Mutations in Patients with Myelodysplastic Syndromes Machado-Neto JA et al. Table 1 - Patient characteristics. MDS patients Age in years: median (range): 63 (26-90) Gender M/F FAB RA/RARS RAEB/RAEBt CMML WHO RCUD/RCMD/RARS/SMD-5q RAEB-1/RAEB-2 CMML1 AML with myelodysplasia-related changes MDS Unclassified Number of individuals 51 30/21 31/8 7/3 2 3/24/8/3 4/2 2 4 1 Abbreviations- FAB: French-American-British; RA, refractory anemia; RARS, refractory anemia with ringed sideroblasts; RAEB, refractory anemia with excess blasts; RAEBt, refractory anemia with excess blasts in transformation; CMML, chronic myelomonocytic leukemia; WHO, World Health Organization; RCUD, refractory cytopenia with unilineage dysplasia; RCMD, refractory cytopenia with multilineage dysplasia; SMD- 5q, MDS associated with isolated del(5q); RAEB-1, refractory anemia with excess blasts 1; RAEB-2, refractory anemia with excess blasts 2; CMML1, chronic myelomonocytic leukemia 1; AML, acute myeloid leukemia. 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- ITD2NPM1+), intermediate (FLT3-ITD2NPM12 or FLT3- ITD+NPM1+), and poor prognosis (FLT3-ITD+NPM12). 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. CLINICS 2011;66(5):793-799 MATERIALS AND METHODS Patients 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 transforma- tion (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 progres- sion and were screened for mutations after disease evolution. Patient characteristics at diagnosis and after disease progres- sion 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-mL reaction volume consisting of 100 ng of genomic DNA, 5 mL of 10X reaction buffer, 2 mL of 50 mM MgCl2, 2.5 units of Taq Abbreviations- MDS: myelodysplastic syndromes; FAB: French-American-British; RA, refractory anemia; RARS, refractory anemia with ringed sideroblasts; RAEB, refractory anemia with excess blasts; RAEBt, refractory anemia with excess blasts in transformation; CMML, chronic myelomonocytic leukemia; WHO, World Health Organization; RCMD, refractory cytopenia with multilineage dysplasia; RAEB-1, refractory anemia with excess blasts 1; RAEB-2, refractory anemia with excess blasts 2; CMML1, chronic myelomonocytic leukemia 1; CMML2, chronic myelomonocytic leukemia 2; AML, acute myeloid leukemia. CMML/CMML2 RAEB/RAEB-1 RAEBt/RAEB-2 LMA/LMA with myelodysplasia-related changes RAEBt/RAEB-2 794 10% 12.5% 10% 69% 20% Mutations after disease progression JAKV617F mutation None None None None MDS patient Case 1 Case 2 Case 3 Case 4 Case 5 CMML/CMML1 RA/RCMD RA/RCMD RARS/RARS RA/RCMD 3% 0% 3% 4% 1% Table 2 - Patient characteristics at diagnosis and after disease progression. Classification at diagnosis (FAB/WHO) Number of blasts at diagnosis Classification after disease progression (FAB/WHO) Number of blasts after disease progression