BackgroundCD56 expression has been associated with a poor prognosis in lymphoid neoplasms, including T-cell acute lymphoblastic leukemia (T-ALL). MicroRNAs (miRNAs) play an important role in lymphoid differentiation, and aberrant miRNA expression has been associated with treatment outcome in lymphoid malignancies. Here, we evaluated miRNA expression profiles in normal thymocytes, mature T-cells, and T-ALL samples with and without CD56 expression and correlated microRNA expression with treatment outcome.MethodsThe gene expression profile of 164 miRNAs were compared for T-ALL/CD56+ (n=12) and T-ALL/CD56- (n=36) patients by Real-Time Quantitative PCR. Based on this analysis, we decided to evaluate miR-221 and miR-374 expression in individual leukemic and normal samples.ResultsmiR-221 and miR-374 were expressed at significantly higher levels in T-ALL/CD56+ than in T-ALL/CD56- cells and in leukemic blasts compared with normal thymocytes and peripheral blood (PB) T-cells. Age at diagnosis (15 or less vs grater than 15 years; HR: 2.19, 95% CI: 0.98-4.85; P=0.05), miR-221 expression level (median value as cut off in leukemic samples; HR: 3.17, 95% CI: 1.45-6.92; P=0.004), and the expression of CD56 (CD56-vs CD56+; HR: 2.99, 95% CI: 1.37-6.51; P=0.006) were predictive factors for shorter overall survival; whereas, only CD56 expression (HR: 2.73, 95% CI: 1.03-7.18; P=0.041) was associated with a shorter disease-free survival rate.ConclusionsmiR-221 is highly expressed in T-ALL and its expression level may be associated with a poorer prognosis.

The vitamin E derivative (þ)a-tocopheryl succinate (a-TOS) exerts pro-apoptotic effects in a wide range of tumors and is well tolerated by normal tissues. Previous studies point to a mitochondrial involvement in the action mechanism; however, the early steps have not been fully elucidated. In a model of acute promyelocytic leukemia (APL) derived from hCG-PML-RARa transgenic mice, we demonstrated that a-TOS is as effective as arsenic trioxide or all-trans retinoic acid, the current gold standards of therapy. We also demonstrated that a-TOS induces an early dissipation of the mitochondrial membrane potential in APL cells and studies with isolated mitochondria revealed that this action may result from the inhibition of mitochondrial respiratory chain complex I. Moreover, a-TOS promoted accumulation of reactive oxygen species hours before mitochondrial cytochrome c release and caspases activation. Therefore, an in vivo antileukemic action and a novel mitochondrial target were revealed for a-TOS, as well as mitochondrial respiratory complex I was highlighted as potential target for anticancer therapy.

10-(Octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC) is an alkylphospholipid that can interact with cell membranes because of its amphiphilic character. We describe here the interaction of ODPC with liposomes and its toxicity to leukemic cells with an ED-50 of 5.4, 5.6 and 2.9 microM for 72 h of treatment for inhibition of proliferation of NB4, U937 and K562 cell lines, respectively, and lack of toxicity to normal hematopoietic progenitor cells at concentrations up to 25 microM. The ED-50 for the non-malignant HEK-293 and primary human umbilical vein endothelial cells (HUVEC) was 63.4 and 60.7 microM, respectively. The critical micellar concentration (CMC) of ODPC was 200 microM. Dynamic light scattering indicated that dipalmitoylphosphatidylcholine (DPPC) liposome size was affected only above the CMC of ODPC. Differential calorimetric scanning (DCS) of liposomes indicated a critical transition temperature (T(c)) of 41.5 degrees C and an enthalpy (H) variation of 7.3 kcal mol(-1). The presence of 25 microM ODPC decreased T(c) and H to 39.3 degrees C and 4.7 kcal mol(-1), respectively. ODPC at 250 microM destabilized the liposomes (36.3 degrees C, 0.46 kcal mol(-1)). Kinetics of 5(6)-carboxyfluorescein (CF) leakage from different liposome systems indicated that the rate and extent of CF release depended on liposome composition and ODPC concentration and that above the CMC it was instantaneous. Overall, the data indicate that ODPC acts on in vitro membrane systems and leukemia cell lines at concentrations below its CMC, suggesting that it does not act as a detergent and that this effect is dependent on membrane composition.

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3065 Poster Board III-2 Deregulations of miRNA expression and function in B-cell acute lymphoblastic leukemia (B-ALL) have been associated with specific recurrent citogenetic abnormalities and clinical outcomes. In contrast, there is few data about miRNAs in T-cell acute lymphoblastic leukemia (T-ALL). We have determined the miRNA expression profile of 48 T-ALL patients' blasts and compared with normal mature T cells. We used the Taqman MicroRNA Assay Human Panel to screen 164 known mature miRNA sequences. Normal CD3+ cells were isolated from peripheral blood of four healthy subjects by immunomagnetic labeling. Total RNA was pooled and reverse transcribed with specific looped RT primers, and expression was evaluated by quantitative real-time PCR (RQ-PCR). Reactions were performed in duplicates and samples with a coefficient of variation greater than 5% were excluded. Furthermore, we considered as differentially expressed those miRNAs with fold change values higher than 10 or lower than 0.1. With this strategy we identified four miRNAs that were hyper-expressed (miR-181a, miR-181b, miR-213 and miR-29b) and three hypo-expressed (miR-150, miR-95, miR-338) in the leukemic pool. In order to confirm our findings, we then performed the analysis of miR-181a, miR-181b and miR-29b expression on 52 individual samples (48 T-ALL and 4 normal T cell samples) using RQ-PCR. Forty-five (93.7%) and 46 (95.8%) of the T-ALL samples presented expression levels of miR-29b and of miRs 181a/181b higher than the maximum detected in the normal samples. The analysis of the predicted targets for these three miRNAs was performed using miRNApath. MAPK signaling was the pathway with the highest number of target genes with 60 genes, of which MAP4K4, FOS, RAP1B, AKT3 and NLK were commonly targeted by all three miRNAs. As deregulation of the MAPK pathway in T-ALL has been previously described, we hypothesized that the hyper-expression of miR-29b, miR-181a and miR181b may be associated with this aberrant MAPK signaling. Disclosures No relevant conflicts of interest to declare.

1439 T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy of immature T cells that accounts about 15% of pediatric and 25% of adult ALL cases. In the last years, several clinical and laboratory features have been described as prognostic markers; nevertheless, with intensification of therapy most of them have lost their predictive value. MicroRNA (miRNA) expression analysis has proved to be an useful tool for identifying specific subsets of cancer patients with relevant cytogenetic, laboratorial and clinical features. The aim of the present study was to determine if miRNAs may be useful markers in T-ALL. First, we performed a supervised analysis comparing the miRNA expression profile of T-ALL blasts from 36 T-ALL/CD56− and 12 T-ALL/CD56+. We selected CD56 as prognostic marker based on our previous report showing that the disease-free survival (DFS) of T-ALL/CD56+ patients was of 28.5 months compared to 69.8 in the CD56− group. Also patients tended to be older and to present normal platelet counts in the T-LLA/CD56+ group. We used the Taqman MicroRNA Assay Human Panel (Applied Biosystems) to perform a screening of 164 knowledge mature miRNA sequences using specific primers and probes according to manufacturer instructions. Total RNA input was normalized based on the geometric means of Ct values obtained from four endogenous RNAs. All reactions were run in duplicate and a coefficient of variation greater than 5% was used as an exclusion factor (seven miRNAs were excluded). The fold change was calculated using comparative 2−δCt method. We have identified a set of 14 miRNAs differentially expressed, of which miR-374 and miR-221 best distinguished T-ALL/CD56+ from T-ALL/CD56− blasts. Based on this profile, we selected miR-221 and miR-374 as potential markers and quantified their expression in the same samples using RQ-PCR. Patients were stratified as high and low expression using the median value as cut off. We detected a significant association between the miR-221 high expression and poorer treatment outcome. On the contrary, miR-374 expression levels were not associated with treatment outcome. We evaluate the impact of age, white blood cell counts, CD56 and miR221 expression on overall survival (OS). Age and miR-221 were the only ones found to be significant. The estimate 5-year OS (mean and confidence interval 95%) was of 67.0 ± 10.3% in the group of patients expressing miR-221 below the cut-off value, whereas this value was of 28.5 ± 14.5% in the alternative group. Even among T-ALL/CD56− patients, the higher expression of miR-221 was significantly associated with poorer outcome. Our data suggest that miR-221 play an important role in T-ALL and its regulation may represent a potential therapeutic intervention. Disclosures: No relevant conflicts of interest to declare.

4802 Caffeic acid phenethyl ester (CAPE) is an active phenolic compound present in propolis obtained from honeybee hives. It is reported to present a spectrum of biological activities including antioxidant, anti-inflammatory and antitumoral. The antitumoral activity of CAPE as evaluated by several studies in vitro and in vivo seems to be related to distinct effects like inhibition of angiogenesis, invasion and metastasis and induction of apoptosis or differentiation of cancer cells. In the scenario of AML the demonstration of CAPE-induced apoptosis or cellular differentiation is restricted to the HL-60 cell line. Our aim was to evaluate the effects of CAPE treatment on primary AML samples as well as APL cell lines NB4 and NB4-R2 (a cell resistant to ATRA-induced differentiation) and on AML cell line Kasumi-1 (representative of core binding factor leukemia with AML1-ETO rearrangement). Proliferation and viability was evaluated by cell count with tripan blue in Neubauer chamber at fixed time intervals. Differentiation was evaluated by flow cytometer determination of CD11b expression. Apoptotic cells were defined as sub-G0 fraction and were evaluated by flow cytometer determination of propidium iodide- DNA fluorescence. Also apoptosis was detected by the annexin-V method. Leishman stained cytospins were used to confirm apoptosis or differentiation. CAPE did not induce differentiation in the cell lines NB4, NB4-R2 or Kasumi-1 and did not alter the differentiation induced by ATRA in NB4 cells. CAPE inhibited the proliferation of AML cell lines in a time and dose dependent fashion. The ED50 in 24h treatment for NB4 cell line (tripan blue) was 32.1 mcg/ml. ED50 (at 24h) for induction of apoptosis in the more sensitive assay using annexin-V in NB4 cells after 24h was 7.5mcg/ml and for Kasumi-1 was 10.2mcg/ml. CAPE (32 mcg/ml) significantly induced apoptosis after 24h in cells from AML patients (n=10), mean (IC95%) of 40.5% (29.26 – 51.76) versus control treated cells 18.16% (12.27 – 24.05); p=0.0004 In order to evaluate the mechanisms of CAPE-induced apoptosis in NB4 cells we performed a microarray analysis after 12 hours treatment (32mcg/ml). The majority of downregulated genes fall into two categories: positive cell cycle regulators and ribosomal genesis / protein traduction. In the other hand, upregulated genes fall into several categories, we point out chemokines and G- protein signalization genes. (Table 1 and 2) The role of IL-8 and Gro chemokines, that signaling by G-protein coupled receptors, has been determined in tumor progression and invasiveness. We are currently investigating the possibility that CAPE exerts an inhibitory effect in chemokine signaling in APL. In conclusion, CAPE-induced apoptosis in AML is associated with the regulation of specific genes. These properties are interesting and need further investigation. Disclosures: No relevant conflicts of interest to declare.