mutation varies significantly between AML subtypes particularly in those subgroups defined by cytogenetic abnormalities. Variation in the frequency of ITD mutations reported (13-27%) may be because of the differences in the size of cohorts or subgroups of cohorts examined in the various studies. Therefore this limitation will be addressed and incidence of these mutation in different cytogenetic groups of AML will further be investigated in future studies. In AML patients, the FLT3-ITD mutations were reported to be associated with increased leukocyte counts at diagnosis. This was observed in our cases harbouring FLT3/ITD mutations as compared with those experiencing wild type FLT3 that is consistent with other reported studies [4]. In this study, significant association between incidence of mutations and patient age was found. Whereas no such correlation between patient age and FLT3 mutation status has been reported in other studies [4].
In conclusion, our results confirmed the significant presence of FLT3/ITD mutations in our adult AML patients. The biology of AMLs is very diverse and varies in different populations Therefore, extensive mutation analysis in different cytogenetic groups with follow-up durations is required to understand the pathogenesis of leukemias and their role as a valuable prognostic marker in our patients. The authors declare no conflicts of interest.

Source of Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-forprofit sectors.
had a combination of FLT3/ITD and D835 mutation in the FLT3 gene. The frequency of mutations in different FAB subtypes in present study was compared with international data as given in Table 2. The data revealed ITD mutation was found in all FAB subgroups among the studied patients. M4 subtype showed most of mutations as compared to other subtypes ( Table 2) whereas majority of the D835 mutant patients (14%) were M1 and M2 type each. ITD mutation was not confined to any gender or age as these were found in all age groups (Table 1). However, statistical significance was achieved in different age groups. Presence of FLT3/ ITD was clearly associated with hyperleukocytosis where WBC counts (mean 150x10 9 /L) were higher in ITD + patients than in FLT3/WT patients (mean 65x10 9 /L) but significance was not achieved. Association of clinical variables with D835 mutations could not be studied due to limited samples though WBC counts were higher (mean 34.8x10 9 /L), but not statistically significant when compared with wild type patients.

Discussion
A number of reports have shown that FLT3/ITD is associated with a poor prognosis in AML patients [7-8-10]. However, in Pakistan, no data exists that investigated the prevalence and prognostic value of the D835 and FLT3 mutations in AML patients with different FAB subtypes. Of the 63 AML patients examined, fourteen patients (22%) showed FLT3/ITD and four patients (6%) showed D835 mutations. None of the patients showed a combination of both FLT3/ITD and D835 mutations. In this study, FAB-M2 was most commonly seen (32%) that has also been reported from another study from Pakistan (32.26%) followed by M1 and M4 (22.58% each) [8]. In contrast, another study from different centre reported AML-M4 as most common FAB subtype in 116 patients studied [11][12]. In this study, the highest rate of FLT3/ITD mutations was detected in FAB-M4 patients while D835 mutations were equally detected in FAB-M1, M2 patients. The incidence of ITD mutations detected in our cohort of AML patients fall within the range of reported international studies [7-8-10]. Among FAB subtypes, FLT3 mutations were more commonly reported within the M2, M3 subtype [13]. However, fairly even distribution of FLT3/ITD mutations across all other FAB subtypes has also been † FAB Subtype ‫٭‬Current study data International data [11] M0