ALBERT

Description: OBJECTIVE: Myelodysplastic syndromes (MDS) are a heterogeneous group with the expansion of a malignant clone. No satisfactory treatment for MDS is available. Sodium valproate (VPA), can induce G0–G1 phase arrest and cell apoptosis, inhibit proliferation of tumor cells in vitro significantly. The effects and possible mechanism of VPA on MUTZ-1 cell line of MDS were studied in this experiment. METHODS: Cell proliferation was determined by MTT assay. Apoptotic morphological features were observed under microscope and transmission electronmicroscope. Cell apoptosis and cell cycle were analyzed by flow cytometry (FCM). The expression of p21WAF1(cyclin-dependent kinase inbihitor) was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. RESULTS: VPA could inhibit the proliferation of MUTZ-1 cells in dose-and time-dependent manners. The typical apoptotic morphological features appeared in cells treated with 4 mmol/L VPA for 72 hours. Condensation of cells and nuclear chromatin, disintegration of nuclear chromatin, and apoptotic body could be observed under microscope. Aggregation and margination of apoptotic nuclear chromatin, cytoplasm condensation, and irregular chromatin masses could be observed under transmission electronmicroscope. The percentage of apoptotic cells which were treated with 1,2 and 4 mmol/L VPA for 72 hours increased from 1.39% to 2.18%, 16.03%, 22.02%, and cell cycle arrest at G0–G1 phase could be caused by VPA as shown by FCM. The expression level of p21WAF1 mRNA and p21WAF1 protein were up-regulated in a dose dependent manner in MUTZ-1 treated with VPA for 72 hours (P

Description: Objective: To prepare functionalized Fe3O4-magnetic nanoparticles(Fe3O4-MNPs) loaded with adriamycin(ADM) or Fe3O4-MNPs co-polymerized with ADM and tetrandrine(Tet) to investigate whether the temperature, time or ratio of drug to nanoparticles influences the polymerization. To study the reversal role that the drug-loaded nano-composites play in K562 and one of its resistant cell line K562/A02 and to learn the reversal mechanism of this kind of treatments so as to provide theoretic evidence for the clinical application of them as resistance modifying agents. Method: The drug-loaded nanoparticles were prepared using mechanical absorption polymerization process in different condition of 4° or 37° for 24h or 48h. To investigate whether Fe3O4-MNPs loaded with ADM and Tet would play a synergetic reverse role in multidrug resistant cell, the drug-loaded nanoparticles by mechanical absorption polymerization were prepared to act with K562 and one of its resistant cell line K562/A02. The survival of cells which were cultured with drug-loaded nano-composites for 48h was observed through MTT assay, the growth inhibition efficacy of cells was calculated then. Using cells under the same condition described before, we took use of fluorescence microscope to measure fluorescence intensity of intracellular ADM at a wavelength of 488nm. P-glycoprotein (P-gp) was analyzed with flow cytometer. The expression of mdr1 mRNA was measured by RT-PCR. Results: The results showed that the growth inhibition efficacy of both the two cells increased with augmenting concentration of Fe3O4-MNPs which were loaded with drugs. The condition of 4° and 48h was significantly better than that of 37° and 24h respectively. Both Fe3O4-MNPs loaded with ADM or Fe3O4-MNPs co-polymerized with ADM and Tet elevated the intracellular ADM accumulation in K562/A02. However, no linear correlation was found between fluorescence intensity of intracellular adriamycin and augmenting concentration of Fe3O4-MNPs in K562/A02. Tet could downregulate the level of mdr-1 gene and decrease the expression of P-gp. Furthermore, Tet polymerized with Fe3O4-MNPs reinforced this downregulation, causing a 100-fold more decrease in mdr1 mRNA level, but did not reduce total P-gp content. Conclusions: Fe3O4-MNPs can load ADM or both ADM and Tet by mechanical absorption polymerization, which depends on proper temperature and time. Furthermore, the drug-loaded nano-composites have the ability in multidrug resistance reversal. Fe3O4-MNPs loaded with ADM or Tet can enhance the effective accumulation of the drugs in K562/A02 and Fe3O4-MNPs loaded with Tet obviously reverse multidrug resistance by reinforcing mdr1 gene downregulation. Functionalized Fe3O4-MNPs loaded with Tet probably have synergetic effect on reversal in multidrug resistance.

Description: Objective To analysis the risk factors for affected patients the long-term persistent survival in 96 patients undergoing Allogeneic Hematopoietic Stem Cell Transplantation, in order to take active prevention and cure measure, and improve trans- plantation curative effect preferably. Methods We retrospectively analysis 96 cases of patients subject to allo-HSCT. The 11 clinical parameters were selected for univariat -e analysis using a Cox regression:age, sex, morbid state, HLA locus, donor type, donor -recipient blood type, conditioning regimen, aGVHD, HC, VOD, IP. Factors that were significant at the 0.1 level on univariate analysis were evaluated by multivariate an- alysis using a Cox regression. The cumulative incidence of aGVHD and patients survival rate were calculated by the method of Kaplan and Meier. Results ninty-five patients achieved sustained donor engraftment except one patients not. The median time of leukocyte engraftment was 13 days. The incidence rates of grades I~IV acute GVHD was 43.75%. grades III~‡WaGVHD was 12.50%. Ten patients relapsed and 38 dead, the overall survival at 5 years was 60.42%. The COX method analysis showed that aGVHD and diease states at transplant were significantly risk factors to improved overall survivals(OS), with relative risk [RR] 2.996 and 2.619, respectively. Conclusion aGVHD and diease states at transplant had significantly improved OS, the key to improvement the outcome 0f allo-HSCT is to reduce the incidence and severity of aGVHD, meanwhile selecting the suitable opportunity for transplantation. We had better do HSCT in CR1to treat that patients with advance refractory and recurrence.

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: Object: The study was aimed to analyze the incidence, risk factors and outcome of invasive fungal infection (IFI) in allogeneic hematopoietic cell transplantation (HSCT) recipients, and to provide evidence for the prevention and treatment of IFI. Method: 79 patients undergoing formed HSCT at this institute from Jan 2005 to Sep 2014 with intact datum, were analyzed retrospectively according to the diagnostic criteria of IFI. The data of age, sex, primary disease, the source of stem cell, HLA-identical situation, ABO matching, conditioning regimen, use of ATG, season, aGVHD, neutropenia less than 0.5×109/L, CMV infection, etiology culture, blood routine test, imaging, relapse of disease and survival were recorded. Moreover, the data of patients with IFI including usage of glucocorticoid and the neutrophil count were recorded when IFI occurred and the blood cells count after IFI. Result: 17 patients has been diagnosed with IFI, in which 13 cases were probable and 4 cases were possible. The infection rate was 21.5%. There were 8 female and 9 male. The median age was 26 years (6-50 years).The total non relapse survival (NRS) rate was 55.6%, The NRS rate in non-infection group was 61.2% and that in infection group was 35.2%. The single-factor analysis showed matching(x2=15.061, p=0.010) and aGVHD (x2=6.983 p=0.008) were risk factors. After IFI, 8 patients died, 2 patients got complete response, 5 patients got partial response and 2 patients got stable response. The death rate was 47%, there was significant difference between two groups. The dose of glucocorticoid before IFI within 30 days was risk factor. Multiple-factor analysis further showed Ⅱ-Ⅳ degree of aGVHD (OR=4.810 p=0.012 CI:1.406-16.459 ) and matching (OR=25 p=0.007 CI:2.380-262.653) were risk factors. Moreover, before IFI, use of methylprednisolone more than 360 mg before infection (OR=0.041 P=0.017 CI:0.003-0.56) was risk factor. Conclusion: Patients with poor matching, unrelated stem cell and Ⅱ-Ⅳ degree of aGVHD are more likely to occur IFI and those who were used more glucocorticoid are likely to die. Therefore, avoiding the risk factors might reduce the incidence rate of IFI and respect using dose of immunosupperssor before infection within 30 days may improve the patients' prognosis. 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: 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