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

Introduction: FLT3 mutations are common genetic changes reported to have prognostic significance in acute myeloid leukemia (AML). Bone marrow/peripheral blood samples of 63 AML Pakistani patients were collected and DNA was isolated. Materials and Methods: The FLT3 internal tandem duplication (ITD) and the D835 activating mutation in the tyrosine kinase domain (TKD) were analyzed by polymerase chain reaction (PCR) Results: Among 63 AML patients, 42 were males and 21 were females with male to female ratio 2.1:1. The age ranged between 15 to 75 years with a median age of 32 years. AML-M2 was the predominant French-American-British (FAB) subtype (32%) followed by M3 (27%), M4 (19%), M5 (6.3%) and M1 (6.3%). The incidence of FLT3/ITD and TKD was 22% and 6.3% respectively. Majority of the FLT3/ITD mutation were detected in AML-M4 (38%) patients while D835 mutation was common in both FAB M1, M2. Presence of mutation was significantly associated with age but significance was not achieved for hyperleukocytosis. Conclusion: This study constitutes the first report from Pakistan reporting significant presence of FLT3/ITD mutations in our adult AML patients with different FAB subtypes Molecular mutation analysis in different cytogenetic groups with follow-up is required to understand the pathogenesis of leukemias and their role as a valuable prognostic marker in our patients.

 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 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 cytogenetically- normal 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 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.

Materials and Methods 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 -20oC 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 37oC 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 χ2 and Fisher’s exact tests were used to analyze differences in the distribution of variables among subsets of patients using SPSS 16.0.

Results Clinical characteristics of Patients

Among 63 AML patients, 42 (67%) were males and 21 (33%) were females with male to female ratio 2:1. The age ranged between 15 to 75 years with a median age of 32 years. Only six patients were above the age of 50 years. Among 63 patients, AML-M2 was the predominant French-American-British (FAB) subtype (32%) followed by M3 (27%), M4 (19%), M1 (9.5%), M1 (6.3%) and M0 (6.3%). Details of clinical characteristics at diagnosis of the 63 de novo AML patients were given in Table 1.

Characteristic Wild Type FLT3 Mutant FLT3
No % No % p
Cases 49 78 14 22
Age (median= 32 years)
≥15- 20 15 31 3 21
21-35 21 43 7 50
36-50 9 18 7 14 0.03
>50 4 8 2 14
Gender
Male 32 65 10 71
Female 17 35 4 29 0.75
Haemoglobin g/dl
≤10 36 73 11 79 0.3
> 10 13 27 3 21
WBC Count x 109/L
≤10 9 18 2 14
>10-50 16 33 4 29 0.14
>50 24 49 8 57
Platelet count x109/L
≤50 36 73 10 71 0.3
>50 13 27 4 29

* Abbreviations, FLT3, fms-like tyrosine kinase 3; AML, acute myeloid leukemia; ITD, internal tandem duplication; WBC, white blood count; FAB, French American British.

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 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 150x109/L) were higher in ITD+ patients than in FLT3/WT patients (mean 65x109/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 34.8x109/L), but not statistically significant when compared with wild type patients.

†FAB Subtype ٭Current study data International data [11]
M0 4/63 (6.3%) 1/4 (25) 0/14
M1 6/63 (9.5%) 1/6 (17) 40/148 (27.0)
M2 20/63 (32%) 3/17 (18) 49/210 (23.3)
M3 17/63 (27%) 5/20 (25) 58/159 (36.5)
M4 12/63 (19%) 3/12 (25) 51/172 (29.7)
M5 4/63 (6.3%) 1/4 (25) 20/81 (24.7)
M6 0/63 0/2 1/15 (6.7)
Total 14/63 (22.2) 219/810 (27.0)

*Data are shown as FLT3/ITD mutation-positive cases/ tested cases (%); † Data shown as prevalence (%) of FAB subtypes in AML 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 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.

Acknowledgements

The authors would like to acknowledge Director, INMOL for permission to use technical facilities and collection of patient data.

Conflict of interest

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-for- profit sectors.

References 9 09 650-665 3 2003 10.1038/nrc1169 Stirewalt Derek L. Radich Jerald P. Nature Reviews Cancer The role of FLT3 in haematopoietic malignancies 1911 1918 10 1996 Nakao M Yokota S Iwai T Kaneko H Horiike S Kashima K et al Leukemia Internal tandem duplication of the FLT3gene found in acute myeloid leukemia 10 10 1605-1609 11 1997 10.1038/sj.leu.2400812 Yokota S Kiyoi H Nakao M Iwai T Misawa S Okuda T Sonoda Y Abe T Kahsima K Matsuo Y Naoe T Leukemia Internal tandem duplication of the FLT3 gene is preferentially seen in acute myeloid leukemia and myelodysplastic syndrome among various hematological malignancies. A study on a large series of patients and cell lines 3074 3080 93 1997 Kiyoi H Naoe T Nakano Y et al Blood Prognostic implication of FLT3 and N-RAS gene mutations in acute myeloid leukemia 9 09 1738-1752 17 2003 10.1038/sj.leu.2403099 Levis M Small D Leukemia FLT3: ITDoes matter in leukemia 1 10 190-195 111 2000 10.1046/j.1365-2141.2000.02317.x Abu-Duhier F. M. Goodeve A. C. Wilson G. A. Gari M. A. Peake I. R. Rees D. C. Vandenberghe E. A. Winship P. R. Reilly J. T. British Journal of Haematology FLT3 internal tandem duplication mutations in adult acute myeloid leukaemia define a high-risk group 4 04 675-683 14 2000 10.1038/sj.leu.2401731 Rombouts WJC Blokland I Löwenberg B Ploemacher RE Leukemia Biological characteristics and prognosis of adult acute myeloid leukemia with internal tandem duplications in the Flt3 gene 200 203 43 1993 Hassan K Qureshi M Shafi S Ikram N Akhtar MJ J Pak Med Assoc Acute myeloid leukemia-FAB classification and its correlation with clinico-haematological features 2001 Sambrook J Russell DW Molecular Cloning, A Laboratory Manual. 3rd ed. New York: Cold Spring Harbor Laboratory Press 1333 1337 12 1998 Kiyoi H Towatari M Yokota S Hamaguchi M Ohno R Saito H et al Leukemia Internal tandem duplication of the FLT3 gene is a novel modality of elongation mutation which causes constitutive activation of the product 26 29 17 2005 Harani MS Adil SN Shaikh MU Kakepoto GN Khurshid M J Ayub Med Coll Abbottabad Frequency of FAB subtypes in acute myeloid leukemia patients at Aga Khan University Hospital Karachi 15 6 09 1752-1759 98 2001 10.1182/blood.v98.6.1752 Kottaridis Panagiotis D. Gale Rosemary E. Frew Marion E. Harrison Georgina Langabeer Stephen E. Belton Andrea A. Walker Helen Wheatley Keith Bowen David T. Burnett Alan K. Goldstone Anthony H. Linch David C. Blood The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy: analysis of 854 patients from the United Kingdom Medical Research Council AML 10 and 12 trials 826a 96 2000 Schnittger S Schoch C Kern W et al Blood FLT3 length mutations in AML: correlation to cytogenetics, FAB-subtype, and prognosis in 652 patients 15 12 06 4326-4335 99 2002 10.1182/blood.v99.12.4326 Thiede Christian Steudel Christine Mohr Brigitte Schaich Markus Schäkel Ulrike Platzbecker Uwe Wermke Martin Bornhäuser Martin Ritter Markus Neubauer Andreas Ehninger Gerhard Illmer Thomas Blood Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia: association with FAB subtypes and identification of subgroups with poor prognosis