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Examination of the FLT3 and NPM1 mutational status in patients with acute myeloid leukemia from southeastern Poland

Introduction Acute myeloid leukemia (AML) is a genetically heterogeneous disease at both the cytogenetic and molecular levels. In AML cells many chromosomal aberrations are observed, some of them being characteristic of a particular subtype of patients, and others being less significant. Besides chromosomal abnormalities, the leukemic cells can have a variety of mutations involving individual genes. The aim of this work was to investigate the frequencies of molecular alterations with the focus on FLT3-ITD and NPM1 mutations in AML patients of different age groups living in a southeastern region of Poland. Material and methods The study group comprised 50 consecutive AML patients. We analyzed bone marrow samples by conventional cytogenetics. Cytogenetic evaluation in selected cases was complemented by the FISH technique. The internal tandem mutation in the FLT3 gene was identified using polymerase chain reaction (PCR), and the NPM1 mutation was assessed by direct nucleotide sequencing. Results The studies using classical cytogenetics showed chromosomal aberrations in 32 (64%) patients. In 18 cases no changes in the karyotype were found by conventional karyotyping. FLT3-ITD mutation was detected in 4 (8%) patients and mutation of NPM1 in 3 patients with AML (6%). Conclusions The incidence of both mutations in our study group was lower than described elsewhere. We have confirmed that FLT3-ITD occurred more commonly in older patients and it was associated with shorter overall survival. By contrast, mutation of exon 12 of the NPM1 gene seems to be a good prognostic factor in AML patients with normal karyotype.


Introduction
Acute myelogenous leukemia (AML) is a hematopoietic proliferative disease in which there is clonal proliferation and accumulation of morphologically and functionally immature blast cells, derived from a precursor, transformed hematopoietic marrow cells with the exception of the lymphoid lineage [1,2]. Acute myelogenous leukemia represents 75-80% of the cases of acute leukemia in adults; in children it occurs in about 15% of all cases [1,3].
Chromosomal instability manifested in the form of chromosomal aberrations is a classic marker of many malignancies derived from the hematopoietic system [4]. The presence of chromosomal aberrations correlates with morphological features and clinical course of leukemia [5,6]. In 40-49% of AML patients cryptic structural changes of chromosomes are present, undetectable by means of standard karyotyping, which results in false-negative cytogenetic test results. Patients with such a normal karyotype (cytogenetically normal AML -CN-AML) represent the largest proportion of AML and are classified in the intermediate risk group, together with carriers of t (9;11) and trisomy 8 (+8) [7]. To better characterize the prognosis in this patient group, it is important to study the FLT3 gene (locus 13q12) and NPM1 gene (locus 5q35) mutational status.

FLT3
The FLT3 gene encodes a receptor tyrosine kinase (fms-like tyrosine kinase 3 -FLT3). Tyrosine kinases are enzymes that direct the phosphorylation of proteins involved in cellular signal transduction. FLT3 receptors are present on the surface of early hematopoietic progenitor cells [8].
The FLT3 ligand through its FLT3 receptor plays an important role in the proliferation and differentiation of hematopoietic progenitor cells as well as in the pathogenesis of AML [4,9]. It is mutated in about 25% of AML patients (all FAB subtypes), either by internal tandem duplications (ITD) of the juxtamembrane domain or by point mutations usually involving the kinase domain (KD) [10,11]. Both types of mutation constitutively activate FLT3, and autophosphorylation of FLT3 receptor activates the intracellular signal pathways responsible for proliferation [9]. Many studies have shown that AML patients with FLT3-ITD mutations have poor cure rates due to relapse [12,13].

NPM1
The NPM1 gene contains 12 exons that encode three isoforms of protein -nucleophosmin (NPM), a phosphoprotein with molecular weight of 37 kDa. It belongs to the chaperone protein family involved in, among other actions, the formation of ribosomes and the distribution of centromeres. It plays a key role in post-transcriptional processing of rRNA and ribosomal submission [14,15]. Nucleophosmin protein maintains genomic stability and controls mechanisms of DNA repair and duplication of centrosomes during mitosis [16]. NPM also interacts with tumor suppressors such as TP53 and ARF [17].
To date, about 50 mutations of the NPM1 gene have been found in AML [18]. They are the most common cryptic alterations in CN-AML, which are associated with distinct molecular, pathological and clinical characteristics. The majority of NPM1 mutations in patients with AML have a fixed location in exon 12; they are mostly insertions and deletions [19]. NPM1-mutated AML was included in the 2008 World Health Organization (WHO) classification of lympho-hemopoietic neoplasms as a provisional entity.
Identification of cryptic genome alterations allows for more precise stratification of AML patients into risk groups. This is why we decided to check for the aforementioned lesions in patients living in southeastern Poland.

Material and methods
The study group consisted of 50 consecutive patients (19 women and 31 men) aged between 18 and 74 years, hospitalized at the Department of Hematooncology and Bone Marrow Transplantation, Medical University of Lublin in the years 2008-2012. The preliminary diagnosis of AML was based on standard FAB (French-American-British) criteria. The follow-up covered a period of 3 years (36 months).
With informed consent (approval number of the University Ethics Committee: KE-254/24/2011) the bone marrow aspirates were collected to heparin and EDTA collection tubes (Sarstedt, Germany), to be used for cell culture and for DNA isolation, respectively. Twenty-four and 72-hour unstimulated cell cultures were terminated conventionally, then GTG and RHG banded chromosomes were analyzed. Cytogenetic evaluation in selected cases was complemented by the fluorescent in situ hybridization (FISH) technique.

NPM1 mutation status study
The determination of all possible mutations in exon 12 of the NPM1 gene was performed using direct sequencing. Primer sequences were selected and the procedure of sequencing was performed according to Döhner et al. [21], except for modified thermal conditions of the first PCR reaction aimed at amplifying the template for sequencing reactions: i. initial denaturation -95°C 5 min, ii. 35 cycles of 95°C 25 s, 57°C 30 s, 72°C 1 min, iii. final elongation -72°C 5 min. HD Advantage polymerase (Clontech, USA) was used for PCR. Amplification products were sequenced using an ABI PRISM 3130 Genetic Analyzer.

Results
The studies using classical cytogenetics showed chromosomal aberrations in 32 (64%) patients. The most common aberrations included deletion of 7q, 5q, 17p, and trisomy of chromosome 8. In 18 (36%) cases no changes in the karyotype were found by conventional karyotyping (cases defined as CN-AML) (Table I). Implementation of the FISH technique allowed for the confirmation of translocation t(15;17) (PML/RARα) in 2 patients; in one patient this aberration was detected only by FISH. Other alterations identified by FISH included: addition of 3q26.1-q26.3 (EVI1 gene amplification) in one patient, addition of 8q23-q24.3 (MYC gene amplification) in 1 patient, addition of 11q23 (MLL gene amplification) in 1 patient, and deletion of 17p13.1 (TP53) in 6 (12%) patients (Table I).
Molecular studies of mutations of the FLT3 gene showed the presence of internal tandem duplication in 4 (8%)patients with AML. In all cases only one allele was affected ( Figure 1). FLT3-ITD mutation was identified in 1 patient with normal karyotype (CN-AML, FAB M1) and in 3 patients with single lesions in the karyotype (2 classified as M4 and 1 as M1) ( Table I).
Studies of mutations in exon 12 of the NPM1 gene showed the presence of type A mutation (956dupTCTG, the tetranucleotide duplication of TCTG at positions 956 to 959 of the reference sequence -GenBank accession number NM_002520) in three patients with normal karyotype; two of them were classified as M5 and one as M4 according to FAB (Figure 2).
The mutation was present only in patients with normal karyotype, but this difference was not statistically significant (p = 0.0826).

FLT3
There are two categories of mutations of the FLT3 gene. The first group is described as FLT-length mutations (FLT3-LM), which are heterogeneous, and may present as insertions, deletions, and so-called internal tandem duplications (ITD) [9]. FLT-ITD usually occurs within exon 14 or 15, and is usually the result of amplification of the region comprising 3 to 400 base pairs [10]. The second group includes missense point mutations, which lead to an increase in the kinase activity [9]. Nucleotide substitutions typically occur within the intracellular tyrosine kinase domain (FLT3-TDK). Both types of mutations (FLT3-LM and missense point mutations) cause FLT3 receptor autoactivation.
Multicenter studies have shown a direct correlation between FLT-ITD mutations and risk of relapse in patients from the intermediate cytogenetic risk group.
In our studies, FLT3-ITD mutation was found in 4 (8%) patients. Studies by numerous authors have shown that this type of mutation is present in 15-35% of patients with AML [14,22,23]. The frequency of FLT3 mutations increases with age, but FLT3-ITD in older AML patients (aged 60 and more) has little effect on the outcome, due to the presence of other adverse changes, such as 11q23 rearrangement [1,11]. In our material, FLT3-ITD mutations occurred in patients aged over 50 years. FLT3-ITD mutation coexisted with a single lesion in the karyotype in 3 patients, it was also found in one patient with a normal karyotype.
Falini et al. [24], while examining a group of 591 AML patients, observed that FLT3-ITD mutations occur twice as often in the cases of CN-AML with simultaneous NPM1 mutations than in the cases without this mutation. However, in our material, no coexisting FLT3-ITD and NPM1 gene mutations were observed.
The results of clinical studies have shown a direct correlation between FLT3-ITD mutations and the risk of disease relapse and shorter survival time in AML patients with normal karyotype. In our study, 2 patients (P34 and P35) with internal tandem duplication of FLT3 died during the 3-year follow-up.
In acute promyelocytic leukemia (M3 according to FAB subtype), a higher incidence of FLT3 mutations is observed [25]. In our study, FLT3-ITD mutations were found only in patients with AML classified as subtype M1 and M4.
Peng et al., examining a group of 150 AML patients, demonstrated a correlation between the presence of FLT3-ITD mutation and the increased number of leukocytes in blood, increased bone marrow blast cell count and increased activity of lactate dehydrogenase [26]. In addition, they sug-     gested that FLT3-ITD might be an independent prognostic factor in acute myeloid leukemia [26].
In the current study we observed an increased number of white blood cells in three patients with FLT3-ITD, which confirms their results.

NPM1
NPM1 mutations substantially affect the course of AML. They include insertions or de-letions resulting in partial or complete loss of tryptophan residues. Abnormal protein encoded by NPM1 changes the location from nuclear to cytoplasmic, which contributes to the development of proliferative disorders [27]. NPM1 mutations are relatively commonly observed in patients with AML. The majority of NPM1 mutations occurring in AML involve exon 12; some rare mutations were described in exons 9, 10 and 11 [28,29]. The most common type of NPM1 mutation in AML is "type A" mutation, reported in 75-80% of cases [24,30]. NPM1 mutations in AML patients are associated with normal karyotype (CN-AML) and are rarely found in patients with chromosomal aberrations [19].
In our research, conducted over 3 years, NPM1 mutations were observed in 3 (6%) patients. During the follow-up we did not record deaths in patients with NPM1 mutations. It is known that the presence of NPM1 mutation augurs favorably and is associated with higher rates of complete remission [29].
In patients with CN-AML and NPM1 mutations, relapse-free survival is longer in comparison with what we observed in patients with NPM1 mutations [1].
Backer et al. in 2010, studying a group of 148 patients aged ≥ 60 years, found that the prognostic significance of NPM1 mutations increases with age, especially in older patients. Those patients who have a mutation in NPM1, especially without concomitant FLT3-ITD mutation, show significantly better prognosis than patients without mutations in the NPM1 gene [31]. Similar conclusions have been presented by many authors [32,33].
In our study, we observed the occurrence of mutations in this gene only in patients with normal karyotype, and their average age was 49 years.  NPM1 mutation is usually present throughout the leukemic cell population, demonstrating its essential role in carcinogenesis [24]. It is stable in the course of the disease and its presence is detected also at relapse even a few years after the initial diagnosis [34]. Mutation reversion is extremely rare and sometimes is associated with a change in karyotype [18,19,34].
Coexistence of NPM1 and FLT3-ITD mutations is relatively common, found in approximately 18% of patients with CN-AML [35]. Thiede et al. [19] found that in patients with NPM1 and co-existing FLT3-ITD, the NPM1 mutation preceded the occurrence of FLT3-ITD.
In our study group, patients carrying both FLT3-ITD and exon 12 NPM1 mutation were not found. The studies by Schneider et al. [36] in a group of 1321 patients with CN-AML showed the lower incidence of FLT3-ITD and NPM1 mutation with age in adult patients. Furthermore, lack of mutations in NPM1 and FLT3-ITD in the elderly suggested that other molecular and clinical risk factors might influence the outcome in this age group.
Our findings in a relatively small group of AML patients from southeast Poland confirm other reports of a better prognosis for CN-AML patients with NPM1 mutation. In the case of our patients with FLT3-ITD mutation its association with older age and shorter survival time was also confirmed. As the incidence of both mutations in our study group was lower as compared to other reports, studies on larger cohorts are required to confirm these results.