Acute Myeloid Leukemia Induction Research Articles

Cdx4 is dispensable for murine adult hematopoietic stem cells but promotes MLL-AF9-mediated leukemogenesis

Cdx4 is a homeobox gene essential for normal blood formation during embryonic development in the zebrafish, through activation of posterior Hox genes. However, its role in adult mammalian hematopoiesis has not been extensively studied and its requirement in leukemia associated with Hox gene expression alteration is unclear. We inactivated Cdx4 in mice through either a germline or conditional knockout approach and analyzed requirement for Cdx4 in both normal adult hematopoiesis and leukemogenesis initiated by the MLL-AF9 fusion oncogene. Here, we report that loss of Cdx4 had a minimal effect on adult hematopoiesis. Indeed, although an increase in white blood cell counts was observed, no significant differences in the distribution of mature blood cells, progenitors or stem cells were observed in Cdx4-deficient animals. In addition, long-term repopulating activity in competitive transplantation assays was not significantly altered. In vitro, B-cell progenitor clonogenic potential was reduced in Cdx4-deficient animals but no significant alteration of mature B cells was detected in vivo. Finally, induction of acute myeloid leukemia in mice by MLL-AF9 was significantly delayed in the absence of Cdx4 in a retroviral transduction/bone marrow transplant model. These observations indicate that Cdx4 is dispensable for the establishment and maintenance of normal hematopoiesis in adult mammals. These results, therefore, outline substantial differences in the Cdx-Hox axis between mammals and zebrafish and support the hypothesis that Cdx factors are functionally redundant during mammalian hematopoietic development under homeostatic conditions. In addition, our results suggest that Cdx4 participates in MLL-AF9-mediated leukemogenesis supporting a role for Cdx factors in the pathogenesis of myeloid leukemia.

PU.1-mediated upregulation of CSF1R is crucial for leukemia stem cell potential induced by MOZ-TIF2

Leukemias and other cancers possess self-renewing stem cells that help to maintain the cancer. Cancer stem cell eradication is thought to be crucial for successful anticancer therapy. Using an acute myeloid leukemia (AML) model induced by the leukemia-associated monocytic leukemia zinc finger (MOZ)-TIF2 fusion protein, we show here that AML can be cured by the ablation of leukemia stem cells. The MOZ fusion proteins MOZ-TIF2 and MOZ-CBP interacted with the transcription factor PU.1 to stimulate the expression of macrophage colony-stimulating factor receptor (CSF1R, also known as M-CSFR, c-FMS or CD115). Studies using PU.1-deficient mice showed that PU.1 is essential for the ability of MOZ-TIF2 to establish and maintain AML stem cells. Cells expressing high amounts of CSF1R (CSF1R(high) cells), but not those expressing low amounts of CSF1R (CSF1R(low) cells), showed potent leukemia-initiating activity. Using transgenic mice expressing a drug-inducible suicide gene controlled by the CSF1R promoter, we cured AML by ablation of CSF1R(high) cells. Moreover, induction of AML was suppressed in CSF1R-deficient mice and CSF1R inhibitors slowed the progression of MOZ-TIF2-induced leukemia. Thus, in this subtype of AML, leukemia stem cells are contained within the CSF1R(high) cell population, and we suggest that targeting of PU.1-mediated upregulation of CSF1R expression might be a useful therapeutic approach.

Attempts to Optimize Induction and Consolidation Treatment in Acute Myeloid Leukemia: Results of the MRC AML12 Trial

Attempts to Optimize Induction and Consolidation Treatment in Acute Myeloid Leukemia: Results of the MRC AML12 Trial

Biphenotypic acute leukaemia: Case reports of two paediatric patients

Biphenotypic acute leukaemia is an uncommon type of leukaemia whose blasts co-express myeloid and B-or T-lymphoid antigens. We describe two cases of paediatric patients with biphenotypic acute leukaemia. A four-year-old female patient was found to have myeloid and B-lymphoid associated antigens in the same blast cells. Cytogenetic analysis showed a Philadelphia (Ph) positivity t (9;22) (q34;q1l1 with rearrangements of M.bcr-Abl (p210). She was treated with combined acute myeloid leukaemia/acute lymphoblastic leukaemia induction therapy followed by autologous stem cell transplantation. The patient died due to the complications of stem cell transplantation procedure. Another patient was a 20-month-old girl with myeloid and T-lymphoid associated antigens in the blast cells and with normal karyotype. She received acute myeloid leukaemia induction therapy. She has never achieved remission. Immunophenotype is essential to establish the diagnosis of biphenotypic acute leukaemia according to the scoring system adopted by the European Group of Immunological Classification of Leukaemia. There is no agreement about uniformity in treatment for the patients with this type of leukaemia. Biphenotypic acute leukaemia is a high risk leukaemia which requires a more intensive treatment. Therapy for every patient with biphenotypic acute leukaemia should depend on their immunophenotype and gene rearrangement profiles.

Attempts to Optimize Induction and Consolidation Treatment in Acute Myeloid Leukemia: Results of the MRC AML12 Trial

To optimize treatment for younger patients with acute myeloid leukemia and high-risk myelodysplastic syndrome by comparing induction options and the number of consolidation courses and whether consolidation should include transplantation. We randomly assigned 1,658 patients younger than age 60 years to receive mitoxantrone/cytarabine/etoposide versus cytarabine/daunorubicin/etoposide and subsequently 1,193 patients to daunorubicin/cytarabine/thioguanine (DAT) where the cytarabine dose was standard (S-DAT) versus double the standard dose (H-DAT). Patients in this randomization were randomly assigned to all-trans-retinoic acid or not. In consolidation, 992 patients were randomly assigned between a total of four courses versus five courses, and 324 patients who were not good risk were randomly assigned to transplantation or chemotherapy as the final course. Complete remission (CR) was achieved in 74% of patients and CR without recovery was achieved in an additional 11%; overall survival (OS) at 8 years was 38%. No differences in CR, relapse-free survival, relapse, or OS were seen between any of the induction randomizations except for a reduction in relapse risk (RR) on the mitoxantrone arm, which was offset by increased myelosuppression and deaths in CR. The addition of a fifth course did not improve OS and may be detrimental in older patients. Although transplantation reduced RR, it did not improve OS for the intermediate-risk group but was probably of benefit in high-risk patients. Several chemotherapy schedules achieved similar remission rates and OS. Four courses of chemotherapy are adequate, but the addition of transplantation as a final course does not improve OS. New agents are required to enhance conventional chemotherapy.

The Use of Purine Analogues Does Not Aggravate Infectious Complications During Induction and Consolidation Therapy of Acute Myeloid Leukemia.

Abstract 1011Poster Board I-33 Background:Addition of purine analogues to standard induction therapy of acute myeloid leukemia (AML) had previously been demonstrated to increase complete remission rate. The aim of this study was to analyze whether the use of cladribine or fludarabine during induction and consolidation increases the risk of infectious complications. Material and methods:118 AML patients, included in two consecutive randomized trials between 1999-2006 in a single centre were analyzed. Induction therapy consisted of daunorubicin + cytarabine (DA-7, n=53) alone or in combination with cladribine or fludarabine (DAC-7 + DAF-7, n=65 ). Consolidation included one course of high-dose AraC + mitoxantrone and one course of high-dose AraC +/- purine analogues. A median age was 45(17-58) years and 48(20-60) years for patients treated with and without purine analogues, respectively. Results:The frequency of neutropenic fever as well as microbiologically documented bacterial, fungal and viral infections during induction and consolidation did not differ between two compared groups - receiving or not purine analogues. Time to infection occurrence and infection duration were similar in both study groups.During induction and both consolidation treatments significant lower values of lymphocytosis were observed in the group of patients treated with purine analogues.There was a slight tendency to increased rate of mucositis for patients treated with purine analogues (60% vs. 44.3%, p=0.07) during induction treatment, while infections affecting skin and soft tissues were significant frequent for patients treated without purine analogues (43.3% vs. 18%, p=0.03) during second consolidation treatment (high dose AraC).The usage of intravenous anti-infectious medications (antibiotics, antifungal, antiviral) and periods of hospitalization did not differ between two groups in this study. Conclusions:We conclude that the use of purine analogues, either cladribine or fludarabine along with conventional induction and consolidation therapy does not aggreviate infectious complications in adults with AML. Disclosures:No relevant conflicts of interest to declare.

Cost Burden Analysis of Ineffective Induction Chemotherapy in Elderly Patients with AML.

Abstract 4531 IntroductionAccording to the Surveillance, Epidemiology, and End Results (SEER) database 12,810 patients will be diagnosed with acute myeloid leukemia (AML) in 2009 with an incidence rate of 3.5 per 100,000 persons in the United States. The SEER data estimates that 55% of those diagnosed are 65 years or older. Standard first line therapy for the treatment of AML consists of at least one round of chemotherapy commonly referred to as induction. A significant portion of those patients who receive standard induction chemotherapy will have leukemia that fails to show a complete response as defined by less than 5% leukemia blasts in the bone marrow with normal tri-lineage hematopoiesis and peripheral blood count recovery (Mueller S, et al. BMC Cancer 2006, 6:143). Age, patient performance status, the presence of secondary AML, cytogenetics, and molecular markers are prognostic for particular cohorts of patients, however, there is currently no means to predict whether an individual's leukemia will or will not undergo a complete response (CR) after the administration of standard AML induction chemotherapy. MethodsA budget impact model was designed to assess the incremental societal cost and incremental cost to a typical national health plan of treating patients who will fail induction therapy. The analysis only evaluated elderly patients over the age of sixty five. Data were gathered from the peer reviewed literature. All costs have been updated to 2009 dollars using the medical care component of the consumer price index. The patient population was determined to be 6,981 patients in 2009 with 30% receiving induction therapy (Menzin J, et al. Arch Intern Med. 2002; 162:1597-1603) and only 38% demonstrating a CR (Appelbaum FR, et al. Blood 2006 107:3481-5) resulting in 1,303 patients failing during induction therapy. The costs were taken from Menzin et al., which used Medicare claims data to determine the associated costs of AML patients during the first two years post diagnosis. In the model Medicare payments are used as a proxy for direct costs to society and a national health plan. These reimbursements are able to capture the costs of the initial hospital visit, lab/diagnostic/radiology, supportive care, drugs, and adverse events through the observation of payments across all appropriate settings of care (inpatient hospitalization, skilled nursing facility, outpatient hospital/clinical, physician's office, home health, and hospice). A patient undergoing chemotherapy incurred costs of $120,468 over two years, while a patient avoiding chemotherapy only incurred costs of $40,720. ResultsThe incremental cost of a patient receiving induction therapy was calculated to be $79,748. For a national health care plan with an assumed average of one million members results in a cost of $283,856 to treat patients whose leukemia would not respond to induction chemotherapy. The overall societal impact of treating this patient population with ineffective therapy is $104 million. ConclusionsPatients who are subjected to ineffective chemotherapy face the cytotoxic effects of the treatment, none of the benefits of treatment response, and impose a significant cost burden to themselves and the healthcare payment system. Diagnostics currently in development that can identify patients at the time of diagnosis with disease unresponsive to therapy may have the ability to alleviate unnecessary costs, while steering patients to better tailored and more effective therapies. Disclosures:Cleary Cohen:Nodality, Inc: Employment. Middlebrook:Nodality Inc: Employment.

Clofarabine for the Treatment of Adult Acute Myeloid Leukemia

Clofarabine, a next-generation deoxyadenosine analog, has demonstrated significant activity in patients with acute myeloid leukemia (AML). The single-agent activity compares favorably with that demonstrated by the current standard antileukemia agents. Clofarabine has been safely and effectively combined with other agents and will probably become an integral part of induction and/or consolidation regimens in AML. Current studies are underway to better define the role of clofarabine in younger and elderly patients with AML, and also explore development strategies for an oral formulation.

Incidence of Acute Myeloid Leukemia and Hepatocellular Carcinoma in Mice Irradiated with 1 GeV/nucleon56Fe Ions

Abstract Estimates of cancer risks posed to space-flight crews by exposure to high atomic number, high-energy (HZE) ions are subject to considerable uncertainty because epidemiological data do not exist for human populations exposed to similar radiation qualities. We assessed the leukemogenic efficacy of one such HZE species, 1 GeV (56)Fe ions, a component of space radiation, in a mouse model for radiation-induced acute myeloid leukemia. CBA/CaJ mice were irradiated with 1 GeV/nucleon (56)Fe ions or (137)Cs gamma rays and followed until they were moribund or to 800 days of age. We found that 1 GeV/nucleon (56)Fe ions do not appear to be substantially more effective than gamma rays for the induction of acute myeloid leukemia (AML). However, (56)Fe-ion-irradiated mice had a much higher incidence of hepatocellular carcinoma (HCC) than gamma-irradiated mice, with an estimated RBE of approximately 50. These data suggest a difference in the effects of HZE iron ions on the induction of leukemia compared to solid tumors, suggesting potentially different mechanisms of tumorigenesis.

Implication of replicative stress-related stem cell ageing in radiation-induced murine leukaemia

Background:The essential aetiology of radiation-induced acute myeloid leukaemia (AML) in mice is the downregulation of the transcription factor PU.1. The causative mutation of the PU.1-endocing Sfpi1 gene consists mostly of C:G to T:A transitions at a CpG site and is likely to be of spontaneous origin. To work out a mechanism underlying the association between radiation exposure and the AML induction, we have hypothesised that replicative stress after irradiation accelerates the ageing of haematopoietic stem cells (HSCs), and the ageing-related decline in DNA repair could affect the spontaneous mutation rates.Methods:Mathematical model analysis was conducted to examine whether and to what extent the cell kinetics of HSCs can be modified after irradiation. The haematopoietic differentiation process is expressed as a mathematical model and the cell-kinetics parameters were estimated by fitting the simulation result to the assay data.Results:The analysis revealed that HSCs cycle vigourously for more than a few months after irradiation. The estimated number of cell divisions per surviving HSC in 3 Gy-exposed mice reached as high as ten times that of the unexposed.Interpretation:The mitotic load after 3 Gy irradiation seems to be heavy enough to accelerate the ageing of HSCs and the hypothesis reasonably explains the leukaemogenic process.

A comparative study of single-dose pegfilgrastim versus daily filgrastim in patients with acute myeloid leukemia

e18005 Background: Prophylactic treatment with recombinant human granulocyte colony stimulating factor (rhGCSF) is used to decrease the severity and duration of neutropenia in patients receiving myelosupressive chemotherapy. It can be used during induction and consolidation chemotherapy in acute myeloid leukemia (AML). Mechanism of action of pegylated GCSF is the same as filgrastim except for delayed clearance. Few studies addressed the use of pegylated filgrasim in AML. Methods: After 24 hours of consolidation chemotherapy, 8 patients in the study arm received single dose of subcutaneous 6 mg pegfilgrastim. The control group received daily filgrastim at a dose of 5 microgram per kg.until absolute neutroophil count of 2 ×109 cells/μL for 2 consecutive days. The neutrophil recovery time, febrile neutropenia and its duration, duration of hospitalization and safety profiles were compared. Results: There were 20 episodes of pegfilgrastim administration. The median age was 41 years in pegfilgrastim arm and 39 years in filgrastim arm. Medium time to neutrophil recovery was 14 days for pegfilgrastim arm and 17 days for filgrastim arm. In the pegfilgrastim arm 12 (60%) episodes of febrile neutropenia occurred compared to 11 (55%) in filgrastim arm. The median duration of febrile neutropenia was 3 days in pegfilgrastim arm versus 4 days in filgrastim arm. The median duration of hospitalization was 15 days in pegfilgrastim arm and 18 days in filgrastim arm. Safety profile and complete remission status did not differ between the two groups. Conclusions: The present study showed favorable profile of pegfilgrastim for netrophil recovery, duration of neutropenia and hospital stay. There was no difference with regard to safety or response to treatment. No significant financial relationships to disclose.

Infant acute bilineal leukemia

Most cases of acute leukemia can be assigned to the myeloid, B or T lineage. There are rare cases of acute leukemia, which cannot be clearly classified, because either blasts express antigens of more than one lineage (acute biphenotypic leukemias) or distinct blast populations of two lineages co-exist (acute bilineal leukemias, aBLL). We present a 10-month-old infant with de novo aBLL, characterized by blasts of monocytic and B-cell precursor lineages. All leukemic cells harbored identical complex MLL gene rearrangement. Despite poor initial response, both to acute lymphoblastic leukemia (ALL) induction treatment and acute myeloid leukemia induction blocks, the child reached complete clinical remission with minimal residual disease negative status and was transplanted. Unfortunately, 16 months from HSCT the patient experienced BM relapse with all blasts characterized by pro-B-ALL immunophenotype. This case report illustrates that aBLL is a very aggressive type of acute leukemia that should be individually treated and monitored, particularly in children less than 1 year of age.

RUNX1/AML1 DNA-binding domain and ETO/MTG8 NHR2-dimerization domain are critical to AML1-ETO9a leukemogenesis

AbstractThe 8;21 translocation, which involves the gene encoding the RUNX family DNA-binding transcription factor AML1 (RUNX1) on chromosome 21 and the ETO (MTG8) gene on chromosome 8, generates AML1-ETO fusion proteins. Previous analyses have demonstrated that full-length AML1-ETO blocks AML1 function and requires additional mutagenic events to promote leukemia. More recently, we have identified an alternatively spliced form of AML1-ETO, AML1-ETO9a, from t(8;21) acute myeloid leukemia (AML) patient samples. AML1-ETO9a lacks the C-terminal NHR3 and NHR4 domains of AML1-ETO and is highly leukemogenic in the mouse model. Here, we report that the AML1 DNA-binding domain and the ETO NHR2-dimerization domain, but not the ETO NHR1 domain, are critical for the induction of AML by AML1-ETO9a. A region between NHR1 and NHR2 affects latency of leukemogenesis. These results provide valuable insight into further analysis of the molecular mechanism of t(8;21) in leukemogenesis.

Optimal induction and post-remission therapy for AML in first remission

Approximately 300,000 patients in the world are diagnosed annually with acute myeloid leukemia (AML). The median age at presentation has been steadily increasing over the past few decades and now is approaching 70 years. Although considerable progress has been made over the past 3 decades in the therapy of AML, two thirds of young adults still die of their disease. The therapy of AML, unlike acute lymphoblastic leukemia (ALL), is based on maximally tolerated induction and post-remission therapy, all given within a few months from diagnosis. While complete remission can be achieved in the majority of young patients, ultimate cure of the disease depends on disease eradication through the administration of post-remission therapy. This is most often done with intensive chemotherapy. Harnessing the immunologic effect of graft-versus-leukemia, as in allogeneic transplantation, has further improved the outcome for many patients. Treatment of older adults, representing the majority of patients with AML, remains quite unsatisfactory. While 40% to 50% can achieve a complete remission, less than 10% are long-term survivors, and the cure rate of older patients has only minimally improved over the past three decades. Significant progress in the treatment of this age group is urgently required. New and targeted agents have much promise, but a definitive clinical role for these has yet to be conclusively established.

Myelodysplastic syndromes and acute myeloid leukemia in cats infected with feline leukemia virus clone33 containing a unique long terminal repeat

Feline leukemia virus (FeLV) clone33 was obtained from a domestic cat with acute myeloid leukemia (AML). The long terminal repeat (LTR) of this virus, like the LTRs present in FeLV from other cats with AML, differs from the LTRs of other known FeLV in that it has 3 tandem direct 47-bp repeats in the upstream region of the enhancer (URE). Here, we injected cats with FeLV clone33 and found 41% developed myelodysplastic syndromes (MDS) characterized by peripheral blood cytopenias and dysplastic changes in the bone marrow. Some of the cats with MDS eventually developed AML. The bone marrow of the majority of cats with FeLV clone33 induced MDS produced fewer erythroid and myeloid colonies upon being cultured with erythropoietin or granulocyte-macrophage colony-stimulating factor (GM-SCF) than bone marrow from normal control cats. Furthermore, the bone marrow of some of the cats expressed high-levels of the apoptosis-related genes TNF-alpha and survivin. Analysis of the proviral sequences obtained from 13 cats with naturally occurring MDS reveal they also bear the characteristic URE repeats seen in the LTR of FeLV clone33 and other proviruses from cats with AML. Deletions and mutations within the enhancer elements are frequently observed in naturally occurring MDS as well as AML. These results suggest that FeLV variants that bear URE repeats in their LTR strongly associate with the induction of both MDS and AML in cats.

Down-Regulation of CEBPA Expression Has a Stronger Impact on Myeloid Differentiation in Human Versus Mouse Cells: Mechanistic Implications for CEBPA Mutations in AML.

Acute myeloid leukemia (AML) is often associated with gene mutations that affect transcription factors regulating differentiation. CEBPA mutations occur in 5–10% of AML cases. About half of those result in the translation of an N-terminal truncated C/EBPα protein, termed p30. While C/EBPα is not required for adult hematopoiesis, its ablation leads to a block in granulocytic differentiation at the blast stage. Earlier studies showed that p30 dominantly inhibits the transcriptional function of wtC/EBPα. We have previously shown that expression of p30 in human but not murine hematopoietic progenitors leads to the accumulation of myeloblasts. Here, we investigated the role of p30 C/EBPα in AML induction by using different dimerizing mutants of C/EBPα. In addition, we sought to determine if down-regulation of CEBPA expression mimicked the effect of p30 in human hematopoietic progenitors. To evaluate the requirement for dimerization and transcriptional activity of the p30 C/EBPα, a mutant was generated in which the first two leucines of the leucine zipper were changed to valine (C/EBPα L12V), which has been previously shown to disrupt C/EBPα function. CD34+ cells isolated from human cord blood were transduced with retroviral vectors expressing either GFP alone, human or murine p30 C/EBPα, or murine p30-L12V and plated into methylcellulose. While murine mutC/EBPα and human mutC/EBPα showed the expected block in granulocytic differentiation and inhibition of erythrocyte lineage commitment (mean number of 0 ± 2 CFU-GM), mutC/EBPα L12V had no effect as shown by the same colony distribution as the control vector (mean number of 20 CFU-GM). This observation implies that mutC/EBPα requires dimerization and/or transcriptional activity for the inhibition of human hematopoietic differentiation. To predict if p30 homodimerization or heterodimerization between p30 C/EBPα and wtC/EBPα is required for this activity, we replaced the leucine zipper of the p30 C/EBPα with the GCN4 exchange mutant to form p30-GZ, which allows homodimerization but not heterodimerization with wtC/EBPα. We demonstrate here that expression of p30-GZ showes an almost identical phenotype as mutC/EBPα arguing that p30 homodimerization is important for its activity while heterodimerization with wtC/EBPα is not necessary. To test the effects of down-regulation of C/EBPα and to compare the induced phenotype with the one induced by p30 C/EBPα, we generated a short-hairpin (sh) RNA against human C/EBPα and cloned it into a retroviral expression vector. Human CD34+ cells from cord blood were transduced with these vectors and evaluated for the down-regulation of C/EBPα mRNA. Expression of C/EBPα shRNA reduced C/EBPα mRNA levels to 25%. Examination of liquid cultures (with GM-CSF, SCF, IL3) by using FACS analysis demonstrated a relative increase in erythroid cells: 32% CD71+/CD13− C/EBPα siRNA versus 7% CD71+/CD13− control vector, and substantial defect in proliferation of myeloid cells (0% CD14 and 4% CD15 C/EBPα siRNA compared with 10% CD14 and 12% CD15 control vector). Most striking were results in methylcellulose cultures, where a dramatic reduction in CFU-GM colonies (mean number of 8 ± 2 CFU-GM C/EBPα siRNA versus 37 ± 4 CFU-GM control vector), but also BFU-E (mean number of 18 ± 3 BFU-E C/EBPα siRNA versus 42 ± 4 BFU-E control vector) was observed in cultures expressing shRNA against C/EBPα. In addition, an almost complete block in terminal differentiation of both monocytic and granulocytic differentiation was observed in cells dispersed from the colonies. Using shRNA technology, we show that C/EBPα is required at two stages of human myeloid differentiation:during early stages of myeloid/erythroid commitment andfor terminal granulocytic and monocytic maturation.In contrast to mouse studies, the block to terminal differentiation could not be relieved by GM-CSF stimulation. This shows that C/EBPα activity is not readily substituted by alternative pathways in human myeloid differentiation. We thus propose that p30 induces myeloid differentiation at early stages of differentiation but cannot promote terminal differentiation. This coupled with reduced absolute levels of C/EBPα leads to accumulation of myeloblasts, a hallmark of AML.

Upfront Allogeneic Stem Cell Transplantation for Remission Induction in High-Risk Acute Myeloid Leukemia Patients within the Randomized Multi- Center Trial AML2003.

Introduction: Allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative treatment option for most high-risk patients with acute myeloid leukemia (AML). However, many high-risk patients regenerate with blasts or relapse early after induction therapy. Thus, consolidation with allogeneic HSCT in first CR is often not possible. Performing upfront allogeneic HSCT for remission induction as part of induction therapy has the potential to circumvent these problems and might furthermore reduce cumulative toxicity in high-risk patients. Therefore, in 2003 we started a prospective multicenter randomized trial that investigates both the feasibility and efficacy of upfront allogeneic stem cell transplantation for remission induction in high-risk AML patients.Methods: The AML2003 study compares in a randomized fashion an intensified treatment approach using upfront allogeneic transplantation in high risk patients as part of the induction therapy (IT) during marrow aplasia achieved by DA (daunorubicin 60 mg/ m2 – day 3–5; cytarabine 100 mg/m2 – day1–7) to a “conventional” treatment strategy, which allows for allogeneic transplantation only in patients achieving remission after two induction courses (DA). To do this, rapid analysis of cytogenetics, FLT3 status and HLA-DNA-typing of the patient and possible family donors is of utmost importance. This “fast search diagnostics” together with routine analyses of morphology and immunophenotyping is accomplished centrally in all enclosed patients. The dose-reduced preparative regimen for upfront allogeneic stem cell transplantation within induction therapy consisted of melphalan 150mg/m2 and fludarabine 150mg/m2.Results: Until the last update we recruited 679 patients <= 60 years with de novo (n=570) or secondary (n=109) AML. Out of 340 patients randomized for an intensified treatment approach we identified 139 patients (41%) with high-risk defined by cytogenetic criteria (n=87), FLT3 status (n=15) or day 15 blast count (n=37). Fast search strategy revealed HLA identical donors (related or unrelated) for 106 patients. Consequently, 78 high-risk AML patients assigned to the intensified treatment strategy received allogeneic transplantation. Upfront allogeneic stem cell transplantation for remission induction was feasible in 28 high-risk AML patients during marrow aplasia after IT1 (n=10) or IT2 (n=18), respectively. Fifteen of these patients received unrelated grafts.Conclusions: These preliminary results show that rapid risk profiling and fast donor-search is feasible in a large multi-center study. This leads to a significant proportion of upfront allogeneic stem cell transplants as part of the induction therapy within the group of high-risk AML patients, which may improve the disastrous prognosis of this group of patients in the future.

Incidence of bacterial and fungal infections in newly diagnosed acute myeloid leukaemia patients younger than 65 yr treated with induction regimens including fludarabine: retrospective analysis of 224 cases

Infections are the major cause of morbidity and mortality in patients with acute myeloid leukaemia (AML). They primarily occur during the first course of induction chemotherapy and may increase the risk of leukaemia relapse, due to a significant delay in consolidation therapy. The intensification of induction chemotherapy and the use of non-conventional drugs such as fludarabine are considered responsible for the increased risk of infections. In this study, we retrospectively analysed the infections occurred in 224 newly diagnosed AML patients </=65 yr, consecutively treated between 1997 and 2002 with an induction regimen including fludarabine, arabinosyl cytosine and idarubicin, with or without etoposide (FLAI/FLAIE), in the context of three multicentric prospective trials (AML97, AML99, AML02). During the induction phase, 146 (65%) patients experienced fever of undetermined origin (FUO), 30 (13%) and 47 (21%) patients had Gram-negative and positive bacteremias, respectively, and 10 (4%) patients developed a probable/proven invasive fungal infection (IFI). The fatality rate for Gram-negative, Gram-positive bacteremias and probable/proven IFI was 10%, 8% and 60% respectively. During consolidation, 75 (35%) patients had FUO, 43 (20%) and 40 (19%) patients had Gram-negative and positive bacteremias, respectively, and 5 (2%) patients developed a probable/proven IFI. The fatality rate for Gram-negative, Gram-positive bacteremias and probable/proven IFI was 14%, 5% and 80% respectively. Interestingly, the overall incidence of microbiologically documented infections during induction was 38% and the incidence of probable/proven IFIs during the induction/consolidation programme was 7%. No infections caused by viruses or opportunistic pathogens were observed neither during induction, nor during consolidation. These data, although retrospectively collected, suggest that fludarabine-based chemotherapy is not associated with an increased incidence of infections, in particular IFIs, compared to conventional regimens commonly used for AML induction.

266 POSTER Apoptosis induction in acute myeloid leukemia by inhibition of MEK and MDM2 is strongly associated with the BH3-only proteins Puma and Bim

This study investigated the effects of C7 and C9 aliphatic (n-heptane, n-nonane), naphthenic (methylcyclohexane, 1,2,4-trimethylcyclohexane (TMCH)) and aromatic (toluene, 1,2,4-trimethylbenzene (TMB)) hydrocarbons on the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in rat brain synaptosome fraction. Methyl mercury (MeHg) was included as a positive control. Exposure of the synaptosomes to the hydrocarbons produced a concentration-dependent linear increase in the formation of the fluorescence of 2′,7′-dichlorofluorescein (DCF) as a measure of the production of ROS and RNS. Formation of RNS was demonstrated by preincubation of the synaptosome fraction with the neuronal nitric oxide synthase (nNOS) inhibitor Nω-nitro-l-arginine methyl ester (L-NAME), which reduced the MeHg and TMCH-stimulated fluorescence by 51% and 65%, respectively. The naphthenic hydrocarbon TMCH showed the strongest potential for ROS and RNS formation in rat brain synaptosomes, followed by TMB, toluene, n-nonane, n-heptane, and methylcyclohexane, respectively. TMCH was selected for mechanistic studies of the formation of ROS. Both MeHg and TMCH induced an increase in intracellular calcium concentration [Ca2+]i as measured with Fura-2. Blockade of voltage-dependent Ca2+ channels with lanthanum prior to stimulation with MeHg and TMCH led to a reduction in the ROS/RNS formation of 72% and 70%, respectively. Furthermore, addition of cyclosporin A (CSA), a blocker of the mitochondrial permeability transition pore (MTP), lowered both the MeHg and TMCH-elevated DCF fluorescence by 72% and 59%. Preincubation of the synaptosome fraction with the protein tyrosine kinase inhibitor genistein lowered the MeHg and TMCH-stimulated fluorescence by 85% and 91%, respectively. Addition of the extracellular signal-regulated protein kinase (MEK)-1 and -2 inhibitor U0126 reduced the fluorescence stimulated by MeHg and TMCH by 62% and 63%. Furthermore, the protein kinase C inhibitor bisindolylmaleimide reduced the fluorescence stimulated by MeHg and TMCH by 52% and 56%. The compound 1-(6-[17beta-3-methoxyestra- 1,3,5(10)-trien- 17-yl]-aminohexyl)-1H-pyrrole-2,5-dione (U73122), which inhibits phospholipase C, was shown to decrease the ROS and RNS formation induced by MeHg and TMCH by 49% and 64%, respectively. The phospholipase A2 (PLA2) inhibitor 7,7-dimethyl eicosadienoic acid (DEDA) reduced fluorescence in response to MeHg and TMCH by 49% and 54%. Simultaneous addition of L-NAME, CSA, and DEDA to the synaptosome fraction totally abolished the DCF fluorescence. In conclusion, C7 and C9 aliphatic, naphthenic, and aromatic hydrocarbons stimulated formation of ROS and RNS in rat brain synaptosomes. The naphthenic hydrocarbon TMCH stimulated formation of ROS and RNS in the synaptosomes through Ca2+-dependent activation of PLA2 and nNOS, and through increased transition permeability of the MTP. Exposure of humans to the naphthenic hydrocarbon TMCH may stimulate formation of free radicals in the brain, which may be a key factor leading to neurotoxicity.

Upregulation of c-myc gene accompanied by PU.1 deficiency in radiation-induced acute myeloid leukemia in mice

High-dose radiation exposure induces acute myeloid leukemia (AML) in C3H mice, most of which have a frequent hemizygous deletion around the D2Mit15 marker on chromosome 2. This region includes PU.1, a critical candidate gene for initiation of leukemogenesis. To identify novel cooperative genes with PU.1, relevant to radiation-induced leukemogenesis, we analyzed the copy number alterations of tumor-related gene loci by array CGH, and their expressions in primary and transplanted AMLs. For the induction of AMLs, C3H/He Nrs mice were exposed to 3 Gy of x-rays or gamma-rays. The genomic alterations of 35 primary AMLs and 34 transplanted AMLs obtained from the recipient mice transplanted the primary AMLs were analyzed by array CGH. According to the genomic alterations and mutations of the 235th arginine of PU.1 allele, we classified the radiogenic AMLs into three types such as Chr2(del) PU.1(del/R235-) AML, Chr2(del) PU.1(del/R235+) AML and Chr2(intact) PU.1(R235+/R235+) AML, to compare the expression levels of 8 tumor-related genes quantitatively by real-time polymerase chain reaction and cell-surface antigen expression. Results. In addition to well-known loss of PU.1 with hemizygous deletion of chromosome 2, novel genomic alterations such as partial gain of chromosome 6 were recurrently detected in AMLs. In this study, we found similarity between cell-surface antigen expressions of bone marrows and those of spleens in AML mice and significantly higher expressions of c-myc and PU.1 expression, especially in the PU.1-deficient (Chr2(del) PU.1(del/R235-)) AML and Chr2(del) PU.1(del/R235+) compared to Chr2(intact) PU.1(R235+/R235+) AMLs. The new finding on upregulation of c-myc and PU.1 in both and hemizygous PU.1-deficient AMLs and different genomic alterations detected by array CGH suggests that the molecular mechanism for development of radiation-induced AML should be different among three types of AML.