Idarubicin Research Articles

Prevalence & Prognosis Value of TET2 Gene Polymorphisms in Childhood Acute Myeloid Leukemia in Taiwan

Abstract 1551 Introduction:Acute myeloid leukemia (AML) is a phenotypically and genetically heterogeneous disease, accounts for 10% of childhood leukemia. The prognosis of children with AML has improved greatly over the past 30 years, and the Taiwan Pediatric Oncology group (TPOG) AML 97A and B protocols had been designed and explored in 1997 for the treatment of AML in Taiwan with complete remission (CR) and overall survival (OS) rates as high as 80–90% and 50–60%. In recent years, molecular research identified an increasing panel of genetic markers in AML, enhancing better risk stratification, modify treatment strategy and improving prognosis. The tet oncogene family member 2 (TET2) gene, a candidate tumor suppressor gene, and the mutations are found in adult AML with prevalence of around 10–20% and is associated to prognosis. However, the report in childhood AML is limited. Here, we assess the prevalence of TET2 gene alterations in childhood AML and to identify its association with prognosis. Method:We enrolled children who were diagnosed AML and visited national Taiwan university hospital between Jan, 1997 to June, 2010. The patients with APL (acute promyeloid leukemia) were excluded due to different treatment protocol. All the children were treated by TPOG AML 97 protocol. The induction therapy consist two courses of cytarabine (Ara-C) and idarubicin (IDR). Patients who achieved CR subsequently received four courses of consolidation therapy consisted of high-dose Ara-C and mitoxantrone or etoposide. The clinical characteristics, such as age, sex, laboratory data, cytogenetics information, relapse, survival time were all collected from TPOG database. DNA was isolated from bone marrow cells at diagnosis and sequence analysis was carried out for TET2 gene. The all data of eligible cases will be collected and analyzed for estimating EFS (event free survival), and OS (overall survival). Survival curves will be estimated by the Kaplan-Meier method. Comparisons were made by Chi-square test for binary variables and t test for continuous variables. For all analyses, the P-values were two-tailed, and a P < 0.05 was considered statistically significant. Results:Total of 56 pediatric AML patients were enrolled. The mean age is 9.07 ± 5.4 (0.01∼17.54) years. There are 34 (60.7%) males. Twenty-four (42.9%) patients had relapse and the overall survival rate is 44.6% (25/56). In this study, there was no nonsense or frameshift mutation, which is frequently identified in adult AML. Total 44 patients (78.6%) present TET2 SNP (single nucleotide polymorphism) and the details are listed in Table 1. There are 18 SNP, and 3 of them (rs72224084, rs58201766, rs59046770) are located in intron. Other 15 SNP are all located in exon. Most of them are located in exon 3 (27 events) and exon 11 (32 events). Only SNP rs3733609 is located in exon 9, and this is synonymous mutation. There are 9 SNP located in exon are not reported in reference yet. Among these, 4 SNP is synonymous mutation. All SNPs are heterozygous, except 4 SNP are homozygous, which are all SNP rs2454206 (I1762V). The clinical characteristics between patients with or without TET2 SNP, including sex, age, white count while diagnosis, chromosome abnormalities, death and relapse are not different between this 2 groups. Kaplan–Meier survival analysis is used to test the correlation between TET2 gene polymorphism to prognosis, including overall survival and event free survival. There was no difference.Table 1TET2 gene alterations in childhood AML. (n=56)RegiondbSNP rs# cluster idcDNA position & SNP alleleAmino acid position & protein residuePatient numberintronrs72224084-/AGATAGAT2rs58201766-/ATGATAGA25rs59046770-/TAGA12Exon 3rs12498609149 C→GP29R2rs6843141715 G→AV218M8983 T→CL307P11485 G→AP474P11889 C→GS609C12667 T→GF868L42503 C→TR814C1rs37969273180 G→AS1039S8rs1116786783179 C→TS1039L1Exon 9rs37336094193 T->CH1380H3Exon 114601 A→GE1513G2rs24542065347 A→GI1762V275502 A→GQ1813Q15387 G→AS1765N15657 C→GA1865G1 Conclusion:As we know, we are the first group to publish TET2 gene alterations in childhood AML in Asian. We conclude the prevalence of TET2 mutations in pediatric AML patients is far lower than in adults and the TET2 polymorphisms are not associated with prognosis. Disclosures:No relevant conflicts of interest to declare.

The role of bioreductive activation of antitumour anthracycline drugs in cytotoxic activity against sensitive and multidrug resistant leukaemia HL60 cells

Clinical usefulness of anthracyclines belonging to bioreductive antitumour drugs is limited by the occurrence of multidrug resistance (MDR). The aim of this study was to examine the role of structural factors of antitumour anthracycline drugs in the ability to undergo bioreductive activation by NADPH cytochrome P450 reductase (CPR) and determine the impact of this activation on increasing the activity especially in regard to MDR tumour cells. It was evidenced that at high NADPH concentration (500μM) anthracyclines having non-modified quinone structure: doxorubicin (DOX), daunorubicin (DR) and idarubicin (IDA) were susceptible upon CPR catalysis to undergo a multi-stage chemical transformation concerning their chromophore part. Additionally, it was evidenced that the modification of the sugar moiety of DOX did not disturb the susceptibility of the obtained derivative (4′-O-tetrahydropyranyl-doxorubicin, pirarubicin, PIRA) to undergo CPR reductive activation. It was also evidenced that the derivatives having modified quinone groupment (5-iminodaunorubicin, 5-Im-DR) were not able to undergo reductive activation by CPR. The high impact of CPR-dependent reductive activation of anthracycline drugs on increasing the activity in regard to sensitive leukaemia HL60 cell line and its MDR sublines overexpressing P-glycoprotein (HL60/VINC) and MRP1 (HL60/DOX) was evidenced. Furthermore, significant changes in binding manner of activated compounds to naked DNA and cellular nucleus in comparison to their non-activated forms were also observed. It could prevent the export of formed adducts out of the cell by MDR proteins and may explain significant increases in intracellular accumulation of these compounds in HL60/VINC and HL60/DOX cells.

Oxidized phospholipid based pH sensitive micelles for delivery of anthracyclines to resistant leukemia cells in vitro

A self-assembled micelle drug delivery system was constructed with an oxidized phospholipid for anthracycline anti-cancer drug delivery. An oxidized phospholipid, 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazPC), was chosen to fabricate micelles via both electrostatic and hydrophobic interactions for delivery of doxorubicin (DOX) and idarubicin (IDA). The formation of ion–pair complexes between PazPC and DOX was first investigated under different pH conditions. Drug-loaded PazPC micelles at a 5:1 molar ratio of lipid/drug at pH 7.0 were then prepared by the solvent evaporation method. The empty and drug-loaded PazPC micelles exhibited a small particle size (∼10 nm) and high encapsulation efficiency. In vitro stability and release profile indicated that the micelles were stable at physiological conditions, but exhibited pH-sensitive behavior with accelerated release of DOX or IDA in an acidic endosome environment. Finally, in vitro uptake and cytotoxicity were evaluated for leukemia P388 and its resistant subline P388/ADR. The drug-loaded PazPC micelles enhanced drug uptake and exhibited higher cytotoxicity in both leukemia cells in comparison to free drugs. In conclusion, we developed a novel pH sensitive oxidized phospholipid-based micellar formulation which could potentially be useful in delivering anthracycline anti-cancer drugs and provide a novel strategy for increasing the therapeutic index while overcoming multidrug resistance for leukemia treatment.

Synergistic cytotoxic effects of rapamycin and idarubicin on human acute T-cell lymphoblastic leukemia Jurkat cells

To investigate the cytotoxic effects of mTOR inhibitor rapamycin (Rapa) and idarubicin (IDA) on human T-cell acute lymphoblastic leukemia Jurkat cell line. The proliferation of Jurkat cells was observed by CCK-8 assay. The combined index was analyzed by Isobologram method. Apoptosis was detected by electron microscopy and flow cytometry with Annexin V/PI staining. Protein expressions of Caspase 3, PARP, Caspase 8, Caspase 9, Akt, p-Akt, P85S6K, p-P85S6K, P70S6K, p-P70S6K, ERK1/2 and p-ERK1/2 were determined by Western blotting. The IC(50) of IDA for Jurkat cells was significantly reduced when combined with 10 nmol/L rapamycin. The combined index was <1. Both electron microscopy and Annexin V/PI staining flow cytometry revealed that rapamycin significantly increased apoptotic sensitivity to IDA. The combination of IDA with rapamycin enhanced the expressions of Caspase 3, PARP, Caspase 8 and Caspase 9. Rapamycin significantly inhibited mTOR signaling upstream Akt and downstream S6K activation triggered by IDA, and the combination of the two agents led to synergistic inhibition of ERK phosphorylation. Combination of IDA with rapamycin exerted a synergistic anti-proliferative effect and promoted apoptosis by both extrinsic and intrinsic apoptotic pathways in Jurkat cells. Inhibition of ERK phosphorylation and mTOR signaling by rapamycin may play a certain role in this synergistic effect.

Enhanced cellular delivery of idarubicin by surface modification of propyl starch nanoparticles employing pteroic acid conjugated polyvinyl alcohol

Enhanced intracellular internalization of the anti-cancer active idarubicin (IDA) was achieved through appropriate surface modification of IDA loaded propyl starch nanoparticles. This was conducted by synthesizing pteroic acid modified polyvinyl alcohol (ptPVA) and employing this stabilizer for formulating the said nanoparticles. Pteroic acid attached at the nanoparticles improved the surface protein adsorption of the nanoparticle, a condition which the nanoparticles would largely experience in vitro and in vivo and hence improve their cellular internalization. Spherical, homogenous IDA nanoparticles (214 ± 5 nm) with surface modified by ptPVA were formulated using the solvent emulsification–diffusion technique. The encapsulation efficiency and drug loading amounted around 85%. In vitro release studies indicated a controlled release of IDA. Safety and efficacy of the nanoparticles was confirmed by suitable cellular cytotoxicity assays. Protein binding studies indicated a higher adsorption of the model protein on nanoparticles formulated with ptPVA as compared to PVA. Cellular uptake studies by confocal laser scanning microscopy revealed a higher cellular uptake of ptPVA stabilized nanoparticles thus confirming the proposed hypothesis of higher protein adsorption being responsible for higher cellular internalization.

Successful treatment of acute promyelocytic leukemia in a patient on hemodialysis

Acute promyelocytic leukemia (APL) has the best prognosis among acute leukemias, but there is little data about APL in patients on hemodialysis. A 64-year-old hemodialysis patient was successfully treated for APL by induction therapy with all-trans retinoic acid (ATRA), three courses of consolidation therapy with Ara-C, mitomycinC (MIT), daunorubicin (DNR), and idarubicin (IDR), and maintenance therapy with ATRA. Complete remission has been maintained for 42months in this patient. With dose modification, ATRA and chemotherapy may be safely given to patients on hemodialysis.

Phase I trial of gemtuzumab ozogamicin in intensive combination chemotherapy for relapsed or refractory adult acute myeloid leukemia (AML): Japan Adult Leukemia Study Group (JALSG)‐AML206 study

In order to investigate better molecular-target therapy for acute myeloid leukemia (AML), we conducted a phase I trial of a combination of gemtuzumab ozogamicin (GO) with conventional chemotherapy. Between January 2007 and December 2009, a total of 19 adult Japanese patients with relapsed or refractory CD33-positive AML (excluding acute promyelocytic leukemia) were enrolled. All registered patients received a standard dose of cytarabine (Ara-C) (100 mg/m(2) × 7 days), combined with either idarubicin (IDR) (10-12 mg/m(2) × 3 days) or daunorubicin (DNR) (50 mg/m(2) × 3-5 days), and then GO (3-5 mg/m(2) ), which was administered 1 day after the last infusion of IDR (IAG regimen) or DNR (DAG regimen). While doses of both GO and IDR and the administration period of only DNR were increased, the dose-limiting toxicity (DLT) was assessed. Among 19 patients (nine in the IAG regimen, 10 in the DAG regimen), the median age was 59 years (range 33-64), and the relapsed/refractory ratio was 13/6. In the therapy using 3 mg/m(2) GO in the IAG or DAG regimen, grade 3/4 leukopenia and neutropenia were observed in all patients, but none had grade 3/4 non-hematological toxicities, except febrile neutropenia. Three patients in the IAG regimen who were administered 5 mg/m(2) GO showed DLT. No patients had veno-occlusive disease or sinusoidal obstructive syndrome. In conclusion, 3 mg/m(2) GO combined with Ara-C and IDR or DNR can be safely administered, and phase II trials should be conducted to investigate the clinical efficacy of the combination therapy.

Abstract 1705: Comparison of uptake mechanisms for different anthracyclines in leukemic cells

Abstract Aims: The mechanisms behind cellular anthracycline uptake are not completely understood. It is of great interest to gain more insight into cellular uptake mechanisms since this could possibly be used to increase the selectivity of the drugs. We therefore compared the uptake of 5 different anthracyclines, daunorubicin (DNR), doxorubicin (DOX), epirubicin (EPI), idarubicin (IDA), and pirarubicin (PIRA) by leukemic cells and also investigated the possible involvement of specific carriers. Methods: HL-60 cells were incubated with increasing concentrations of anthracyclines for 1 hour and cellular drug uptake was determined. The cells were also preincubated with various protein inhibitors or nucleosides and then with the anthracyclines after which drug uptake was determined with HPLC. Apoptosis was determined with propidium iodine staining and flow cytometry. Results: Of all anthracyclines studied DNR, IDA and PIRA had the highest cellular uptake with a sharp increase in uptake at extracellular concentrations &amp;gt; 1 µM. Uptake of DOX, DNR and IDA was greatly reduced at 0°C. Suramin, a purinergic-2-receptor inhibitor, strongly inhibited the uptake of all anthracyclines except PIRA and dipyridamole, a nucleoside transport inhibitor, only inhibited the uptake of DNR. The addition of nucleosides reduced the uptake of DNR, IDA and PIRA. Conclusion: Our results suggest different uptake mechanisms for various anthracyclines. We found evidence for carrier mediated uptake mechanisms, the data support the involvement of the nucleoside transporter family. Furthermore, our results also indicate an involvement of purinergic-2-receptor signalling in anthracycline uptake. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1705. doi:10.1158/1538-7445.AM2011-1705

Idarubicin-intensified BUCY2 regimens may lower relapse rate and improve survival in patients undergoing allo-SCT for high-risk hematological malignancies: a retrospective analysis

We conducted a retrospective study to evaluate the outcome of 94 consecutive patients with high-risk hematological malignancies who received allo-PBSCT, following idarubicin (IDA)-intensified BUCY2 (IDA-BUCY2) myeloablative conditioning regimens (n=53) and BUCY2 conditioning regimens (n=41). IDA 15 mg/m(2) once daily was administered by continuous infusion on days -11 to -9, followed by BU, 3.2 mg/kg in divided doses daily, on days -6 to -4, and i.v. injection of CY, 1.8 g/m(2) once daily on days -3 to -2 in the IDA-BUCY2 group. The relapse rate in patients in the IDA-BUCY2 and BUCY2-conditioning regimens group was 18.9 and 39%, respectively (P=0.030). There was no significant difference in terms of TRM. The cumulative probabilities of OS and disease-free survival at 2 years for patients conditioned with the IDA-BUCY2 and BUCY2 regimens were 65.3% vs 46.8% (P=0.038), and 63.5% vs 43.4% (P=0.025), respectively. Multivariate analysis showed that IDA-BUCY2 regimens and limited chronic GVHD were the only two factors resulting in improved survival and reduced relapse rate. This retrospective study suggests that IDA-intensified BUCY2 may be substituted for BUCY2 as conditioning regimen for patients with high-risk hematological malignancies.

Micellar electrokinetic chromatographic analysis for in vitro accumulation of anthracyclines enhanced by inhibitors of cell membrane transporter-proteins in cancer cells

Cell membrane transporter-proteins have been partly implicated in lowering the accumulation of drugs in cancer cells, leading to multidrug resistance (MDR). Two cancer cell lines, A549 and RDES, were continuously exposed to subclinical concentration (250 nM) of anthracyclines and micellar electrokinetic chromatography was used to investigate their in vitro accumulation after treatment with inhibitors of membrane transporter-proteins. The four anthracylines [doxorubicin (DOX), epirubicin (EPI), daunorubicin (DNR), and idarubicin (IDA)] were separated within a short analysis time of less than 15 min in borate buffer (80 mM, pH 9.22) containing sodium taurodeoxycholate (35 mM), 2-hydroxypropyl-γ-cyclodextrin (3.5% wt/v), and sodium dodecylsulfate (20 mM). Laser-induced fluorescence was used for detection of the anthracyclines. Three inhibitors, verapamil, cyclosporine A and probenecid, were examined by adding each inhibitor independently or two inhibitors simultaneously to the culture medium. It was found that independent use of each inhibitor leads to more efficient accumulation than combined use of verapamil and probenecid. In addition, the results show that effect of inhibitors on the accumulation of anthracyclines depended on type of cell: in RDES, inhibitors enhanced accumulation of all four anthracyclines, while in A549, inhibitors showed different accumulation behavior for each anthracycline. Generally higher accumulation of anthracyclines was observed in RDES cells than A549, as evidenced by dead cells (7-16%) after 24 h of continuous exposure to subclinical concentration.

Anthracyclines and their metabolism in human liver microsomes and the participation of the new microsomal carbonyl reductase

Anthracyclines (ANTs) are widely used in the treatment of various forms of cancer. Although their usage contributes to an improvement in life expectancy, it is limited by severe adverse effects—acute and chronic cardiotoxicity. Several enzymes from both AKR and SDR superfamilies have been reported as participants in the reduction of ANTs. Nevertheless all of these are located in the cytosolic compartment. One microsomal reductase has been found to be involved in the metabolism of xenobiotics—11beta-HSD1, but no further information has been reported about its role in the metabolism of ANTs. The aim of this study is to bring new information about the biotransformation of doxorubicin (DOX), daunorubicin (DAUN) and idarubicin (IDA), not only in human liver microsomal fraction, but also by a novel human liver microsomal carbonyl reductase that has been purified by our group. The reduction of ANTs at C-13 position is regarded as the main pathway in the biotransformation of ANTs. However, our experiments with human liver microsomal fraction show different behaviour, especially when the concentration of ANTs in the incubation mixture is increased. Microsomal fraction was incubated with doxorubicin, daunorubicin and idarubicin. DOX was both reduced into doxorubicinol (DOXOL) and hydrolyzed into aglycone DOX and then subsequently reduced. The same behaviour was observed for the metabolism of DAUN and IDA. The activity of hydrolases definitely brings a new look to the entire metabolism of ANTs in microsomal fraction, as formed aglycones undergo reduction and compete for the binding site with the main ANTs. Moreover, as there are two competitive reducing reactions present for all three ANTs, kinetic values of direct reduction and the reduction of aglycone were calculated. These results were compared to previously published data for human liver cytosol. In addition, the participation of the newly determined human liver microsomal carbonyl reductase was studied. No reduction of DOX into DOXOL was detected. Nevertheless, the involvement in reduction of DAUN into DAUNOL as well as IDA into IDAOL was demonstrated. The kinetic values obtained were then compared with data which have already been reported for cytosolic ANTs reductases.

Metformin Selectively Suppressed the Proliferation of AML Stem Cells through Increased Phosphorylation of AMP-Activated Protein Kinase

AbstractAbstract 4771 Background:Leukemic stem cell (LSC) has been accused to play a pivotal role in pathogenesis of hematological malignancy such as acute myeloblastic leukemia (AML). AML stem cell (ASC) is known as CD34+CD38– leukemic cell population. Yet, despite their critical importance, much remains to be learned about selectively targeting ASC. Recently metformin selectively targets breast cancer stem cells, and acts together with chemotherapy to block tumor growth. Thus, the current study investigated whether metformin selectively inhibits AML cells and AML stem cells directly or indirectly. Materials and Methods:ASC showing CD34+CD38– phenotype were selectively sorted from primary bulk AML cells and KG-1 AML cell lines using single cell sorter (BD FACS Aria cell sorter, BD Biosciences, USA). ASC from primary AML samples and cell lines were cultured in serum media with each 100 ng/mL of stem cell factor, Flt-3, thrombopoietin and G-CSF. Metformin (1,1-dimethylbiguanide hydrochloride) and idarubicin (IDA) were dissolved and diluted with high purity distilled water in various concentrations (0.1 mmol/L to 30 mmol/L). Morphological change, cell count, viability and apoptosis using annexin V flowcytometry were checked after treatment of metformin and IDA. Phosphorylation of AMPK was assayed by western blotting and mitochondrial membrane potential was measured using MitoTracker green dye. Results:The frequency and proportion of ASC varied according to FAB subtypes. The mean proportion of ASC from AML M2, M4 and M3 showed 25.1±22.5% (mean±SD), 15.1±16.6% and 6.5±3.3%, respectively. The mean proportion of ASC from KG-1 cell lines was 78.6±6.2%. Total cell count of ASC was gradually decreased after treatment of metformin in dose-time dependent manner. Overall viability of AML cell lines and ASC disclosed significant decrease after treatment of metformin. The proportion of apoptosis was increased in time and dose dependent manner after 24-hr treatment of metformin. Mitochondrial membrane potentials were increased after metformin exposure at dose dependent manner. Moreover, when combined with lower concentration of IDA, more profound selective killing effect on AML cell lines and ASC was observed. Western blot analysis showed that the treatment of AML cell lines and ASC with 30 mmol/L of metformin resulting in the increased phosphorylation of AMP-activated protein kinase (AMPK) at approximately 1.5-fold over total AMPK. Conclusion:Metformin selectively kills AML cells as well as AML stem cells, and acts together with chemotherapeutic drugs to eradicate leukemic cells and their stem cells. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Treatment Results of Intensive Chemotherapy for Higher-Risk Myelodysplastic Syndrome (HR-MDS) and Acute Myeloid Leukemia Related to MDS (MDS-AML)

AbstractAbstract 2934 Background:Since MDS is more prevalent in the elderly, use of intensive chemotherapy is considered to be difficult. However, granulocyte colony-stimulating factor (G-CSF) and macrophage colony-stimulating factor (M-CSF), use of clean room and development of promising antifungal agents have resulted in dramatically enhanced safety of post-chemotherapy control of elderly patients. Thus, we attempted to use intensive chemotherapy in HR-MDS and MDS-AML patients. Objectives:To evaluate, in HR-MDS and MDS-AML patients, the efficacy and safety of remission induction therapy and post-remission therapy that are standard treatment for de-novo AML in our department.This study enrolled 213 consecutive patients initially treated at our department between 2000 and 2010 who suffered MDS-related disease and whose survival was expected to be less than several months with supportive therapy alone. Almost all of the patients had ≥20% myeloblasts. The age of the patients ranged from 16 to 93 years (median: 70 years). They comprised 2 with good prognosis, 107 with intermediate prognosis and 104 with poor prognosis based on chromosomal findings. Methods:Remission induction therapy consisted of behenoyl-ara-C (BHAC) 350 mg/m2 (300 mg/m2 for patients aged ≥70 years) over 10 days and idarubicin (IDA) 12 mg/m2 (10 mg/m2 for patients aged ≥70 years) over 4 days. Additional etoposide 100 mg/m2 over 4 days was given if bone-marrow examination on Day 15 revealed residual myeloblasts of ≥5%. The efficacy of the therapy was evaluated after the first course. Patients showing maintained remission received 8 courses of post-remission therapy over 11 months. This post-remission therapy included high-dose cytarabine (2 g/m2, or 1g/m2 for patients aged ≥60 years) (HDAC) × 10 and mitoxantrone (MIT) 7mg/m2 × 3 given initially after remission. Maintenance/intensification therapy mainly consisted of BHAC 350 mg/m2 × 4 plus aclarubicin (ACR) 20 mg/body × 6 or IDA 10 mg × 1 alternately every 5 weeks. Outpatient maintenance therapy consisted of M-CSF over 7 days after the end of chemotherapy, followed by administration of G-CSF until neutrophil recovery. Patients were admitted to a clean room if WBC became <1000/mm3. As post-remission therapy, 90 patients received HDAC-MIT, while 50 did not. A total of 28 patients received bone-marrow transplantation during chemotherapy. Results:CR and PR were achieved in 146/213 (68%) and 43/213 (20%) patients, while 14/213 (7%) showed no response (NR) and 10/213 (5%) died during chemotherapy (CD). CR rate was comparable between patients aged ≥70 years (76/110, 69%) and those aged <70 years (70/103, 68%). Chemotherapy death (CD) occurred in 9/110 (8%) patients aged ≥70 years compared to 1/103 (1%) patients aged <70 years. In terms of prognosis by chromosomal type, CR was achieved in 2/2 (100%), 79/107 (74%) and 65/104 (63%) patients with good, intermediate and poor prognosis, respectively. NR was shown in 3/107 (3%) patients with good prognosis compared to 11/1074 (11%) patients with poor prognosis. The overall survival (OS) in the whole patients ranged 1 to 122 months (median: 11 months), and the event free survival (EFS) in patients achieving CR ranged 1 to 109 months (median: 8 months). Six patients received no treatment after remission because of refusal of post-remission therapy or poor P. S. Among patients receiving post-remission therapy, EFS and OS in 75 patients with intermediate prognosis were 12 and 15 months, respectively, while those in 65 patients with poor prognosis were 6 and 10 months, respectively. EFS in those with and without HDAC-MIT were 8 and 7 months, respectively, showing no difference. OS in 28 patients receiving bone-marrow transplantation ranged 6 to 122 months (median: 17 months). A total of 22 patients (16 with chemotherapy alone and 6 with chemotherapy and bone-marrow transplantation) survived for more than 5 years. There was only one chemotherapy death during post-remission therapy. Conclusion:The results of this single-center clinical study indicate that major improvement of supportive therapy may allow introduction of remission safely and at a high probability in elderly patients with HR-MDS or MDS-AML. There remains a challenge since the median OS in patients with remission was still as short as around 1 year. One of the future challenges is how to use intensive chemotherapy in combination with distinctive new antileukemics such as lenalidomide. Disclosures:No relevant conflicts of interest to declare.

Potent antitumor activities of recombinant human PDCD5 protein in combination with chemotherapy drugs in K562 cells

Conventional chemotherapy is still frequently used. Programmed cell death 5 (PDCD5) enhances apoptosis of various tumor cells triggered by certain stimuli and is lowly expressed in leukemic cells from chronic myelogenous leukemia patients. Here, we describe for the first time that recombinant human PDCD5 protein (rhPDCD5) in combination with chemotherapy drugs has potent antitumor effects on chronic myelogenous leukemia K562 cells in vitro and in vivo. The antitumor efficacy of rhPDCD5 protein with chemotherapy drugs, idarubicin (IDR) or cytarabine (Ara-C), was examined in K562 cells in vitro and K562 xenograft tumor models in vivo. rhPDCD5 protein markedly increased the apoptosis rates and decreased the colony-forming capability of K562 cells after the combined treatment with IDR or Ara-C. rhPDCD5 protein by intraperitoneal administration dramatically improved the antitumor effects of IDR treatment in the K562 xenograft model. The tumor sizes and cell proliferation were significantly decreased; and TUNEL positive cells were significantly increased in the combined group with rhPDCD5 protein and IDR treatment compared with single IDR treatment groups. rhPDCD5 protein, in combination with IDR, has potent antitumor effects on chronic myelogenous leukemia K562 cells and may be a novel and promising agent for the treatment of chronic myelogenous leukemia.

Fatty acid based hyperbranched polymeric nanoparticles for hydrophobic drug delivery

Background: In recent years nano-sized dendrimer/hyperbranched polymers gained importance in drug delivery applications. Objective: In this study, a novel fatty acid-based hyperbranched resin (HBR) was synthesized and used for tamoxifen (TAM) and idarubicin (IDA) delivery. Methods: The core of the HBR was dipentaerythritol, and the branching was provided by dimethylolpropionic acid. The molecule was terminated by ricinoleic acid. Chemical and structural characterization of the resin was carried out and then drug-loading experiments were performed. Conclusion: The loading efficiencies were found to be 73.3% for TAM and 74% for IDA. The Fourier transform infrared spectroscopy analysis showed that TAM physically bounded onto the resin whereas IDA interacted chemically. Controlled release in phosphate buffer was improved by Pseudomonas sp. lipase and sodium dodecyl sulfate. The release rates decreased with the increase of loading concentrations. The cytotoxicity analyses were carried out on MCF-7 breast cancer cells for both drug-free and drug-loaded HBR. Drug-free particles did not have significant toxicity. Drug-loaded nanoparticles caused higher levels of cell death than pure drugs.

Inclusion of chemotherapeutic agents in substituted β-cyclodextrin derivatives

The stabilities of the inclusion compounds of three chemotherapeutic agents, camptothecin (CPT), docetaxel (DOC) and idarubicin (IDA), plus a model compound 1,4-dihydroxyanthraquinone (DHA) with several β-cyclodextrin (β-CD) derivatives were investigated by solubility measurements, isothermal titration microcalorimetry and fluorescence anisotropy measurements. Ionic heptakis-(6-deoxy-6-thioethers) of β-CD were found to exhibit very high binding potentials for these drugs making them to good candidates for advanced drug delivery.

Randomized trial of response-oriented individualized versus fixed-schedule induction chemotherapy with idarubicin and cytarabine in adult acute myeloid leukemia: the JALSG AML95 study

A multicenter, prospective, randomized study was conducted to compare a response-oriented individualized remission induction therapy with a standard fixed-schedule induction therapy, using idarubicin (IDR) and cytarabine (Ara-C), in adult patients with acute myeloid leukemia (AML). Newly diagnosed patients with AML of age less than 65 were randomly assigned to receive either of the two schedules. Both groups received IDR (12 mg/m2) for 3 days and Ara-C (100 mg/m2) for 7 days. In the individualized group, if the bone marrow on day 8 did not become hypocellular with less than 15% blasts, patients received additional IDR for one more day and Ara-C for 2 or 3 more days. Patients achieving complete remission (CR) received the same post-remission therapy. The CR rate was 79.4% for the individualized group (n = 209) and 81.9% for the fixed group (n = 221) (p = 0.598). At a median follow-up of 81 months, 7-year predicted overall survival was 37% for the individualized group and 39% for the fixed group (p = 0.496), and 7-year predicted event-free survival was 22% for the individualized group and 23% for the fixed group (p = 0.546). Thus, the present study could not demonstrate any advantage of a response-oriented individualized induction therapy over a fixed-schedule induction therapy in this protocol setting.

A Phase I/II Trial of Gemtuzumab Ozogamicin in Combination with Cytarabine and Anthracyclines (Daunorubicin or Idarubicin) for CD33-Positive Refractory or Relapsed AML Patients Younger Than 65 Years. Results of Phase I Parts of the JALSG-AML206 Study.

Abstract 4145 INTRODUCTION:Gemtuzumab ozogamicin (GO) is an antibody-targeted chemotherapeutic agent consisted of a humanized anti-CD33 monoclonal antibody conjugated to a calicheamicin. Several trials evaluated the efficacy of GO in combination with cytotoxic chemotherapy for treating refractory/ relapsed acute myeloid leukemia (AML) or as front-line therapy. However, there is a still open question what is optimal dose or timing of GO as well as optimal partner of antileukemic drugs. In order to investigate better molecular-target therapy for AML, we initiated phase I/II trial named JALSG AML206 study. PATIENTS and METHODS:The primary goal of this study is to evaluate the safety and the antileukemic activity of combination of GO, cytarabine (Ara-C) and either daunorubicin (DNR)[DAG regimen] or idarubicin (IDR)[IAG regimen] in patients with relapsed/refractory CD33-positive AML. Phase I part was dose-finding study for GO, IDR and DNR. Eligibility criteria included age (20-64yrs), AML(except APL) in first relapse more than 6 months from induction therapy or refractory to the first induction therapy, ECOG performance status 0-2, CD33 expression >20%. All registered patients were received standard dose of Ara-C(100 mg/m2/CIV) on days 1-7 combining GO with either DNR or IDR. GO was planned to administer one day after last administration of DNR or IDR. In the DAG regimen, DNR (50 mg/m2/IV) on days 1-3 + GO(3 mg/m2/DIV) on day 4 as level-1, DNR(50 mg/m2/IV on days 1-4 + GO(3mg/m2/DIV) on day 5 as level-2, and DNR(50 mg/m2/IV) on days 1-5 + GO(3mg/m2/DIV) on day 6 as level-3 were administered. In the IAG regimen, IDR(10 mg/m2/IV) on days 1-3 + GO(3mg/m2/DIV) on day 4 as level-1, IDR(12 mg/m2/IV) on days 1-3 + GO(3 mg/m2/DIV) as level-2, and IDR(12 mg/m2/IV) on days 1-3 + GO (5 mg/m2/DIV) on day 4 as level-3 were administered. Dose and schedule level indicated above was increased using a standard 3+3 design. RESULTS:Nineteen patients (10 patients in the DAG, 9 patients in the IAG) have enrolled: median age was 59 yrs (range 33-64) and 53% were male. Eight patients were refractory and 11 patients were relapsed AML. Cytogenetic risk (according to CALGB): poor 19%, intermediate 63%, and good 19%. The median value of blasts in the bone marrow before treatment was 93%(range 39-100%). No DLT was observed at levels 1 and 2 in both the DAG and the IAG regimens. At level 3 in the DAG, no DLT was observed although one patient had Grade 4 neutropenia for 4 weeks due to progression of AML. In the IAG regimen, 3 of 3 patients had a DLT at level 3; these were Grade 3/4 infection due to severe neutropenia and prolongation of Grade 4 neutropenia independent to progression of leukemia. Two patients had Grade 3 of increase of AST/ALT, but none had VOD/SOS. Among 15 patients assessable for response, 6 patients (4 in DAG, 2 for IAG) (40%) attained CR, and 6 patients had hematologic improvement. One patient died of CNS bleeding within 30-days of therapy due to progression of AML. CONCLUSION:The MTD of GO just after administration of anthracycline combining with standard Ara-C was 5 mg/m2. Addition to standard Ara-C, GO (3 mg/m2) on day 6 with dose-dense DNR(50 mg/m2 × 5 days) and GO(3 mg/m2) on day 4 with IDR(12 mg/m2 × 3days) can be safely administered. Antileukemic activity of these level-3s of DAG and IAG regimens will be evaluated in phase 2 part of this study. Disclosures:No relevant conflicts of interest to declare.

Incidence, Clinical Features and Outcome of Patients with Differentiation Syndrome in Acute Promyelocytic Leukemia Treated with All-Trans Retinoic Acid (ATRA) Alone Versus ATRA Plus Idarubicin Versus ATRA Plus Arsenic Trioxide.

Abstract 1044Poster Board I-66 Background:Differentiation Syndrome (DS) in patients (pts) with acute promyelocytic leukemia (APL) remains a source of significant morbidity and mortality. DS is reported in 2-27% of pts with newly diagnosed APL treated with all-trans retinoic acid (ATRA) alone or in combination with idarubicin (IDA). More recently, arsenic trioxide (ATO) has been used in combination with ATRA as frontline therapy to improve rates of complete remission (CR) and overall survival (OS). It has been postulated that with the use of ATO, the risk of DS may decrease. AimTo describe the incidence, characteristics and outcome of differentiation syndrome with various modalities of ATRA-based therapy used for APL. Methods:We reviewed the records of 167 pts with newly diagnosed APL treated at our institution from 1992-2009 with three regimens: ATRA + IDA (Group 1), liposomal ATRA (Group 2) and ATRA plus ATO (Group 3). Patients in Group 1 (n=52; 1992-1997) received induction with ATRA 45mg/m2 orally daily in two divided doses until CR and IDA 12mg/m2 IV daily for 4 days. Group 2 (n=34; 1997-2000) received liposomal ATRA at 90mg/m2 IV every other day until CR. Patients in Group3 (n=82; 2002-2009) received 45mg/m2 ATRA orally daily in two divided doses, 9mg/m2 gemtuzumab ozogamicin if the WBC count exceeded 30 ×109/L in the first 4 weeks of therapy, ATO 0.15mg/kg/day IV starting on day 10 in 47 patients and on day 1 in 35 patients, and methylprednisolone (50 mg daily for 5 days) to prevent DS. A diagnosis of DS was made by the presence of: dyspnea, unexplained fever, weight gain, peripheral edema, unexplained hypotension, acute renal failure or congestive heart failure, and particularly by a chest radiograph demonstrating interstitial pulmonary infiltrates, or pleuropericardial effusion [Sanz MA, Blood. 2009;113(9):1875-91].Patients with ≥4 features were classified as having severe DS and those with ≤3 mild DS. No single sign or symptom was considered sufficient for diagnosis of DS. Patients with a final diagnosis of pneumonia, sepsis, diffuse alveolar hemorrhage and decompensated heart failure were not considered to have DS. Patients who developed DS ≤7 days of starting therapy with ATRA were classified as “early DS” and others as having “late DS”. Results:Forty one patients (24%) were diagnosed with DS: 14 (27%) in Group 1, 12 (35%) in Group 2, and 15 (18%) in Group 3. Baseline characteristics of patients with DS in each group are shown in Table. Dyspnea, weight gain and pulmonary infiltrates were the most common features of DS in all groups. The median number of days to develop DS after starting ATRA was 3 (1-15) in Group 1, 5 (2-18) in Group 2 and 10 (1-18) in Group 3. ATRA was held in 8 pts (57%) in Group 1, 9 pts (75%) in Group 2, and 8 pts (53%) in Group 3. Intravenous corticosteroids were used for treatment of all patients with DS. CR was achieved in 7 (50%) pts in group 1, 10 (83%) in Group 2 and 14 (93%) in Group 3. The number of patients who died during induction therapy was 6, 2 and 1 in Groups 1, 2 and 3 respectively. There were no deaths directly attributable to DS in any groups. Three-year survival was 65% for pts with DS and 83% for those without DS (p-value: 0.07). Conclusion:The incidence of DS is higher when ATRA alone is used as frontline therapy for APL. With ATRA + ATO (and prophylaxis with corticosteroids) there is a trend for decreased frequency and more delayed occurrence of DS. The severity of DS appears lower for patients not receiving chemotherapy with ATRA. With adequate management, a diagnosis of DS during induction therapy for APL does not influence outcomes independent of therapy.CharacteristicsGroup 1Group 2Group 3TotalDS14/52 (27%)12/34 (35%)15/82 (18%)41/167 (24%)Median age, yrs (range)42 (22-67)37 (10-77)45 (18-74)43 (10-77)Sex (M/F)8/67/58/723/18ECOG*0-16 (43)6 (50)13 (87)25 (61)2-38 (57)6 (50)2 (13)16 (39)WBC (K/μL)< 107 (50)8 (67)9 (60)24 (59)≥ 107 (50)4 (33)6 (40)17 (41)Platelets (K/μL)< 406 (43)9 (75)9 (60)24 (59)≥ 408 (57)3 (25)6 (40)17 (41)SeverityMild7 (50)7 (58)6 (40)20 (49)Severe7 (50)5 (42)9 (60)21 (51)Time course*Early12 (86)8 (67)6 (40)26 (63)Late2 (14)4 (33)9 (60)15 (37)Median time to develop DS*, days3 (1-15)5 (2-18)10 (2-18)4 (1-18)ATRA held8 (57)9 (75)8 (53)25 (61)Dialysis*3 (21)0 (0)1 (7)4 (10)Ventilation*6 (43)1 (8)2 (13)9 (22)*p-value <0.05; otherwise all NS Disclosures:Ravandi:Cephalon: Consultancy, Honoraria.

High Remission Rates and Long-Term Survival in a High Percentage of Patients Achieved by Intensive Chemotherapy for AML and Further Improvement of Supportive Care.

Abstract 1052Poster Board I-74 Background:Until recently, intensive chemotherapy for acute myeloid leukemia (AML) did not necessarily lead to high success rates, partly because of deaths from infections due to the associated long-term neutropenic phase. However, the advent of effective antifungal agents or the use of granulocyte colony-stimulating factor (G-CSF) or macrophage colony-stimulating factor (M-CSF) has definitely reduced deaths from chemotherapy and has improved the results of treatment with intensive chemotherapy also in elderly patients. Objectives:The complete remission rate after remission induction therapy and the event-free survival (EFS) after postremission therapy were investigated in 165 patients (99 men and 66 women) with untreated de novo AML (excluding subtype M3) who were consecutively registered in a single institution between March 2001 and March 2009. The patients' ages ranged from 16 to 94 years (median: 59 years). There were 3 patients with M0, 18 patients with M1, 25 patients with M2, 25 patients with t(8;21), 35 patients with M4, 12 patients with M4Eo, 35 patients with M5, 10 patients with M6, and 2 patients with M7. Methods:Remission induction therapy consisted of 10 days of behenoyl-ara-C (BHAC) at 350 mg/m2 (300 mg/m2 for patients 70 years or older) and 4 days of idarubicin (IDA) at 12 mg/m2 (10 mg/m2 for 70 years or older). Further, if bone marrow examination revealed 5% or more residual blast cells on day 15, etoposide was additionally administered at a dose of 100 mg/m2 for 4 days. The efficacy of the remission induction therapy was evaluated after 1 course of treatment. The patients who had achieved remission underwent 9 courses of postremission therapy, which lasted 11 months. The details are omitted, but therapy with high-dose (2 g/m2 [1 g/m2 for patients 60 years or older]) cytarabine (HDAC)×10 plus 7 mg/m2 of mitoxantrone (MIT) ×3 was performed during the 1st and 9th courses. No HDAC was performed on the elderly aged above 75 years old. The intensive therapy with Aclarubicin (ACR) of 20 mg/body for 14 days and the maintenance therapy with a combination of BHAC 350 mg/m2×4 with ACR or IDA were repeated alternately every 6 weeks. Chemotherapies other than remission induction therapy and HDAC were performed in an outpatient clinic, and if the patients with the WBC decreasing to 1000/mm3 were hospitalized in the biological clean room. M-CS was administered for 7 days after the day following the end of chemotherapy, and subsequently G-CSF was administered until the WBC becomes to be 1000/mm3. Results:Complete remission (CR) was achieved in 143 of the 165 patients overall (86.7%), 113 of the 123 patients 69 years or younger (92.7%), and 29 of the 42 patients 70 years or older (69.1%). During the remission induction therapy, death occurred in 6 of the 165 patients overall (3.6%), 2 of the 123 patients 69 years or younger (1.6%), and 4 of the 42 patients 70 years or older (9.5%). The EFS in patients with CR was 61.5% at 8 years in patients 69 years or younger, while it was 26.9% at 5 years in patients 70 years or older. There was only a case of death due to chemotherapy during postremission therapy. Seven patients underwent bone marrow transplantation during the first remission, and 6 of these patients have been enjoying EFS. Conclusion:Improvement in supportive care has enabled safe intensive chemotherapy. The patients with good or intermediate prognosis were clearly improved by the present preliminary treatment at a single institution, but the patients with poor prognosis still highly require bone marrow transplantation in the future. Disclosures:No relevant conflicts of interest to declare.