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versão impressa ISSN 1807-5932

Clinics vol.66 no.5 São Paulo  2011 



Screening for hotspot mutations in PI3K, JAK2, FLT3 and NPM1 in patients with myelodysplastic syndromes



João Agostinho Machado-Neto; Fabiola Traina; Mariana Lazarini; Paula de Melo Campos; Katia Borgia Barbosa Pagnano; Irene Lorand-Metze; Fernando Ferreira Costa; Sara T Olalla Saad

Hematology and Hemotherapy Center, National Institute of Blood, University of Campinas, São Paulo, Brazil




INTRODUCTION: Myelodysplastic syndromes encompass a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenia and a tendency to progress toward acute myeloid leukemia. The accumulation of genetic alterations is closely associated with the progression of myelodysplastic syndromes toward acute myeloid leukemia.
OBJECTIVE: To investigate the presence of mutations in the points most frequent for mutations (hotspot mutations) in phosphatidylinositol-3-kinase (PI3K), Janus kinase 2 (JAK2), FMS-like tyrosine kinase 3 (FLT3) and nucleophosmin (NPM1), which are involved in leukemia and other cancers, in a population of Brazilian MDS patients.
METHODS: Fifty-one myelodysplastic syndromes patients were included in the study. According to French-American-British classification, the patients were distributed as follows: 31 with refractory anemia, 8 with refractory anemia with ringed sideroblasts, 7 with refractory anemia with excess blasts, 3 with refractory anemia with excess blasts in transformation and 2 with chronic myelomonocytic leukemia. Bone marrow samples were obtained and screened for the presence of hotspot mutations using analysis based on amplification with the polymerase chain reaction, sequencing, fragment size polymorphisms or restriction enzyme digestion. All patients were screened for mutations at the time of diagnosis, and 5 patients were also screened at the time of disease progression.
RESULTS: In the genes studied, no mutations were detected in the patients at the time of diagnosis. One patient with chronic myelomonocytic leukemia was heterozygous for a Janus kinase 2 mutation after disease progression.
CONCLUSIONS: These results show that hotspot mutations in the PI3K, JAK2, FLT3 and NPM1 genes are not common in MDS patients; nevertheless, JAK2 mutations may be present in myelodysplasia during disease progression.

Keywords: Hematopoietic Disorder; Acute Leukemia; Myelodysplasia; Mutations; Bone Marrow.




Myelodysplastic syndromes (MDS) encompass a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, refractory cytopenia and a tendency to progress to acute myeloid leukemia (AML).1 Low-risk MDS present high levels of intramedullar apoptosis, whereas high-risk MDS show a decrease in apoptosis, an increase in cell proliferation and a high frequency of evolution to AML.2,3 The accumulation of genetic alterations is closely associated with the progression of MDS toward AML, and efforts are being made to determine the significance of various genetic aberrations in patients with MDS.4-6 The same occurs for liver adenomatosis,7 Rubinstein-taybi syndrome8 and hemochromatosis.9

The Phosphatidylinositol-3-kinase (PI3K) and Janus kinase 2 (JAK2) signaling pathways are involved in numerous cellular processes, such as proliferation, apoptosis and differentiation.10-12 Mutations in the catalytic subunit of PI3K are frequently observed in several cancers, including AML.4,5,13 Hotspot mutations occur in exon 9 (E542 and E545) and in exon 20 (H1047), resulting in increased PI3K/Akt activity.5,14-17 One somatic mutation in the JAK2 gene (V617F) has been identified in myeloproliferative disorders such as polycythemia vera (PV) and myelofibrosis.18

FMS-like tyrosine kinase 3 (FLT3) is a tyrosine kinase receptor that plays an important role in the proliferation and differentiation of hematopoietic progenitors.19 Nucleophosmin (NPM1) is a key regulator of hematopoesis that shuttles between the nucleus and cytoplasm. NPM1 mutations often result in the predominant localization of the protein to the cytoplasm, leading to destabilization of p14ARF and to the inhibition of p53.20 Internal tandem duplications (ITDs) in FLT3 and NPM1 mutations are frequent events in the development of AML, and are associated with prognosis. According to Gale et al,21 it is possible to identify 3 prognostic groups based in the presence or absence of FTL3 and NPM1 mutations: good (FLT3-ITD-NPM1+), intermediate (FLT3-ITD-NPM1- or FLT3-ITD+NPM1+), and poor prognosis (FLT3-ITD+NPM1-). Furthermore, a point mutation in exon 20 of the FLT3 gene (FLT3-D835) has been described in a case of AML.19

Mutations in PI3K, JAK2, FLT3 and NPM1 have been described in cases of MDS; however, additional studies are necessary to clarify their role in this disease. In this context, the objective of this work was to investigate the occurrence of the hotspot mutations E542, E545 and H1047 in PI3K, V617F in JAK2, ITDs and D835 in FLT3 and exon 12 mutations in NPM1 in MDS patients in a Brazilian population.




DNA samples were obtained from bone marrow aspirates of 51 patients diagnosed with de novo MDS. According to the French-American-British (FAB) classification,22 the patients were classified as follows: 31 cases of refractory anemia (RA), 8 cases of refractory anemia with ringed sideroblasts (RARS), 7 cases of refractory anemia with excess blasts (RAEB), 3 cases of refractory anemia with excess blasts in transformation (RAEBt), and 2 cases of chronic myelomonocytic leukemia (CMML). Using the World Health Organization (WHO) 2008 classification guidelines,23 there were 3 cases of refractory cytopenia with unilineage dysplasia (RCUD), 23 cases of refractory cytopenia with multilineage dysplasia (RCMD), 8 cases of refractory anemia with ring sideroblasts (RARS), 3 cases of MDS associated with isolated del(5q) (MDS-5q), 7 cases of refractory anemia with excess blast-1 (RAEB-1), 3 cases of refractory anemia with excess blast-2 (RAEB-2) and 4 cases of AML with multilineage dysplasia. Samples were obtained at the time of diagnosis, and none of the patients had received any cytotoxic drugs or growth factors for MDS treatment. Patient characteristics are shown in Table 1. Additionally, among the 51 patients evaluated at the time of diagnosis, 5 patients presented disease progression and were screened for mutations after disease evolution. Patient characteristics at diagnosis and after disease progression are shown in Table 2. Samples were collected at the Hematology and Hemotherapy Center of the University of Campinas, Brazil. All patients who contributed to this study provided informed written consent, and the National Ethical Committee Board approved the study.



Nucleic acid isolation

Genomic DNA was extracted from mononuclear bone marrow cells with the GFXTM Genomic Blood DNA Purification Kit (Amersham Biosciences, Piscataway, USA), according to the manufacturer's instructions.

Detection of FLT3-ITD and NPM1 mutations

Identification of FLT3-ITD and NPM1 exon 12 mutations was performed using polymerase chain reaction (PCR) and analysis of fragment size. PCR was performed in a 50-μL reaction volume consisting of 100 ng of genomic DNA, 5 μL of 10X reaction buffer, 2 μL of 50 μM MgCl2, 2.5 units of Taq polymerase and 200 nM each of the forward and reverse primers (Table 3). The reaction conditions were set as follows: 5 minutes of denaturing at 94°C followed by 35 cycles of 20 seconds at 92°C, 30 seconds at 57°C and 45 seconds at 72 °C, with a final step at 72°C for 7 minutes. After dilution (1:20) in water, 1 μL of each PCR product was mixed with 9 μL of Hi-Di formamide (Applied Biosystems, Foster City, CA) and 0.5 μL of GeneScan 500-ROX size marker, and the mixture was denatured for 5 minutes at 95°C. Samples harboring the mutation were identified based on the areas under the curves representing the wild-type (FLT3:397 bp and NPM1:294 bp) and mutated alleles (FLT3-ITD.397 bp and NPM1.294 bp).



AML patients with FLT3-ITD or NPM1 mutations were used as positive controls.

Detection of the JAK2 V617F and FLT3-D835 mutations

Identification of JAK2 and FLT3 genotypes was performed using PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. PCR was performed in a 50-μL reaction volume consisting of 100 ng of genomic DNA, 5 μL of 10X reaction buffer, 2 μL of 50 mM MgCl2, 2.5 units of Taq polymerase and 200 nM each of the forward and reverse primers (Table 3). The reaction conditions were set as follows: 5 minutes of denaturing at 94°C followed by 35 cycles of 30 seconds at 92°C, 30 seconds at 57°C and 50 seconds at 72°C, with a final step at 72°C for 7 minutes. For RFLP analysis, JAK2 and FLT3 PCR products were digested with BsaXI or Eco321 (New England Biolabs, Hitchin, UK), respectively, according to the manufacturer's protocol, and visualized on a 2.5% agarose gel. The normal genotype for JAK2 was represented by a 460-bp fragment, and the heterozygous genotype was represented by 460-bp, 241-bp and 189-bp fragments, whereas the homozygous mutant genotype produced 241-bp and 189-bp fragments. For FLT3-D835, the normal genotype was represented by 68-bp and 46-bp fragments, and the heterozygous genotype was represented by 114-bp, 68-bp and 46-bp fragments, whereas the homo-zygous mutant genotype produced only a 114-bp fragment. AML or PV patients with FLT3-D835 and JAK2 V617F mutations, respectively, were used as positive controls.

Detection of the PI3K E542, E545 and H1047 mutations

Screening for PI3K mutations was performed by sequencing PCR products. PCR was performed in a 50-m L reaction volume consisting of 100 ng of genomic DNA, 5 mLof10X reaction buffer, 2 mL of 50 mM MgCl2, 2.5 units of Taq polymerase and 200 nM each of the forward and reverse primers (Table 3). The reaction conditions were set as follows: 5 minutes of denaturing at 94°C followed by 35 cycles of 30 seconds at 94°C, 50 seconds at 63°C and 55 seconds at 72°C, with a final step at 72°C for 7 minutes. Sequencing reactions were performed in both directions with the ABI PRISM BigDye terminator version 3.0 cycle sequencing kit, according to the manufacturer's instructions, using either one of the primers used for amplification (Table 3). After ethanol-sodium acetate precipitation, samples were analyzed on the ABI PRISM 3100 Genetic Analyzer.



PI3K mutation analysis

Samples from 51 MDS patients were screened for PI3K mutations; all 51 samples were screened at diagnosis, and 5 were screened again after disease progression. We examined exons 9 and 20, as a previous report has shown that over 75% of the PI3K mutations found in a large number of cancers are present in these exons.19 The sequencing of PCR products showed the absence of mutations in exons 9 and 20 of the PI3K gene in all MDS patients. PCR products and the sequences of exons 9 and 20 are presented in figure 1.

FLT3-ITD and NPM1 mutation analysis

Forty-six MDS patients were screened for FLT3-ITD and NPM1 exon 12 mutations at diagnosis, and 5 of these patients were also screened at the time of disease progression. AML patients with the FLT3-ITD or NPM1 mutations were used as positive controls. Analysis of DNA samples from the MDS patients showed that all samples included fragments of normal size, indicating the absence of mutations (figure 2).

JAK2 V617F and FLT3-D835 mutation analysis

Fifty-one MDS patients were screened for JAK2 V617F, and forty-seven were screened for FLT3-D835 at the time of diagnosis. Five patients were screen after disease progression.

One PV patient with JAK2 V617F and one AML patient with FLT3-D835 were used as positive controls. RFLP analysis showed the absence of JAK2 and FLT3-D835 mutations in all MDS patients at the time of diagnosis. Interestingly, we observed the presence of the JAK2 V617F mutation in one patient with CMML after disease progression (case 1; Table 2). Figures 3 and 4 represent the RFLP analysis of the JAK2 V617 and FLT3-D835 mutations, respectively.



Acute leukemia results from a combination of mutations and changes in protein function that lead to an increase in proliferation and defects in differentiation and apoptosis.24 Although FLT3 and NPM1 mutations have been described with great frequency in cases of AML,25,26 these mutations were not detected in the MDS patients included in this study. As the presence of these mutations was investigated at the time of diagnosis, screening during disease progression could be interesting. Pinheiro and colleagues27 have reported the acquisition of the FLT3-ITD mutation in 2 of 50 MDS patients included in their study one year after diagnosis. These patients later progressed toward AML, suggesting that the acquisition of this mutation may be related to leukemic transformation.

JAK2 mutations were not found in the MDS patients in this study at diagnosis. Interestingly, the JAK2 V617F mutation was identified in one CMML patient after disease progression. Initially, this patient presented with fewer than 5% bone marrow blasts and lacked the JAK2 V617F mutation. We observed the presence of the JAK2 V617F mutation during disease progression, with increased white blood cell (WBC) counts and bone marrow blasts (at diagnosis: 9000 WBC/L, 3% bone marrow blasts; at disease progression: 60000 WBC/ L, 10% bone marrow blasts). JAK2 mutations occur in 10% of CMML cases and are associated with clinical and morphological features.28 The JAK2 V617F mutation leads to constitutive activation of the JAK2/STAT3 pathway and aberrant signaling, resulting in growth factor independence, increased proliferation and differentiation failure.29 In light of the frequency of these events during MDS progression, our results suggest that the acquisition of JAK2 mutations may be involved in disease progression and should be investigated in more cases of MDS evolution. This finding is in agreement with other authors.30,31 A recent publication by Malcovati et al.31 reported 3 patients who evolved from RARS withnormal platelet counts and wild-type JAK2 to RARS-T with JAK2 mutation at the time of transformation.

Mutations in exons 9 and 20 of the PI3K gene are frequently described in cancer.4,5,13 However, we did not observe the presence of these mutations in the MDS patients included in this study. Constitutive activation of PI3K occurs in AML and high-risk MDS patients at diagnosis32-34, and mutations in exons 9 and 20 result in constitutive activation of this protein.5,14-17 The presence of PI3K mutations in AML5 justifies the evaluation of these mutations in a larger number of MDS patients, as they represent a possible factor involved in disease progression.

The presence or absence of these mutations has prognostic value in AML,21 and therefore, the investigation of similar mutations in other myeloid diseases such as MDS could be interesting in the context of developing targeted therapies. The PI3K/Akt pathway has already been targeted in acute leukemia, and specific PI3K inhibitors, such as LY294002, have been tested in vitro.35 Other members of the PI3K signaling pathway have also been investigated as targets for leukemia treatment. Clinical studies with rapamycin analogues, which inhibitor mTOR, are currently in phase II AML trials, alone or in combination with other chemotherapeutics.36 Furthermore, FLT3 inhibitors have shown therapeutic activity in AML patients with FLT3 mutations,37 and selective JAK2 inhibitors have been tested in patients with JAK2 mutations.38

In summary, our study has shown that mutations in the JAK2, FLT3, NPM1 and PI3K genes are not common in patients with MDS at diagnosis and that JAK2 mutations may occur in MDS during disease progression. Further studies may be helpful to understand the involvement of genetic changes and the impact of these mutations in MDS progression and in different subgroups of patients with the disease.



The authors would like to thank Raquel S. Foglio for English review. This work received financial support from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP).

Contributions: João Agostinho Machado-Neto contributed to the selection of patients, carried out all experiments and participated in the writing of the manuscript; Fabiola Traina contributed to the selection of patients, clinical follow-up of the patients, analysis of the results and the writing of the manuscript; Mariana Lazarini provided technical assistance with the experiments and participated in the writing of the manuscript; Paula de Melo Campos contributed to the selection ofpatients and clinical follow-up of the patients; Katia Borgia Barbosa Pagnano contributed to the clinical follow-up of the patients and with the techniques to detect the FLT3-ITD and NPM1 mutation; Irene Lorand-Metze was responsible for the morphological diagnosis of myelodysplastic syndrome in the patients included in this study; Fernando Ferreira Costa contributed to the analysis of the results; Sara T. Olalla Saad was the principal investigator.



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Received for publication on November 30, 2010; First review completed on January 4, 2011; Accepted for publication on February 11, 2011



E-mail: Tel.: 55 19 3289-1089



This article has received corrections asked by the editor on 2011 in agreement with the ERRATUM published in Volume 66 Number 7. (

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