ALBERT

Beschreibung: Abstract 5114 The hypoxia-inducible factor (HIF) is a transcriptional factor with important roles in tumor biology, which activates transcription of genes encoding glucose transporters, glycolytic enzymes, and vascular endothelial growth factor. The VHL (von Hippel-Lindau) gene normally regulates ubiquitin mediated proteolysis of HIF-1a. VHL inactivation blocks HIF-1 proteolysis, resulting in increased HIF-1 expression. In the previous study, the expression of HIF-1a was detected in children ALL and NHL. To clarify the possible involvement of the HIF-1a and VHL gene in the development and progression of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), we analyzed the mutation and methylation of the VHL gene in 98 patients (54 AML and 44 MDS). The mutations in VHL gene was examined by direct exons sequence and the methylation status of the promoter region was determined using methylation-specific polymerase chain reaction (MSP). In addition, we examined the expression of HIF-1a in 44 of these patients (25 AML and 18 MDS) through the immunoblotting. VHL mutations were identified only in one patient with AML. However, no mutation was detected in the coding region of the VHL gene in MDS. Meanwhile the methylation of VHL gene was not found in the AML and MDS. But the expression of HIF-1a was found in 10 (66.7%) of 25 patients with AML and 5 (38.5%) of 18 patients with MDS. There is no significant correlation between the expression of HIF-1a and the VHL gene. Our primary date support that the HIF-1a may be involved in the development and progression of AML and MDS, but is not associated with the VHL gene. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Abstract 1515 Background: Decitabine is a prototype for epigenetic therapy in cancer which targets DNA methyltransferase. While it was known that decitabine monotherapy has been associated with a relative low rate of complete remission rates in acute myeloid leukemia and myelodysplastic syndrome. Several groups have attempted to increase the response rate with decitabine-based therapy by developing combinations. In the preliminary experiments, we investigated the effect of anti-leukemia drugs (idarubicin, daunorubicin, cytarabine, thalidomide and homoharringtonine) in combination simultaneously or sequentially with decitabine of inhibiting proliferation of acute myeloid leukemia cell lines. The result showed that sequential combination of decitabine and idarubicin induced a significant synergistic effect in cell apoptosis, inhibition of cell proliferation. With the gene chip technology, we found that the gene expression of Wnt pathway changed obviously after the treatment of DAC combined with IDA. Further we confirm that sequential combination of DAC and IDA caused depression of wnt pathway in vitro. In this study, we evaluate the anti-leukemic effect and the combination mechanism of DAC in sequential combination with IDA in vivo. Methods: The AML cell line U937 (14×107 cells per animal) were injected subcutaneously into the right flank of 6 to 8-week old NOD-SCID mice. After tumor growth, decitabine(0.5mg/kg/day) were injected intraperitoneal of a five consecutive days followed by a three days of idarubicin(0.5 mg/kg/day). The tumor growth inhibition effect was evaluated by microPET to reveal the synergistic effect of decitabine and idarubicin. Using TUNEL method and electron microscopy, we detected the apoptosis of leukemic cells from tumor bearing mice. The expression of β-catenin which is the key gene of Wnt/β-catenin pathway was also examined by immunochemistry. We then detected the expression of protein levels of the other genes(cyclinD1, c-myc, SFRP1,HDPR1 and DKK3) of Wnt/β-catenin pathway in tumor cells of tumor-bearing mice by western blot. Results: In vivo, the effect of sequential combination of decitabine and idarubicin was initially examined in a subcutaneous AML mouse model. The AML cell line U937(1×107 cell per animal) was transplanted subcutaneously into the right flank of 2-week-old female NOD-SCID mouse. The cell line developed into a rapidly growing tumor. Treatment was initiated in 7th days after injection of the leukemic cells, included with 5 days of decitabine alone, 3 days of idarubicin alone and 5 days of decitabine followed by 3 days of idarubicin. A significant inhibition of tumor growth was demonstrated with microPET in animals treated with sequential combination of decitabine and idarubicin compared with the other treatment and control group animals. The apoptosis of tumor cells was also significantly increased in the sequential combination treatment group detected by TUNEL and electron microscope. We also found that the sequential treatment group induced synergistic effect in re-expression or up-expression of the Wnt inhibitor genes (SFRP1, HDPR1 and DKK3) in tumor cells. Furthermore, the down-steam genes of wnt pathway including β-catenin, c-myc and cyclinD1 were down-regulated which suggest depression of wnt/β-catenin pathway. These results suggest that sequential combination of decitabine and idarubicin has a powerful anti-leukemic effect not only in vitro but also in vivo. Conclusion: Our results demonstrate decitabine in sequential combination with idarubicin in mouse model of AML and suggest that this combination may be of clinical value in the treatment of patients with AML. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Background: DNA methyltransferase inhibitors sensitize leukemia cells to chemotherapeutics. Previous studies have indicated decitabine sequentially combined with idarubicin was an effective treatment for myeloid neoplasms. We therefore conducted a phase 2 study of combination of decitabine followed by low-dose idarubicin plus cytarabine in acute myeloid leukemia (AML) evolving from myelodysplastic syndrome (MDS) and higher risk MDS. Methods: The study was a single-arm, prospective, multicenter study. Patients with AML evolving from MDS and refractory anemia with excess blasts (RAEB-2) high-risk MDS based on the 2008 WHO classification were included. The treatment plan consisted of decitabine 20 mg/m2 daily for 3 consecutive days on days 1-3, followed by idarubicin 6mg/m2 for 3 consecutive days on days 4-6, and cytarabine 25mg/m2 every 12 hours for 5 days on days 4-8. Granulocyte colony stimulating factor ( G-CSF) was given from day 4 and discontinued when neutrophil count increased to 1.0 × 109/L. In case remission was achieved after the first course, a similar second remission-induction course was given, to be followed by either an allogeneic hematopoietic stem cell transplantation (allo-HSCT) or subsequent consolidation courses, which was at the investigator's discretion. Results: A total of 71 patients were enrolled and treated, among whom 44 (62.0%) had AML evolving from MDS, 55 (77.5%) were previously untreated, 12 (16.9%) had an adverse karyotype, and 6 (10.0%) had TP53 mutations. 40 (56.3%) patients achieved a major response, including 20 (28.2%) patients with complete remission (CR) and 20 (28.2%) patients with complete remission with incomplete blood count recovery (CRi). The major response (CR+CRi) rate was 63.6% in AML patients and 44.4% in MDS patients (p=0.142). The median overall survival (OS) was 22.4 months (95% confidence interval (CI) ,17.8-27.0). The median OS was 24.2 months (95% CI 18.0-30.4) and 20.0 months (95% CI 11.2-28.7) for patients with AML and MDS (p=0.347), respectively. The median progress free survival (PFS) for the entire cohort was 12.1 months (95% CI 3.9-20.4). The median PFS was 17.6 months ( 95% CI 13.3-22.0) and 6.8 months (95% CI 0.1-13.6) for patients with AML and MDS (p=0.151), respectively. Patients who achieved CR/CRi had superior OS: 26.4 months ( 95% CI 17.6-35.2) vs. 20.0 months (95% CI 6.2-33.8) in non CR/CRi patients (p=0.042). Also patients who achieved CR/CRi had prolonged PFS: 17.8 months (95% CI 8.6-27.0) vs. 8.6 months (95% CI 3.5-13.8) in those not achieving CR. Patients with platelet doubling achieved significantly higher CR/CRi rate (24/24(100%) vs. 16/47(34.0%), p50×109/L of 25 days (range: 8-41). Conclusions: The regimen of decitabine priming followed by low-dose idarubicin plus cytarabine appears to be highly effective and has an acceptable safety profile in patients with AML evolving from MDS or RAEB-2. Further controlled studies are needed to confirm the activity of this regimen, and could help to provide an alternative option for induction therapy in this population. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Abstract 3628 PURPOSE: In the preliminary experiment, we investigated the effect of anti-leukemia drugs (idarubicin, daunorubicin cytarabine, thalidomide, homoharringtonine) in combination with decitabine of inhibiting growth of myeloid leukemic cell lines. The result showed notable synergistic effect of sequential combination of decitabine and idarubicin. In searching the associated mechanism, we assessed the effect of sequential combination of decitabine and idarubicin in apoptosis, cell cycle arrest, and depression of wnt pathway. Methods: Myeloid leukemia cells (U937, HEL and SKM-1) were treated with decitabine alone, idarubicin alone, and decitabine sequential with idarubicin to determine their impact on cellular proliferation, cell cycle regulation, apoptosis and the regulation of wnt pathway by detecting DNA methylation status, mRNA level and protein expression. Results: The sequential combination of decitabine and idarubicin produced synergistic inhibition of myeloid leukemia cells growth by apoptosis and cell cycle arrest. Sequential treatment with both agents induced synergistic effect on decreasing methylation status of wnt antagonists (SFRP1, HDPR1 and DKK3) which result in re-expression or up-expression of these genes. Furthermore, we found that the down-steam genes of wnt pathway including c-myc and β-catenin were down-regulated. Conclusion: Taken together, these findings showed that decitabine sequentially combined with idarubicin has synergistic anti-leukemia effect, which suggest clinical potential in the treatment of myeloid leukemias. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Abstract 5048 Background and Objectives: Myelodysplastic syndrome (MDS) is one of the most threatening hematological malignancies. Recently, the epigenetic changes have been recognized in the MDS, and some research found that the aberrant DNA methylation had close relationship with the MDS. Meanwhile, some studies showed that the activation of the Wnt signaling pathway and abnormal methylation of the Wnt antagonists had close relationship with hematological malignancies, such as AML AALL Aand CLL, but less is known in MDS. In our research, we studied the methylation status of Wnt antagonists (DKK1, DKK3, HDPR1, WIF-1, SFRP1 and SFRP4) in the patients with MDS and evaluated the role of them in the pathogenesis and progression of MDS, with providing a new theory support for clarifying the complicated pathogenesis and progression of MDS. Methods: The methylation status of Wnt antagonists (DKK1, DKK3, HDPR1, WIF-1, SFRP1 and SFRP4) in pretreatment bone marrow samples from 53 patients with MDS was measured by methylation-specific polymerase chain reaction (MSP). On the other hand, we collected the clinical materials of the patients with MDS and follow up the patients. Then the correlation between methylation and clinical features as well as prognosis of MDS patients was analyzed byχ2 test and Kaplan-Meier method. Results: In 53 bone marrow samples, the methylation frequencies of the Wnt antagonists were as follows: SFRP4 for 62.3 % (33/53), DKK1 for 45.3 % (24/53), HDPR1 for 34.0 % (18/53), SFRP1 for 15.1 % (8/53), DKK3 for 9.4 % (5/53), and WIF-1 for 5.7 % (3/53). After analyzing individual tumor suppressor gene, clinical parameters and prognostic information, it was found that the patients with the percentage of BM blast above 5% had higher methylation frequency of HDPR1 than the patients with the percentage of BM blast bellow 5% (44.4%∼a13.3%, P=0.034); besides, the methylation frequency of HDPR1 exhibited significant differences in the MDS subtypes (P=0.019), and was significantly correlated with the WPSS (P=0.037); In addition, Kaplan-Meier survival curve indicated that the mean overall survival of patients with methylation was significantly shorter than that of patients without HDPR1 methylation (457.3 days vs. 919.6 days, P=0.037) (Fig.1). Conclusion: The methylation of the Wnt antagonists (DKK1, DKK3, HDPR1, WIF-1, SFRP1 and SFRP4) were observed in patients with MDS and their methylation frequencies were different. The aberrant methylation of HDPR1 may play an important role in the progression and prognosis of MDS. Disclosures: No relevant conflicts of interest to declare.