ALBERT

Description: Abstract 224 During hematopoiesis, all-trans-retinoic acid (ATRA), a natural derivative of vitamin A, has been shown to induce both myelomonocytic progenitor/stem cell differentiation and self-renewal. Although these opposing effects are likely to be partly due to developmental differences, it has been shown that pro- and anti-differentiation effects of ATRA are mediated by distinct retinoic acid receptor isotypes (RARα and RARγ, respectively). With the exception of acute promyelocytic leukemia (APL), ATRA treatment as a single agent has not been successful in other types of acute myeloid leukemia (AML). We have previously hypothesized that one of the underlying reasons for poor response of non-APL AML to ATRA (pan-RAR agonist) is aberrant expression and/or activities of RAR isotypes favoring RARγ and cell growth versus differentiation. Consistently, we have reported that expression of RARα isoforms, particularly ATRA-inducible RARα2, are down-regulated in AML (Blood. 2008; 111:2374). Epigenetic analysis of patient samples revealed that relative to normal CD33+ cells, the loss of RARα2 in AML is associated with a diminution in levels of histone histone H3 lysine 4 dimethylation (H3K4me2) on the ATRA-responsive RARA2 promoter (a modification associated with transcriptional activation). Interestingly, the H3K4me1/me2 demethylase LSD1/KDM1 (AOF2) is highly expressed in AML patients (www.proteinatlas.org). A number of small molecules that target this enzyme (LSD1i) are in development and, collectively, these data predict that the use of LSD1i will facilitate induction of expression of genes that are required for differentiation of AML cells. In this study we used tranylcypromine (TCP, a monoamine oxidase used as an antidepressant and anxiolytic agent in the clinical treatment of mood and anxiety disorders, respectively), which functions a time-dependent, mechanism-based inhibitor of LSD1. Here we show that TCP unlocked the ATRA-driven therapeutic differentiation response in non-APL AML cell lines including the TEX cell line, which is derived from primitive human cord blood cells immortalized by expression of the TLS-ERG oncogene. TEX cells are 〉90% CD34+, respond poorly to ATRA and mimic features of primary human AML and leukemia initiating cells (Leukemia. 2005; 19:1794). Consistent with this, ATRA/TCP treatment increased differentiation in primary patient samples. ATRA alone had in general only small effects in primary AML samples and TCP showed minimal activity in most cases. Furthermore, shRNA-mediated knockdown of LSD1 confirmed a critical role for this enzyme in blocking the ATRA response in AML cells. The effects of ATRA/TCP on AML cell maturation were paralleled by enhanced induction of genes associated with myelomonocytic differentiation, including direct ATRA targets. LSD1i treatment did not lead to an increase in genome-wide H3K4me2, but did increase H3K4 dimethylation of myelomonocytic differentiation-associated genes. Importantly, treatment with ATRA/TCP dramatically diminished the clonogenic capacity of AML cells in vitro and engraftment of cells derived from AML patients in vivo, suggesting that ATRA/TCP may also target leukemic stem cells. These data strongly suggest that LSD1 may, at least in part, contribute to AML pathogenesis by inhibiting the normal function of ATRA in myelomonocytic development and pave the way for effective differentiation therapy of AML. Disclosures: No relevant conflicts of interest to declare.

Description: Histone modifications play a crucial role in the regulation of gene expression by activating or inactivating transcription. The Polycomb Group Protein Enhancer of Zeste Homologue 2 (EZH2) mediates trimethylation of histone H3K27, thereby inducing gene silencing. Overexpression of EZH2 has been reported to be associated with metastases and cancer progression in solid tumors like breast cancer or prostate cancer. However, loss of function mutations or deletions of EZH2 occur in myeloid malignancies and T-ALL. These mutations result in a poor prognosis. The aim of this study was to analyze the relevance of histone modifications for therapy resistance in AML. FLT3-ITD positive MV4-11 leukemic cells that were continuously cultured in media containing the kinase inhibitor PKC412 became resistant not only to PKC412 but also to standard chemotherapeutics Cytarabin (AraC) and Daunorubicin. Western blot analysis identified an almost complete loss of H3K27me3 in resistant MV4-11 cells (MV4-11R). This was accompanied by loss of EZH2 protein in the MV4-11R compared to the sensitive MV4-11. To test for acquisition of drug resistance due to reduced H3K27me3 levels, lentiviral knock-down (KD) of EZH2 was performed in the sensitive MV4-11 leading to diminished H3K27me3 levels. Knock-down cells showed resistance to the apoptosis-inducing effects of PKC412 compared to scrambled controls. Furthermore, resistance to standard chemotherapeutics AraC and Daunorubicin could be also observed in MV4-11 KD cells compared to control. To verify whether diminished levels of H3K27me3 can cause a more general, FLT3-ITD-independent drug resistance, knock-down of EZH2 was performed in FLT3-WT AML cell lines HL60, Kasumi-1 and ML-1. Again, this led to resistance to the standard chemotherapeutics AraC and Daunorubicin. In order to investigate the regulation of EZH2 in MV4-11R, promoter methylation and microRNA expression analysis was performed revealing no regulation of EZH2 expression via both mechanisms. Instead, the reduction of EZH2 protein expression was depended on posttranslational mechanisms that could be counteracted by CDK1-inhibitors. CDK1-inhibitors restored EZH2 protein and H3K27 trimethylation levels as well as drug sensitivity. By analyzing EZH2 mRNA expression of 220 primary diagnosed AML patients, a trend towards low EZH2 mRNA expression and poor overall as well as relapse free survival could be demonstrated. Thus, EZH2- and H3K27me3 protein expression were also analyzed by immunohistochemistry in bone marrow biopsies from AML patients (N=126). H3K27me3 and EZH2 protein expression correlated closely (r= 0.9, p

Description: Abstract 893 Introduction: A European collaborative harmonization study involving 61 laboratories is being conducted under the auspices of the European Treatment and Outcome Study (EUTOS) for CML that aims to facilitate reporting of molecular BCR-ABL quantification results according to the International Scale (IS). The aim of this analysis was to investigate the effectiveness of this process and specifically the stability of conversion factors (CF) over time. Methods: The currently accepted way of adopting the IS is to establish and validate a laboratory-specific CF which is then used to convert local results to the IS. For round 1, preliminary CFs were calculated by centrally distributing standard samples containing 10–20 million WBC approximating to 10%, 1%, 0.1%, and 0.01% BCR-ABL IS. Rounds 2 and 3 were employed to refine the CF calculations using 25–30 CML patient samples from each participating laboratories covering a range of BCR-ABL levels between 0.01% and 10%. Log BCR-ABL values for the same samples were compared between reference and local laboratories applying the Bland-Altman bias plot. In order to judge the stability of each laboratory`s methodology, a CF index (ratio of round 3 CF divided by round 2 CF) was calculated and evaluated according to its capability to achieve optimum concordance of results. Results: Of the 61 laboratories participating in round 1, evaluable patient samples have been provided to date by 56 and 30 laboratories in rounds 2 and 3, respectively. Of the 30 laboratories with complete data, 12 had stable CFs (defined as a CF index within 0.75–1.33) whereas 18 laboratories were outside this range. Comparison of the CFs derived from round 2 with those derived from round 3 revealed better and more consistent concordance between laboratories with stable CFs compared to those with unstable CFs. For the 12 stable laboratories, 79% (round 3 CF) vs 79% (round 2 CF) of the samples were within a 2-fold range (0.5–2.0) and 93% vs 89% were within a 3-fold range (0.33–3.0). For the 18 unstable laboratories, 74% vs 55% of the samples were within a 2-fold range (0.5–2.0), p=0.0005 and 92% vs 77% were within a 3-fold range (0.33–3.0), p=0.0005. 2 of 12 laboratories with stable CFs and 8 of 18 laboratories with unstable CFs indicated changes in either one or more components of their procedures (cDNA synthesis, PCR platform, RQ-PCR protocol) that may have impacted on their CFs. Conclusion: These data indicate that CFs may be unstable in some laboratories even in the absence of significant changes to laboratory protocols. Further, it supports the need for continuous revalidation of CFs. In laboratories with unstable CFs we suggest revalidation within 3 to 6 months whereas those with stable CFs should be assessed on a yearly basis. We also suggest that laboratories with unstable CFs need to rigorously examine their internal processes to identify potential sources of variation. Disclosures: Müller: Novartis: Honoraria, Research Funding. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Description: Abstract 2325 Background: The medical backbone of GvHD prophylaxis has been considered the use of a calcineurin inhibitor (CNI) like cyclosporine or tacrolimus in combination with methotrexate (MTX) or mycophenolate mofetil (MMF). The mammalian target of rapamycin (mTOR) inhibitor everolimus (RAD001, Certican®) is a novel immunosuppressive drug and beside sirolimus approved in solid organ transplantation. Recently, it was reported that mTOR-inhibitors in combination with CNI showed promising clinical effectiveness in innovative prevention strategies of acute GvHD after allogeneic stem cell transplantation (SCT). In this single centre retrospective analysis, we report the outcome on patients (pts), treated interventional with everolimus due to severe toxicity induced by CNI-based GvHD prophylaxis. Patients and Methods: Ten pts (3 acute myeloid leukaemias, 1 myelodysplastic syndrome, 1 acute lymphoblastic leukaemia, 4 Non-Hodgkin-lymphomas) underwent allogeneic blood SCT between 7/2007 and 3/2010. The median age was 51 years [range: 24–64] with 6 pts in advanced stages of their disease. Myeloablative conditioning was used in 3 pts (12 G TBI/120mg/kg cyclophosphamide (cyclo)] and reduced-intensity conditioning in 7 pts [5× treosulfan/fludarabine (flu), 1× busulfan/flu and 1× amsacrine/cytarabine/flu followed by 4 G TBI/cyclo]. HLA-matched family donors in 4 pts and unrelated donors in 6 pts donated on day 0 a median of 4.73 × 106 kg per bodyweight CD-34 positive stem cells. GvHD-prophylaxis consisted of standard CNI+MTX in 3 pts, CNI+MMF in 6 pts and CNI+MMF+MTX in 1 patient. Antithymocyte globulin as in vivo T-cell depletion was given in 7 pts as part of the reduced intensity conditioning regime. Results: Everolimus (0.75 mg Certican ® twice a day orally) was individual started after a median of 14 days [range: 6–16] after SCT. Primary reasons for stopping CNI (median day +10 [range 1–15]) were nephrotoxicity (CTC ≥ grade 3) in 7 pts and neurotoxicity (CTC ≥ grade 3) in 3 pts. Two pts received steroids in addition to everolimus temporally, 5 pts MMF and 3 pts steroids+MMF. The intended plasma therapeutic level of everolimus was 3–8 mg/l. All patients showed neutrophil engraftment on median day +18 [range: 8–20] and platelet engraftment on day +21 [range: 10–88]. Acute GvHD ≥ grade II (Glucksberg/Consensus NHI) occurred in 3pts (30%) (2× grade II, 1× grade III) or IBMTR-Index-Score ≥ B (2×B, 1×C). Chronic GvHD limited disease score grade I (Consensus NIH) was observed in 3 pts (n=1 overlap syndrome and n=2 de-novo cGvHD). Two out of 7 high risk pts for CMV reactivation developed viral activation. Until day +100 3 pts stopped interventional GvHD prophylaxis due to oral bleeding on day +46 (n=1) and assumed TMA on day +57/+64 (n=2). The period of everolimus administration was 140 days on average [range: 19–509]. As per August 2010 6 pts are alive resulting in a median cumulative overall survival of 58%. ALL-relapse had to be observed on day +312 in one patient. Fatal casualties occurred on day +19 by multiorgan failure (MOD), on day +103 by pneumonia, on day +211 by questionable pulmonary embolism and on day+ 411 by MOD and limited cGvHD. Conclusions: A delayed CNI-free GvHD-prevention strategy with the m-TOR inhibitor everolimus is feasible and efficacious in a series of patients after allogeneic SCT. The manageable toxicity profile in our cohort with pre-existing organ dysfunction at treatment onset mandates further evaluation of everolimus after SCT. A prospective multicentre phase II trial of everolimus as intended early replacement for CNI after SCT with a focus on tolerability is scheduled to start soon. Disclosures: Sayer: Novartis Pharma: Honoraria, Research Funding. Off Label Use: Everolimus -Certican (R)- is only approved as a immunosuppressive drug in solid organ transplantation. Hochhaus: Novartis: Consultancy, Research Funding.

Description: Abstract 2272 Background and Aim: Dialysis based approaches can provide rapid removal of dabigatran in cases of emergency due to its low protein binding of ∼35%. However the in vitro properties of these filtration devices have not yet been characterized in detail. This study in the porcine system (both in vitro and in vivo) was performed to evaluate dabigatran elimination by hemodialysis and activated charcoal perfusion as compared to normal renal elimination. Methods: Porcine blood (5L) was supplemented with 1000 ng/mL dabigatran and circulated through an circuit of tubing allowing attached to an activated charcoal filter (Absorba 300 C, Gambro). Further supplementation of dabigatran allowed the determination of maximum binding capacity of the filter. A similar set up was used to also test dialysis (Polyflux 140H, Gambro) and determine the dependence of the flow rate on dabigatran removal. Dialysate flow rates were increased up to 500 ml/min. Anesthetized pigs (Domestic swine, female, ∼60 kg) were attached to an activated charcoal column or a High-Flux hemodialysis filter with a blood flow rate of 200 ml/min. Animals were given an initial i.v. infusion of dabigatran (7.5 mg in 15 min) and then reduced to 5 mg/hr to achieve steady state dabigatran over 1hr. Infusion was then stopped and elimination methods were applied over 4 hrs. An observation time of 1 hr followed. Dabigatran plasma levels were quantitated with diluted thrombin time. Preliminary settings/flow rates were obtained in vitro using 5L citrated porcine whole blood exposed to different AC or HD conditions. Results: Activated charcoal completely removed dabigatran within 1 hr from the 5L whole blood supplemented with 1000 ng/mL dabigatran, with a clearance rate of 100%. By repeatedly reapplying dabigatran, it was shown the active charcoal filter had a maximum binding capacity of ∼30 mg drug. Upon saturation there was no further clearance of dabigatran. Hemodialysis removed dabigatran with increasing clearance rates depending on dialysate flow rates (100 ml/min-35%, 200 ml/min-60%, 300 ml/min-65%) reaching a plateau of ∼65%. Further increases of dialysate flow to 500 ml/min had no further effect on drug clearance. Initial plasma levels of dabigatran ranged between 200–450 ng/mL after 60 min infusion in pigs. Exposure to activated charcoal or hemodialysis (dialysate flow 300 ml/min) resulted in 75–80% reduction in circulating dabigatran after 1 hr as compared to ∼25% reduction untreated controls after 1 hr. After 2 hrs dabigatran levels were below the detection limit using both elimination methods. Conclusions: Dabigatran can effectively be removed from the circulation in this in vivo porcine model using dialysis based approaches, which results in a restoration of blood coagulation. Active charcoal perfusion was fast and effectively removed dabigatran, but may be saturated if dabigatran plasma levels are too high (human body load for 150 mg dose in steady state is ∼14g). Stationary hemodialysis with sufficiently high dialysate flows achieves similar results in this model without saturation limitations; however, the set up for dialysis is much more specialized than the simpler approach of activated charcoal filtration. Disclosures: Formella: Boehringer Ingelheim: Employment. Clemens:Boehringer Ingelheim (Anticoagulant Therapy): Employment. van Ryn:Boehringer Ingelheim: Employment. Schenk:Boehringer Ingelheim: Research Funding.

Description: Abstract 3271 Introduction: Hemophagocytic Lymphohistiocytosis (HLH) is a rare and potentially life-threatening disorder in adults. Patients (pts) with fever of unknown origin, lymphadenopathy, deteriorating performance status, encephalopathy and elevated systemic inflammatory parameters frequently show no clear cut diagnostic criteria specific for HLH. Among adults, late onset of inherited HLH is possible, but acquired HLH triggered by infection, neoplasia or autoimmune disease is more frequent than in pediatrics. Due to the variability of clinical presentation, large-scale unreporting can be assumed. We therefore commenced a German registry for HLH pts, aiming to sensitize clinicians and to establish an HLH network as starting point for research and clinical guideline activities. Methods: We performed a retrospective multicenter case study in Germany. Diagnostic criteria were derived from the pediatric revised HLH-04 protocol. Treatment and outcome parameters were evaluated. Results: In 29 adult pts registered so far, median age was 46 years (18–81), 13 pts were female. HLH in 10 pts (34%) was malignancy-associated (aggressive B-cell lymphoma, n=6; B-CLL n=1; AML n=2; melanoma n=1), infection-associated (IHLH) in 13 pts (45%), 2 pts (7%) had connective tissue disease (CTD), in 1 pt HLH developed after therapeutic plasma exchange for TTP and in 3 pts (10%) the cause could not be determined. The main pathogen in IHLH pts was EBV (9/29, 31%) followed by other viruses (CMV n=1; HSV n=1; VZV n=1; Parvovirus B19 n=1). Of the 13 IHLH pts, 4 pts showed an EBV-reactivation post-allogeneic stem cell transplantation (SCT) for acute leukemia (n=3) and severe aplastic anemia (n=1) and 1 pt a systemic VZV infection after autologous peripheral blood SCT for aggressive B-cell NHL. 2 pts showed an EBV- and a lymphoma-triggered HLH respectively during late pregnancy. In 2 pts with EBV-associated HLH sequencing revealed mutations in XIAP and XLP-1 genes. The median time from onset of symptoms to diagnosis was 14 days (range 1–60 days). Most pts received polyvalent immunoglobulin (20/29, 69%) and HLH-04 based therapy, including steroids (14/29, 48%), cyclosporine (6/29, 21%), and etoposide (10/29, 34%). 5 pts (17%) received rituximab and 3 pts (10%) R-CHOP for lymphoma. 2 pts each (7%) received alemtuzumab and cyclophosphamide. One patient underwent allogeneic SCT but died. With a median follow up of 413 days (range, 54–1204 days), 12 pts (41.3%) survived. 2 pts were lost to follow-up. Median survival after diagnosis was 151 days (range, 14–1204+ days). Conclusions: Adult pts with HLH in our cohort showed a heterogeneous clinical syndrome most frequently triggered by EBV. HLH represents a final common pathway of an aberrantly activated immune system responding to a variety of triggers. If not recognized and treated early, HLH in adults has a very poor prognosis. Clinical registries need to include adult HLH pts in order to facilitate basic and clinical research to improve outcome of this frequently fatal immune response syndrome. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3468 The development of drug resistance is a common feature in AML that occurs towards classical cytotoxic drugs as well as novel kinase inhibitors. Mutations in primary drug targets explain a significant fraction of acquired drug resistance but the mechanisms of resistance still remain unknown in most patients. About 20–30% of all AML patients possess the mutant FLT3-ITD leading to a constitutive activation of the FLT3 kinase. Flt3-mutations can be targeted by treatment with tyrosine kinase inhibitors. Resistance to these kinase inhibitors is an increasing phenomenon whose mechanisms are not entirely understood today. As epigenetic mechanisms are shown to play an important role in leukemia pathogenesis they are also likely to influence drug resistance. To elucidate the potential role of epigenetic mechanisms in the development of drug resistance towards kinase inhibitors we used a PKC412 partially resistant clone (MV4-11R) of the AML cell line MV4-11, which harbors a homozygous FLT3 internal tandem duplication (ITD) mutation. An initial screening for histone modifying enzymes revealed a downregulation of EZH2 on mRNA as well as protein level compared with the parental cell line. The reduction of EZH2, a H3K27 methyltransferase, in MV4-11R is furthermore correlating with globally diminished H3K27me3 levels. ChIP-Seq experiments using H3K27me3 antibody revealed differences in histone 3 K27 methylation at specific promoter sites between the parental and resistant MV4-11. To test for an increased drug resistance due to reduced EZH2 protein levels lentiviral knock-down of EZH2 was performed in the MV4-11 parental cell line and three individual knock-down cell clones were investigated for their drug resistance potential. These knock downs all showed elevated IC50 values as well as resistance towards the apoptosis-inducing effects of PKC412 compared with the scrambled shRNA cells. Furthermore, EZH2 protein levels of 5 FLT3-ITD-positive AML patient samples were determined by Western Blot, samples were treated with PKC412 for 3 days and cell survival was assayed. Using this approach higher EZH2 levels in patients could also be associated with a higher sensitivity to PKC412 pointing to a putative role of EZH2 in the development of PKC412 resistance in vivo. As EZH2 has been shown to interact with DNMTs in the context of the Polycomb Repressive Complex 2 and 3 (Viré et al., Nature 2006), we analyzed whether parental and resistant MV4-11 differ in their DNA methylation pattern. To identify hyper-/hypomethylated genes in MV4-11R, we applied the Illumina 27k Methylation BeadChip approach as well as Reduced Representation Bisulfite Sequencing (RRBS) for a genome wide CpG methylation analysis. In particular genes associated with apoptosis pathways and signal transduction were hypermethylated in MV4-11R cells compared to the parental cell line. Based on the observation of DNA methylation changes between the parental cell line and MV4-11R, treatment with the demethylating agent 2-deoxy-5-azacytidine (Aza dC) was conducted to investigate recovery of drug sensitivity. Incubation with 250 nM Aza dC for 5 days could restore the sensitivity of MV4-11R towards PKC412 as shown in proliferation and apoptosis assays. Using the Affymetrix Human Gene 1.0 ST Array platform we identified 110 genes whose expression was reactivated after treatment of MV4-11R with Aza dC, predominantly genes playing a role in signal transduction, apoptosis pathways and cell cycle. A cell cycle analysis of the MV4-11 and MV4-11R cells indeed showed that the resistant cell line is cycling less than the parental one, which possibly also favors the resistance development towards PKC412. Summarized, our data show that a loss of EZH2 accompanied by DNA methylation changes lead to PKC412 resistance in a FLT3-ITD AML cell line model possibly reflecting a way of acquisition of drug resistance in patients. Disclosures: Thiede: Novartis: Lectures, Research Funding. Müller-Tidow:Novartis: Research Funding.

Description: Acquired hemophilia A (AHA) is a rare autoimmune disorder caused by neutralizing autoantibodies against coagulation factor VIII (FVIII:C). Immunosuppressive treatment may result in remission of disease over a period of days to months. Until remission, patients are at high risk of bleeding and complications from immunosuppression. Prognostic parameters to predict remission and the time needed to achieve remission could be helpful to guide treatment intensity, but have not been established so far. GTH-AH01/2010 was a prospective multicenter cohort study using a standardized immunosuppressive treatment protocol. The primary study endpoint was time to achieve partial remission (PR, defined as FVIII:C activity 〉50 IU/dl after cessation of any hemotherapy for 〉24h, and no active bleeding). Secondary endpoints were time to achieve complete remission (CR, defined as PR plus negative FVIII:C inhibitor, steroid tapered to