ALBERT

Description: Abstract 1871 Introduction: PI3K/AKT pathway is involved in cell growth, proliferation and apoptosis. A key downstream effector is the phosphorylated serine-threonine Akt (p-AKT). Constitutive activation of PI3K/AKT has been observed in solid tumours and leukemic cells. Inhibition of PI3K/AKT activity, results in apoptosis in cell lines (CL) after treatment with different compounds, e.g. deguelin, a natural product from the leguminous Mundulea sericea, with antitumour effects. Aims: To evaluate PI3K/AKT activation in MDS patients and its therapeutic potential in MDS. Methods: PI3K/AKT activation was evaluated by flow cytometry (FC) using an alexa-fluor 488-antibody Ser 473 p-AKT (Cell Signalling Technology). A triple immunostaining procedure using CD45-PerCP and CD34-PE was used for p-AKT expression in CD34+ primary samples. The p-AKT activity was determined using Kolmogorov-Smirnov test (D). CD34+ cells from healthy donors and Jurkat cells were used as negative and positive controls respectively. Apoptosis (determined by Annexin V and PI/7AAD) and cell cycle arrest (using RNAse and PI) were determined following treatments with LY294002 (50uM), and deguelin (100-500nM) in P-39 myeloid leukemia cell line, with constitutive PI3K/AKT activation. Apoptosis was determined in bone marrow mononuclear cells and CD34+ cells from MDS patients with the same treatments. To evaluate in vivo activity of deguelin, we used a xenotransplant model. Briefly, NODSCID mice were injected intrafemurally with P-39 CL and 12 days post transplant a three week-course of treatment, every other day, was started (deguelin 4mg/Kg, n=3 vs vehicle, n=3). Results: P-39 CL showed constitutive PI3K/AKT activation with levels significantly higher than in CD34+cells from controls (median±SD= 0.73. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3240 Introduction: Treatment of adult acute lymphoblastic leukemia (ALL) has shown only modest improvements over the last 2 decades, with overall survival of 15% to 40%. The mitogen-activated protein kinase (MAPK) signaling cascade and the phosphoinosytol-3 phosphate/AKT (PI3K/AKT) pathways are involved in proliferation and differentiation of hematopoietic cells. It has been reported that those pathways are frequently activated in solid tumors and acute myeloid leukemia. However, their role in adult ALL is still uncertain. Better understanding of such pathways is necessary for development of novel therapeutic strategies. Aims: To evaluate the phopho-ERK and phospho-AKT protein expression in ALL at diagnosis and to correlate with biological and clinical parameters. Material and Methods: Twenty eight patients (median age 33y, 14–69y) with ALL at diagnosis were studied. Bone marrow and/or peripheral blood mononuclear cells (PBMC) (10 fresh and 18 cryopreserved cells at diagnosis) were stained using phospho-ERK and phospho-AKT/alexafluor 488 monoclonal antibodies (Cell Signaling Technology, Beverly, MA) and their expression was evaluated by flow cytometry (FACScalibur cytometer and CELL Quest program - BDB, San Jose, CA). The monoclonal antibodies CD19, CD10, CD3, CD45, IgM, CD34, CD7, CD2 were used for leukemic cells characterization by four-colour staining. Healthy donor PBMC and Jurkat cell line were used as controls: normal T lymphocytes are negative for p-ERK and Jurkat cell line express p-ERK and p-AKT in low levels. Samples were analyzed for constitutive expression of p-ERK and p-AKT and also after cell activation by phorbol-myristate-acetate (PMA). The expression of these proteins was evaluated by Kolmogorov-Smirnov test using fluorescence ratio between control isotype and phospho-protein (D). p-ERK and p-AKT expression was also evaluated in fresh and frozen samples of the same patients (2 cases) and similar results were obtained. In addition, patients were evaluated for multidrug resistance (MDR) through p-glycoprotein (PGP) expression and Rhodamine (Rh) efflux test and minimal residual disease (MRD) detection at the end of induction by flow cytometry. Results: Twenty cases were B-ALL (EGIL B-I 3, B-II 9, B-III 8, B-IV 5) and 3 T-ALL. Median WBC count was 25.3×109/L (2.3-373×109/L). The expression of p-ERK and p-AKT varied and the median value of p-ERK expression was D = 0.16 (0.01-0.80) and p-AKT median D = 0.08 (0.00-0.63). Considering these values as cutoff there was no difference regarding the patients` age and WBC count at the diagnosis between the positive and negative groups. In regards to EGIL subtypes, p-ERK expression was higher in T-ALL [median 0.50 (0.18-0.54)] than in B-precursor ALL [median 0.14 (0.01-0.80)] (p=0.03). Conversely p-AKT expression was similar in all cases, although high levels were observed among BIII cases. The frequency of Rh efflux was 88% in pERK negative cases and 66% in positive group but there was no difference on PGP expression. On the contrary, PGP expression and Rh efflux were more frequently seen in p-AKT positive cases (88% and 100%, respectively) than p-AKT negative ones (63% and 60%). MRD analysis was performed in seven patients. Two cases presented detectable MRD (〉0.01%) and both were p-ERK positive and p-AKT negative. Interestingly all five MRD (-) cases were p-ERK negative and p-AKT positive. In addition, PMA test showed p-ERK activation of normal T lymphocytes and the expression was increased in 52% of ALL cases upon treatment. Conclusion: MAPK and PI3/AKT activation varied among ALL patients. MAPK pathway showed to be more activated in T-ALL than in precursor-B ALL. The functional analysis of these pathways can address the role in ALL pathogenesis. Both pathways may be potential therapeutic targets for novel therapies. (Support: FAPESP proc.09/51002-8). Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: Multiparameter flow cytometry immunophenotyping (FC) has shown the presence of phenotypic abnormalities in both CD34 precursors and maturing myeloid and erythorid cells in patients with myelodysplastic syndromes (MDS). However, detection of megakaryocytic dysplasia by FC still remains a challenge. Aims: To evaluate the potential utility of FC analysis of megakaryopoesis and platelet immunophenotype in MDS patients for the diagnosis and prognostic evaluation of the disease. Methods: A total of 54 peripheral blood (PB) samples corresponding to 39 patients with MDS and 15 controls (6 healthy subjects, 3 reactive cytopenias, 3 patients with May-Heglin syndrome and 3 myeloproliferative disorders) were collected in sodium citrate. In all cases, the median amount of expression of CD31, CD34, CD36, CD41a, CD41b, CD42a, CD42b, CD61, CD62P, CD63, PAC-1 and Fibrinogen receptor was evaluated on circulating platelets as refleted by the median fluorescence intensity (MFI). Morphologic megakaryocytic dysplasia was evaluated in parallel in bone marrow samples from the same MDS individuals. Results: Morphological megakaryocytic dysplasia was detected in 49% of MDS patients who showed a shorter median overall survival (50 ± 8 vs 107 ± 12 months; p = 0.04). The following FC abnormalities were detected in circulating PB platelets of MDS patients: increased FSC and SSC (36%), overexpression of CD36 (18%), CD31 (36%), CD41b (20%), CD42b (2.5%) and CD61 (20%) and underexpression of CD36 (10%) and CD61 (5%). One case of aberrant CD34 expression was detected. A total of 27 (69.2%) patients presented at least one of these abnormalities by FC. Eighty percent of patients with morphological dysplasia and 64% without showed immunophenotypic abnormalities. According to IPSS, 6 patients constituted a high risk group (HRG) (Int-2 and High) and 28 a low risk group (LRG) (Int-1 and Low). Platelet phenotypic aberrations were detected in 83% of the HRG and in 75% of the LRG. Immunophenotypic abnormalities were not detected in myeloproliferative disorders. All May-Heglin cases and 2 reactive cytopenias presented overexpression of at least one of the following: FSC, SSC, CD31, CD36, CD41a, CD41b, CD42b and CD61. Deficient expression of CD36, CD61 and aberrant expression of CD34 were unique to MDS. Conclusion: Our results show the presence of immunophenotypic abnormalities in PB platelets from MDS patients, the abnormally decreased reactivity for CD36 and CD61 together with aberrant CD34 expression being of potential diagnostic utility in MDS.