ALBERT

Description: Background: Prophylactic transfusion of platelets provides a good protection for the implementation of invasive procedures and the prevention of bleeding events during chemotherapy and hematopoietic stem cell transplantation in patients who are suffering from hematological diseases. The issues that what is the optimal dose of platelet transfusion and how to monitor platelet transfusion efficiency are important due to the short storage time of platelet products, rising clinical demands, and decreasing donors. It is worthy that there are amount of studies have been conducted in the past to explore factors that may affect the clinical efficacy of platelet transfusion and characteristics of patients under these different transfusion effects. While, previous studies failed to exactly address the problems of clinical needs for platelet transfusion. Methods: The aim of our study is to develop a model to evaluate the efficacy of platelet transfusion and the statistic method of machine learning algorithm (ML) was involved. The differences between this algorithm and traditional methods are that the former one can continuously be learning from the data and form a self-training model, therefore ML is more accurate than traditional artificial models and generally independent of the model and parameters themselves. We further take the multi-layer fully connected layer neural network model (MLNN) into our consideration because it simulates the multi-layer interconnection of human nervous systems, which is suitable for processing inaccurate and fuzzy information. In our study, the establishment of a neural network model was used to make a multiple-dimension analysis between factors affecting platelet transfusion efficacy and platelet count added value correction index (aCCI), and explore the correlation among these influencing factors as well. Results: The study utilized the data relative to 1840 platelet transfusions performed in 460 patients with hematological diseases. The participants ranged in age from 16 to 92, and the median age was 59.5. There were 199 females and 261 men. The whole data was divided to 2 parts, 2/3 of them were analyzed as a training set and the others were used for validating. We selected 30 factors (including patient-related factors and transfusion product-related characteristics) that may affect the efficacy of platelet transfusion except the storage time of platelet (all data 〈 2 days), and established a model for predicting platelet transfusion efficacy based on the volume of platelet transfusion. After the model was established, it was tested for goodness of fit, and the results showed that the LOSS value tended to be stable. Conclusions: The establishment of this model may not only be used for predicting the platelet count after platelet transfusions in patients and the amount of platelets that need to be transfused, but also provide supports for the solution of related problems. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 4859 Objectives: Signal transducer and activator of transcription 5 (STAT5) protein is one of the concernful part of STATs families. The constitutive STATs activation has been especially showed in transformation by fusion genes in leukemias. Methods: In this study, we aimed to investigate whether the genetic polymorphisms in the STAT5 gene are associated with the treatment outcomes of Ara-C-based chemotherapy regimens in Acute Myeloid Leukemia (AML) patients. 152 AML patients in a Chinese sample were enrolled in our study. Peripheral blood samples were analyzed by matrix assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF-MS). Results: The results showed that the frequencies of the T/T genotype in rs2293157 and rs1135669 were higher in unfavorable and intermediate group respectively (P=0.023 and P=0.033). The frequency of patients with T/T genotype of rs1135669 was significantly higher in complete remission (CR) group. Conclusions: We concluded that the T/T genotype of rs1135669 might be an important marker for therapeutic efficiency of Ara-C-based chemotherapy in AML patients, especially in Chinese population. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4858 Objectives: In the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway signaling pathway, the STAT3 is one of the most prominent predictable factors for cancer and leukemia. STAT3 activation might promote cellular transformation and therefore have an important role in human tumors. This study was aimed to investigate the relationship between the STAT3 polymorphisms and the treatment responses of AML in the Chinese population. Methods: We tested three single nucleotide polymorphisms (SNPs) with 130 Acute Myeloid Leukemia (AML) patients. Genomic DNA was isolated from peripheral blood and assayed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF). Results: The results showed there were strong relationship between unfavorable cytogenetic, partial remission (even no remission) and the frequencies of GG genotype in rs9909659 (P=0.01 and 0.03) and the patients less than 45 years old was significant association with GA/AA genotype (P=0.01). But associations were not confirmed about event-free survival and leukemia-free survival in the Chinese population studied. Conclusions: It is clear that the AML patients with GG genotype in rs9909659 are not sensitive to standard chemotherapy method Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3969 Objective: Although many strategies have been explored to overcome the multidrug resistance (MDR) in leukemia which has rendered many currently available chemotherapeutic drugs ineffective, the results have been disappointing to the obstacle. The aim of this study was to investigate whether the new strategy of combining drug-loaded nanoparticles (Nps) and ultrasound (US) would show useful effects on the reversal of MDR in human leukemia cell line K562/A02. Methods: In this study, daunorubicin (DNR), a frequently chemotherapeutic agent known to cause DNA damage and induce apoptosis and cell death, was loaded on the TiO2 Nps which is chemically stable, environmental friendly, and shows weak or non cytotoxic to apply as the nano-drug carrier. The MDR leukemia K562/A02 cells were treated with the DNR-loaded TiO2 Nps drug carrier and US exposure. Then, we examined the effectiveness of delivering DNR into the MDR leukemia K562/A02 cells with the electrochemical studies, observed the bio-effects on the cell viability by MTT assays, investigated the induced apoptosis, and assessed the reversal ability and the mechanism of MDR by combining the drug-loaded Nps and US. Results: We observed good biocompatibility of the therapeutic approach. When the K562/A02 cells were incubated with DNR only, the cathodic current decreased by only 10% normalized to the DNR (10 μg/mL) standard, indicating less DNR was absorbed by the MDR cells. The cathodic current decreased by 39% and 63% in the presence of US or DNR-loaded TiO2 Nps, respectively. In comparison, when the cells were treated by the novel strategy of US mediated drug-loaded Nps crossing cell membranes, the cathodic current of DNR in the supernatant decreased greatly by 82% and became the minimal, which suggesting the least amount of DNR remained outside the MDR cells in this case and the largest uptake into the cells by this new strategy. These observations demonstrated that the remarkable synergistic effect of the novel strategy facilitated the accumulation of DNR in the MDR K562/A02 cells. In addition, our MTT assay illustrated comparative sensitization of the MDR K562/A02 cells under the treatment of US or drug-loaded Nps, but especially enhanced effect by combining drug-loaded Nps and US. The resisting fold of the MDR leukemia K562/A02 became obviously lower, decreasing from 58.71 to 16.69. The fresh evidence from caspase-3 immunocytochemistry demonstrated that the strategy could induce the apoptosis in the cells as well. Conclusion: It was therefore concluded that the strategy could have good reversal ability of MDR in tumor. These findings reveal that the reversal of MDR in tumor by US mediated drug-loaded Nps crossing cell membranes could represent promising approach in cancer therapy. Disclosures: No relevant conflicts of interest to declare.

Description: Background: MCM7 is one of the subunits of the MCM2-7 complex that are essential for DNA replication licensing and control of cell cycle progression. It also participates in mRNA transcription and DNA damage regulation. It is found that MCM7 was highly expressed in many cancer cells including Leukemia. Thus, lentivirus-mediated siRNA targeting MCM7 was used to suppress its endogenous expression in K562 cells and develop a novel therapeutic strategy for leukemia. Method: Lentivirus-mediated siRNA targeting MCM7 were designed and infected K562 cells. Down-regulation of MCM7 mRNA and protein expression in K562 cells were determined by Real-time PCR and Western blot analysis. The proliferation of the transfected K562 cells was assessed by CCK8 assay. Cell cycle progression and apoptosis were calculated by flow cytometry. The related protein levels of the transfected K562 cells were detected by Western blot. The stably transfected K562 cells were injected into nude mice. The transplanted tumors were measured and weighed, then Western blot and Immunocytochemistry were used to evaluated the expression of the proteins for mechanism. Results: The mRNA and protein expression of MCM7 in the stably transfected K562 cells decreased with the percentage of 92.3±1.88 and 91.18%±0.93 respectively. Compared with control and negative control groups, the proliferative rate of K562 cells was significantly reduced after LV-MCM7-RNAi infection. The cell cycle progression of K562 cells was blocked, with a massive increase in the percentage of cells in the G0/G1 phase and decrease in S phase. The apoptosis rate of the transfected K562 cells was significantly higher. PLK1 and pPLK1 protein were down-regulated in response to MCM7 inhibition; P53 and bax protein were up-regulated simultaneously. In vivo, the tumor size and weight dramatically decreased in the MCM7-RNAi group than in negative control group. PLK1, pPLK1 proteins in transplanted tumors were down- regulated and P53,bax were up-regulated correspondingly. Conclusions: Lentivirus-mediated MCM7 shRNA has effectively down regulated the expression of MCM7 gene on mRNA and protein levels. Moreover, the silence of MCM7 has led to the significant decrease in proliferation, blocking the cell cycle progression of K562 cells in vitro. Simultaneity, the apoptosis of the leukemia cell was enhanced. Both of the functions above cause the inhibition of the tumor growth in nude mice. Overall, these results suggest that the proliferation and cell cycle of K562 cells could be regulated by silencing MCM7 gene which is a promising gene therapeutic method to treat gastric cancer. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 4947 Objective: The aim of this study was to investigate the expression of survivin and the apoptosis induced by DNR and BrTet in the leukemic cells K562/A02. Methods: In a typical experiment, the K562/AO2 cells were treated with daunorubicin (DNR), 5-bromotetrandrine (BrTet), or DNR and BrTet for 48 hours, and the cells treated without any drugs were used as control group. Cell proliferation was analyzed by MTT assay. Cells apoptosis and the concentration of DNR within the cells were measured by Flow cytometry (FCM). The expressions of mRNA and protein of survivin were determined by semi-quantitative reverse transcription PCR (RT-PCR) and Western blot, respectively. Results: The results of MTT assay indicated that DNR and BrTet were both able to inhibit the proliferation of K562/AO2 cells in dose-dependent manner. The fresh evidence from flow cytometry showed that a higher apoptosis rate could be induced and a higer concentration of DNR could be detected in K562/AO2 cells by DNR and BrTet as compared with those by DNR or BrTet in the same concentrations(P

Description: Objective The aim of this study is to investigate and analysis the effect of ultrasound potentiate the cytotoxicity of adriamycin and reverse drug resistance on leukemia drug resistance K562/Adm cell line in vitro, to find out the mechanism of the reverse effect of ultrasound exposure. Methods Human leukemia adriamycin resistant strain K562/Adm as target cells, were treated by adriamycin singly in group Adm, by ultrasound exposure singly in group US and by adriamycin prior to ultrasound exposure in group Adm+US. The dosages that didn’t attribute to cell killing immediately were detected. Trypan blue dye exclusion test and MTT assay were used to determine the sensitivity of K562/Adm. Wright’s staining and transmission electron microscope were used to detect the apoptosis and structure change of K562/Adm cells. Flow cytometry was used to analysis intracellular drug concentration and electron microscopic scanning was used to observe the membrane change. Immunocytochemistrial method was used to evaluate the expressions of P-gp. Results At 0.17W•cm−2 and lower acoustic intensity, ultrasound didn’t result in K562/Adm acute cells destruction; and 0.5 W•cm−2 ultrasonic intention could make cell killed rapidly after K562/Adm cells were irritated by ultrasound exposure singly. Significant differences were obtained between ultrasound treated and untreated cells in the presence of various concentrations of adriamycin. If the same concentration of cytotoxic agents were used, more cells were killed if sonication was applied. ultrasound for 30s at 20kHz, 0.17W•cm−2 intensity almost could not damage K562/Adm cells but dramatically decrease adriamycin concentration which induce cell achieve IC50;There were some morphological alterations in cells irradiated by ultrasound, Nearly all the treated cells by ultrasound exhibited small holes with diameter about 1~2 μm in the K562/Adm cell surface; the intracellular adriamycin accumulation in group Adm+US were prompted compared with Adm group and controlled group. Many apoptotic phenomena were observed in Adm+US group, show many vesicle and the form of apoptotic body, but there were no change in US group compared with controlled group. And the expression of P-gp protein had no significant difference between before and after ultrasound eradiated. Conclusions Higher ultrasound intensity could make K562/Adm cells killed rapidly, and lower ultrasonic level could potentiate the cytotoxicity of adriamycin to K562/Adm cells and reverse drug resistance on K562/Adm cells. Ultrasonic cavitation and sonoporation are the main mechanisms of the synergism between adriamycin and low-level sonication; the ultrasonically induced increase in intracellular drug accumulation. The expression of P-gp had no change in US group compare with controlled group.

Description: Objective: To establish a method for quantitative analysis of hematopoietic chimerism by polymerase chain reaction (PCR) based on short tandem repeat (STR) locies. To investigate the correlation between the kinetics of chimerism and hematologic engraftment, graft rejection, disease relapse and graft versus host disease (GVHD) after allogeneic nonmyeloablative peripheral blood stem cell transplantation. To guide implementation of therapy at an early stage and to improve patients life quality. Method: Cell dilution experiments were performed by mixing mononuclear cells (MNCs) obtained from peripheral blood samples of unrelated individuals to test the sensitivity, accuracy and linearity of the assay. Quantitative assessment of hematopoietic chimerism was performed by short tandem repeat-polymerase chain reaction(STR-PCR), polyacrylamide gels, silver staining and analyzed by Image Analysis System. 28 patients received nonmyeloablative stem cell transplantation were evaluated. The conditioning regimen included fludarabine 30mg/(m2·d)×6d, busulphan 4mg/(kg·d)×2d, CTX 600mg·d−1×2d, ±Ara-C. Peripheral blood were collected before and after transplantation in different period and the chimerism were analysed by this method. Results: Sensitivity varied from 1.25% to 5% depending on the STR locies being tested. vWA and D16S539 appeared better sensitivity from 1.25% to 2.5% than other locies. The quantitative results showed a linear correlation between the percent chimerism calculated and the DC proportion mixed(R2 =0.97~0.98, P

Description: A functional polymer composed of PLGA, PLL and PEG was synthesized, which was used as carrier material for fabricating drug delivery system of nanoparticles. PLGA-PLL-PEG nanoparticles simultaneously loaded with daunorubicin (DNR) and tetrandrine (Tet) were prepared in order to inhibit MDR activity and enhance the antitumor activity of DNR. A modified double-emulsion solvent evaporation/diffusion method was used to increase the incorporation of DNR (hydrophilic) and Tet (hydrophobic) into PLGA-PLL-PEG nanoparticles (NPs). The influence of various processing parameters on particle size and drug loadings were investigated systematically, such as the molecular weight, such as the molecular weight and concentration of PLGA-PLL-PEG, volume ratio of acetone to dichloromethane, PVA concentration in the external aqueous phase, the volume ratio of internal aqueous phase to external aqueous phase and the surfactants of internal aqueous phase. The particle size of the nanoparticles produced by optimized formulation and preparation was 213.0±12 nm (n = 3) with low polydispersity index (0.075 ± 0.023, n = 3). Transmission electron microscopy (TEM) examination showed that the morphology of the prepared nanoparticles was spherical in shape with a smooth surface. The drug loadings were 3.63±0.15% for DNR and 4.27±0.13% for Tet (n = 3). The entrapment efficiencies were 70.23±1.91% for DNR and 86.5±0.7% for Tet (n = 3). The release of DNR and Tet were sustained over one week. The PLGA-PLL-PEG-NPs formulation was potentially useful for hydrophilic and hydrophobic drugs that require efficient delivery to cancer cells. Disclosures: No relevant conflicts of interest to declare.

Description: In multiple myeloma (MM), the hypoxic environment is an important factor causing tumor angiogenesis which is strongly correlated to disease progression and unfavorable outcome by activating the key transcription factor, hypoxia-inducible factor-1α (HIF-1α). Gambogic acid (GA) is the major active ingredient of gamboge, which has been shown to possess antitumor effect by in vitro and in vivo study. However, the underlying molecular mechanism that whether GA inhibits tumor angiogenesis remains not well understood. In this study, we investigated the effects of GA on expression of HIF-1α, and its downstream target gene vascular endothelial growth factor (VEGF) in human MM U266 cells. We found that hypoxia induced increase in the level of HIF-1α subunit protein and activated the PI3K/Akt/mTOR pathway. Moreover, the treatment with GA markedly decreased HIF-1α and VEGF expression under hypoxic condition. Mechanistic studies exhibited that GA inhibited the production of HIF-1α by reducing phosphorylation of Akt and mTOR in U266 cells. Furthermore, in vivo study revealed that intravenous injection of GA once every other day for two weeks could suppress tumor volumes by antiangiogenesis activity. Taken together, our results identify GA suppress hypoxia-activated pathways that linked to MM progression, at least partly, by the inhibition of PI3K/Akt/mTOR signaling pathway. Therefore, GA may be a new potent therapeutic agent against human MM cells. Disclosures No relevant conflicts of interest to declare.