Prognostic Factor In AML Research Articles

AML Treatment By the AXL Inhibitor Bemcentinib in Combination with Cytarabine Shows Clinical Efficacy Related to TNFα and Cytotoxic Immune Cells: A Single-Cell Translational Study from the BGBC003 Trial

Acute myeloid leukemia can be treated by intensive chemotherapy. However, this treatment is unsuitable for a subset of patients because of age and/or co-morbidities. Such patients benefit from hypomethylating agents and venetoclax but frequently develop resistance to this treatment regimen. This calls for the development of novel drugs that can prolong survival in AML patients unfit for intensive chemotherapy. The receptor tyrosine kinase AXL is a negative prognostic factor in AML. Its activation promotes survival, chemoresistance and proliferation of AML blasts. In addition, it is also present on innate immune cells and contributes to the formation of immunosuppressive environments. Together, this makes AXL a promising target for the treatment of AML. Bemcentinib is a first-in-class, orally available, selective inhibitor of AXL. Here, we report a single-cell translational sub-study in a sub-cohort of patients unfit for intensive chemotherapy (B2+B5) from the phase Ib/II clinical trial BGBC003 (NCT02488408). This sub-cohort was treated by a combination of bemcentinib and low-dose cytarabine (LDAC). To gain first insights into the mechanisms underlying treatment response, we conducted a translational study evaluating 13 participating patients. Among 32 patients evaluated for efficacy, 8 patients responded to their treatment (objective response rate: 25%). Among patients who received previous therapy ( i.e. relapsed/refractory patients), objective response rate was 18.5% (5/27). The median overall survival was 8.0 months, with a notable survival benefit (median overall survival of 24.8 months) in responders. Relapsed/refractory patients exhibited a median overall survival of 7.8 months. These data highlight that bemcentinib-LDAC benefits a considerable subset of AML patients unfit for intensive chemotherapy. In order to gain mechanistic insights into the efficacy of bemcentinib-LDAC, we profiled cells from bone marrows of 13 participating patients (6 responders, 7 non-responders, according to best response) using single-cell transcriptomics and multi-omics (CITE-seq). Cell type annotation highlighted various immune cell populations next to AML blasts. Successful treatment was associated with stronger TNFα signaling in blasts before treatment. A tight link between TNFα and AXL could subsequently be established in vitro as several AML cell lines up-regulated expression of AXL upon exposure to TNFα. Inhibiting AXL in these cell lines using bemcentinib increased expression of TNFα. This indicates a potential negative feedback loop between these two players. Furthermore, cytotoxic immune cells (CD8 + effector T cells, γδ T cells and natural killer cells) from responders displayed evidence for increased pro-inflammatory signaling upon bemcentinib-LDAC treatment. This included TNFα signaling programs next to IFNα and IFNγ signaling programs. Thus, our data indicate that bemcentinib-LDAC promotes the activity of such cytotoxic cells. In line with this, we observed increasing crosstalk between immune cells upon bemcentinib-LDAC treatment in responders. Importantly, cells from non-responders generally exhibited the opposite trend, highlighting the aforementioned cell types and pathways as factors that distinguish responders from non-responders. Together, our results indicate a potential role of TNFα and cytotoxic immune cells in the successful application of bemcentinib-LDAC. In conclusion, our findings warrant further clinical development of bemcentinib-LDAC for AML patients unfit for intensive chemotherapy. Additionally, extended research on TNFα and cytotoxic immune cells may lead to a better understanding of what mechanisms cause a treatment response. This may ultimately enable an accurate selection of patients who will benefit from bemcentinib-LDAC.

IL-37 Was Involved in Progress of Acute Myeloid Leukemia Through Regulating IL-6 Expression.

BackgroundInterleukin-37, which was discovered in 2000, is a natural suppressor of immune and inflammatory responses. Recent studies reported that IL-37 was abnormally expressed in several tumor patients, including those with hepatocellular carcinoma, gastric cancer, lung cancer, colon cancer, epithelial ovarian cancer, and multiple myeloma. However, the expression and potential function of IL-37 in leukemia remain unknown.ObjectiveThe aim of this study was to evaluate IL-37 as a prognostic factor and its possible mechanism of action.MethodsPolymerase chain reaction products were analyzed by agarose gel electrophoresis and were purified and subsequently sequenced by a genetic testing laboratory. Human PBMC was purified from whole blood samples by using Ficoll-Paque PLUS. The concentrations of human IL-37 and human IL-6 were measured using enzyme-linked immunosorbent assay (ELISA) kits.ResultsIL-37, especially isoform b and d, was expressed in the bone marrow of AML, CML, ALL, and CLL. Importantly, IL-37 expression was downregulated in newly diagnosed AML patients and restored in patients in complete remission. Moreover, a significant association was found between IL-37 expression and NPM1 mutation or possible prognosis evaluated by karyotype and gene mutation. Further analysis revealed that IL-37 expression was negatively correlated with IL-6 expression. With regard to the mechanism, recombinant human IL-37 could suppress IL-6 expression stimulated by LPS in PBMC of AML patients.ConclusionOur study suggested that IL-37 may be an important prognostic factor in AML and is involved in AML via the IL-6 signaling pathway, indicating that IL-37 is an innovative research strategy for AML pathogenesis and therapy.

Molecular Predictors and Effectiveness of Measurable Residual Disease (MRD) Eradication with Chemotherapy and Allogeneic Stem Cell Transplantation for Acute Myeloid Leukemia

Molecular Predictors and Effectiveness of Measurable Residual Disease (MRD) Eradication with Chemotherapy and Allogeneic Stem Cell Transplantation for Acute Myeloid Leukemia

Cytogenetic Pattern in Adult Patients with de novo Acute Myeloid Leukaemia: A Single Centre Study in Bangladesh

Background: Cytogenetic analysis performed at diagnosis is considered to be the most important prognostic factor in AML. Objective: The purpose of this study was to observe the pattern of cytogenetic abnormalities in adult patients with de novo AML. Method: Total fifty-two newly diagnosed de novo AML patients were selected for the study. Six cytogenetic abnormalities including t(8;21), t(15;17), inv(16), BCR-ABL1, FLT3-ITD & NPM1 mutations were detected by Real-Time PCR. Results: In this study, 36 (69.2%) patients showed different cytogenetic abnormalities. The t(15;17) was found to be the most common. t(15;17), t(8;21) and inv(16) were found only in M3, M2 and M4 FAB subtypes, respectively. Significant association was found with increasing age and cytogenetic risk groups. BCR-ABL1 mutation showed significant relation with increased age. FLT3-ITD mutation showed significant association with increased WBC count and inv16 was significantly associated with relatively less bone marrow blast percentage. Conclusion: Cytogenetic study should be performed routinely in all cases of AML for proper diagnosis, prediction of prognosis and implementation of effective therapeutic measures.

Final Analysis of the Dose Escalation, Expansion and Biomarker Correlations in the Ph I/II Trial BGBC003 with the Selective Oral AXL Inhibitor Bemcentinib (BGB324) in Relapsed/Refractory AML and MDS

Final Analysis of the Dose Escalation, Expansion and Biomarker Correlations in the Ph I/II Trial BGBC003 with the Selective Oral AXL Inhibitor Bemcentinib (BGB324) in Relapsed/Refractory AML and MDS

Predictive and Prognostic Markers in Adults With Acute Myeloid Leukemia: A Single-Institution Experience

BackgroundAcute myeloid leukemia (AML) is a heterogeneous malignancy with diverse genetic abnormalities, clinical presentations, and outcomes. Known predictive and prognostic factors in AML include age, performance status, comorbidities, cytogenetics, and molecular mutations. Identifying prognostic and predictive factors can inform the choice of induction therapy and outcomes prediction. Patients and MethodsA retrospective review was performed of 137 adult AML patients from 2010 to 2015. Predictors of complete remission (CR) and overall survival (OS) were determined for patients treated with 3+7 (3 days of anthracycline and 7 days of cytarabine) or hypomethylating agent. Variables associated with CR or OS were assessed using univariate Cox regression and a multivariate Cox model. ResultsThe average age was 65 years and 91 patients (66%), sample size is 137 patients had primary AML. Patients in the 3+7 induction group were younger, had a higher bone marrow blast percentage, and more de novo AML compared with those in the hypomethylating agent group (P < .001, P < .001, P = .005, respectively). Univariate logistic regression for CR showed a significant association between age (P < .001), choice of induction (P < .001), and monosomy (P = .015), although only induction with 3+7 (P < .001) and absence of monosomy (P = .042) remained significant in multivariate analysis. Univariate Cox regression indicated that age (P = .003), AML status (de novo or secondary; P = .0277), choice of induction (P = .030), and monosomy (P = .010) had a significant association with OS. Only younger age (P = .018) and absence of monosomy (P = .022) were predictive of OS in multivariate Cox analysis. ConclusionPositive predictors of CR in adult AML include absence of monosomy and induction treatment with 3+7; whereas positive predictors of OS are younger age and absence of monosomy.

High expression of ETS2 predicts poor prognosis in acute myeloid leukemia and may guide treatment decisions

BackgroundETS2 is a downstream effector of the RAS/RAF/ERK pathway, which plays a critical role in the development of malignant tumor. However, the clinical impact of ETS2 expression in AML remains unknown. MethodsIn this study, we evaluated the prognostic significance of ETS2 expression using two relatively large cohorts of AML patients.ResultsIn the first cohort, compared to low expression of ETS2 (ETS2low), high expression of ETS2 (ETS2high) showed significant shorter OS, EFS and RFS in the current treatments including the allogeneic HCT group (n = 72) and the chemotherapy group (n = 100). Notably, among ETS2high patients, those received allogeneic HCT had longer OS, EFS and RFS than those with chemotherapy alone (allogeneic HCT, n = 39 vs. chemotherapy, n = 47), but treatment modules play insignificant role in the survival of ETS2low patients (allogeneic HCT, n = 33 vs. chemotherapy, n = 53). Moreover, gene/microRNA expression data provides insights into the biological changes associated with varying ETS2 expression levels in AML. The prognostic value of ETS2 was further validated in the second AML cohort (n = 329).ConclusionsOur results indicate that ETS2high is a poor prognostic factor in AML and may guide treatment decisions towards allogeneic HCT.

Immunophenotypic Profile in Adult Patients with Acute Leukemia Association with Clinical Feature: Fluorescence Cytometry Quantitative Analysis

Antigen surface markers represent as the new prognostic tool for detection of acute leukemia. To investigate the prevalence expression of lymphoid and myeloid antigen lineage in acute leukemias. This study included 100 acute leukemias patients. Specimens were selected from consecutive patients who had sufficient material available. Among the 100 patients in which a detailed history, hematological, clinical and immunophenotyping analysis were performed. This study was showed distribution of immunophenotyping characters between studied AML and ALL cases. The most abundant immunophenotyping features in acute myeloid leukemia were cMPO, CD33, CD117, CD13, CD14 and CD64, while the most abundant immunophenotyping features in acute lymphoblastic leukemia were CD19, CD79a, TdT, CD20, CD10 and CD34. cMPO which act as independent prognostic factor for AML, CD10 and TdT can used as independent prognostic factor for differentiate between ALL and AML.

Time Profile of Time-Dependent Area Under the ROC Curve for Survival Data

In the setting of survival analysis, the time-dependent area under the receiver operating characteristic curve (AUC) has been proposed as a discrimination measure of interest. In contrast with the diagnostic setting, the definitions of time-dependent sensitivity and specificity are required. This paper evaluates the time-dependent profile of the resulting AUC(t), which has not been previously assessed. We show that, even when the effect of a binary biomarker on the hazard rate is constant, the value of AUC(t) varies over time according to the prevalence of the marker. The Time-profile of the continuous biomarker is illustrated with numerical integration, and data on several prognostic factors in AML are examined.

ARC Is Regulated by MAPK and PI3K and Confers Drug Resistance and Survival Advantage to AML in vitro and in vivo

Abstract Abstract 893 ARC (Apoptosis repressor with caspase recruitment domain) is a unique antiapoptotic protein that has been shown to suppress the activation of both intrinsic and extrinsic apoptosis. We previously reported that ARC is one of the most potent adverse prognostic factors in AML and that high ARC protein expression predicted shorter survival and poor clinical outcome in patients with AML (Carter BZ et al., Blood 2011). Here we report how ARC is regulated and its role in inhibition of AML apoptosis and in cell survival. We provide evidence that ARC expression is regulated by MAPK and PI3K signaling. Inhibition of MAPK and PI3K pathways decreased ARC mRNA and protein levels in AML cells. ARC expression in AML cells is upregulated in co-cultures with bone marrow-derived mesenchymal stromal cells (MSCs) and the upregulation is suppressed in the presence of MAPK or PI3K inhibitors. To investigate the role of ARC in apoptosis resistance in AML, we generated stable ARC overexpressing (O/E) KG-1 and stable ARC knock down (K/D) OCI-AML3 and Molm13 cells and treated them with Ara-C and agents selectively inducing intrinsic (ABT-737) or extrinsic (TRAIL) apoptosis. We found that ARC O/E cells are more resistant and ARC K/D cells more sensitive to Ara-C, ABT-737, and TRAIL-induced apoptosis: EC50s of Ara-C, ABT-737, or TRAIL treatment at 48 hours for ARC O/E KG-1 and control cells were 1.5 ± 0.1 μM vs. 83.5 ± 4.6 nM, 2.2 ± 0.2 μM vs. 60.2 ± 3.1 nM, or 0.97 ± 0.03 μg/mL vs. 0.17 ± 0.08 μg/mL, respectively and for ARC K/D OCI-AML3 and control cells were 0.33 ± 0.02 μM vs. 3.4 ± 0.2 μM, 0.24 ± 0.01 μM vs. 1.3 ± 0.1 μM, or 0.13 ± 0.09 μg/mL vs. 0.36 ± 0.03 μg/mL, respectively. Bone marrow microenvironment is known to play critical roles in AML disease progression and in protecting leukemia cells from various therapeutic agent-induced apoptosis. Leukemia cells were co-cultured with MSCs in vitro study to mimic the in vivo condition. ARC was found to be highly expressed in MSCs and stable ARC K/D MSCs were generated. AML cell lines and primary patient samples were co-cultured with ARC K/D or control MSCs and treated with Ara-C, ABT-737, or TRAIL. Interestingly, ARC K/D MSCs lost their protective activity for leukemia cells treated with these agents. EC50s for OCI-AML3 cells co-cultured with ARC K/D or control MSCs for 48 hours treated with Ara-C, ABT-737, or TRAIL were 1.0 ± 0.04 μM vs. 4.5 ± 0.2 μM, 0.15 ± 0.06 μM vs. 0.53 ± 0.02 μM, or 1.4 ± 0.8 μg/mL vs. 8.1 ± 0.3 μg/mL, respectively. In addition, ARC O/E KG-1 cells grew faster and ARC K/D OCI-AML3 and Molm13 cells and ARC K/D MSCs grew slower than their respective controls. We then injected KG-1 cells into mice and found that NOD-SCID mice harboring ARC O/E KG-1 had significantly shorter survival than mice injected with the vector control KG-1 (median 84 vs. 111 days) as shown in the figure. Download : Download high-res image (24KB) Download : Download full-size image Collectively, results demonstrate that ARC plays critical roles in AML. ARC is regulated by MSCs through various signaling pathways in AML cells, protects leukemia cells from apoptosis induced by chemotherapy and by agents selectively inducing intrinsic and extrinsic apoptosis. ARC regulates leukemia cell growth in vitro and in vivo. The results suggest that ARC is a potential target for AML therapy. In addition, targeting ARC in MSCs suppresses microenvironmental protection of AML cells. Disclosures: No relevant conflicts of interest to declare.

Novel Cryptic Collagen IV Mediated Activation of Discoidin Domain Receptor 1 (DDR1) in Bone Marrow Microenvironment May Promote Migration in Myeloid Leukemia.

Abstract 2429Acute myeloid leukemia (AML) represents a heterogeneous group of malignancies with huge variability in clinical course and response to therapy. Discovery of new prognostic factors in AML is enhancing our understanding of disease biology and helping to identify new therapeutic targets. Previously, we showed for the first time that Discoidin Domain Receptor 1 (DDR1), a class of collagen-activated receptor tyrosine kinase, was highly expressed on bone marrow derived CD33+ leukemic blasts of AML patients, however, its functional role was not known. Here in, we attempt to understand the role of the bone marrow microenvironment in promoting DDR1 activation and its subsequent functional role in migration of leukemic cells. Initial screening of several leukemic cell lines for DDR1 expression identified K562 as an optimal system for further studies due to high DDR1 expression. In addition to native collagen IV, the current studies also tested for the first time the ability of the cryptic collagen IV in activation of DDR1. Proteolytic remodeling of the collagenous extracellular matrix (ECM) have been shown to result in generation of cryptic collagen epitopes, which under normal physiologic conditions, are masked in its 3-dimensional structure. Previously, the functional cryptic site of collagen IV was shown to be highly expressed within the extracellular matrix (ECM) of malignant tumors and within the sub-endothelial basement membrane of tumor-associated blood vessels. Presently, exposure of K562 cells to denatured collagen IV resulted in statistically significant (p<0.045) migration rates compared to native collagen IV. Further, exposure of denatured collagen IV lead to increased activation of DDR1 in comparison to native collagen IV as assessed via phosphorylation of DDR1 at activation sites of Y792, Y796 and Y797 respectively using phospho-DDR1 sandwich ELISA approach. Importantly, co-incubation with HU-IV 26, a previously characterized monoclonal antibody against cryptic collagen IV epitope significantly (p<0.0003) decreased the phospho-DDR1 levels. Interestingly co-incubation with HU-IV 26 did not significantly alter the migration rates between native and denatured collagen IV. To test the role of matrix metalloproteinases (MMPs) in DDR1 induced migration, MMP2 and MMP9 levels were assayed via zymogram and western blots using conditioned media to assess levels of secreted MMPs. Overall, active and inactive MMP-2 levels were higher when exposed to native or denatured collagen IV compared to serum-free only culture conditions. Currently these outcomes suggest a possible feed forward loop for collagen IV-DDR1-MMP2-cryptic collagen IV in promoting migration of leukemic cells. We next examined the levels of collagen IV and cryptic collagen IV in bone marrow derived leukemic blasts of AML patients (n=24) using HU-IV 26 and a native collagen IV antibody via generating a tissue microarray (TMA) and performing immunohistochemistry (IHC). In comparison to control samples (n=9), native collagen and cryptic collagen IV levels were found in significantly high levels (p<0.019 & p<0.034 respectively). Most importantly, the levels of cryptic collagen IV were significantly higher than native collagen (p<0.05) in AML patients. Significantly, the DDR1 levels were concurrently elevated in the same AML patients with high cryptic collagen IV as examined via TMA and IHC. Taken together, for the first time we demonstrate that cryptic collagen IV is a potent activator of DDR1 and high levels of cryptic collagen IV is expressed in leukemic cells of AML patients. Further understanding of this phenomenon may lead to the development of therapeutic agents that directly modulate the bone marrow microenvironment and attenuate leukemogenesis. Disclosures:No relevant conflicts of interest to declare.

ARC Is Regulated by MAPK and PI3K and Confers Drug Resistance and Survival Advantage to AML in Vitro and in Vivo

Abstract 893ARC (Apoptosis repressor with caspase recruitment domain) is a unique antiapoptotic protein that has been shown to suppress the activation of both intrinsic and extrinsic apoptosis. We previously reported that ARC is one of the most potent adverse prognostic factors in AML and that high ARC protein expression predicted shorter survival and poor clinical outcome in patients with AML (Carter BZ et al., Blood 2011). Here we report how ARC is regulated and its role in inhibition of AML apoptosis and in cell survival.We provide evidence that ARC expression is regulated by MAPK and PI3K signaling. Inhibition of MAPK and PI3K pathways decreased ARC mRNA and protein levels in AML cells. ARC expression in AML cells is upregulated in co-cultures with bone marrow-derived mesenchymal stromal cells (MSCs) and the upregulation is suppressed in the presence of MAPK or PI3K inhibitors.To investigate the role of ARC in apoptosis resistance in AML, we generated stable ARC overexpressing (O/E) KG-1 and stable ARC knock down (K/D) OCI-AML3 and Molm13 cells and treated them with Ara-C and agents selectively inducing intrinsic (ABT-737) or extrinsic (TRAIL) apoptosis. We found that ARC O/E cells are more resistant and ARC K/D cells more sensitive to Ara-C, ABT-737, and TRAIL-induced apoptosis: EC50s of Ara-C, ABT-737, or TRAIL treatment at 48 hours for ARC O/E KG-1 and control cells were 1.5 ± 0.1 μM vs. 83.5 ± 4.6 nM, 2.2 ± 0.2 μM vs. 60.2 ± 3.1 nM, or 0.97 ± 0.03 μg/mL vs. 0.17 ± 0.08 μg/mL, respectively and for ARC K/D OCI-AML3 and control cells were 0.33 ± 0.02 μM vs. 3.4 ± 0.2 μM, 0.24 ± 0.01 μM vs. 1.3 ± 0.1 μM, or 0.13 ± 0.09 μg/mL vs. 0.36 ± 0.03 μg/mL, respectively.Bone marrow microenvironment is known to play critical roles in AML disease progression and in protecting leukemia cells from various therapeutic agent-induced apoptosis. Leukemia cells were co-cultured with MSCs in vitro study to mimic the in vivo condition. ARC was found to be highly expressed in MSCs and stable ARC K/D MSCs were generated. AML cell lines and primary patient samples were co-cultured with ARC K/D or control MSCs and treated with Ara-C, ABT-737, or TRAIL. Interestingly, ARC K/D MSCs lost their protective activity for leukemia cells treated with these agents. EC50s for OCI-AML3 cells co-cultured with ARC K/D or control MSCs for 48 hours treated with Ara-C, ABT-737, or TRAIL were 1.0 ± 0.04 μM vs. 4.5 ± 0.2 μM, 0.15 ± 0.06 μM vs. 0.53 ± 0.02 μM, or 1.4 ± 0.8 μg/mL vs. 8.1 ± 0.3 μg/mL, respectively.In addition, ARC O/E KG-1 cells grew faster and ARC K/D OCI-AML3 and Molm13 cells and ARC K/D MSCs grew slower than their respective controls. We then injected KG-1 cells into mice and found that NOD-SCID mice harboring ARC O/E KG-1 had significantly shorter survival than mice injected with the vector control KG-1 (median 84 vs. 111 days) as shown in the figure. [Display omitted] Collectively, results demonstrate that ARC plays critical roles in AML. ARC is regulated by MSCs through various signaling pathways in AML cells, protects leukemia cells from apoptosis induced by chemotherapy and by agents selectively inducing intrinsic and extrinsic apoptosis. ARC regulates leukemia cell growth in vitro and in vivo. The results suggest that ARC is a potential target for AML therapy. In addition, targeting ARC in MSCs suppresses microenvironmental protection of AML cells. Disclosures:No relevant conflicts of interest to declare.

Analysis of the IDH1G105 (SNPrs11554137) Polymorphism in 961 AML Patients and in a Large Cohort of 475 Healthy Controls

Abstract 2518 Introduction:Mutations in IDH1 and IHD2 at arginines 132 and 140 or 172, respectively, have recently been shown to play an important role in AML. Also the IDH1105GGT minor allele of the IDH1G105 (SNPrs11554137) polymorphism that is localized in the same exon as the IDH1R132 mutation has recently been reported to be an adverse prognostic factor in AML (JCO: 14, 2356–2364, 2010). Aim: We aimed at further delineating the frequency and impact of the IDH1105GGT minor allele in AML and also analyzed a healthy control cohort. Methods:IDH1G105 (SNPrs11554137) was analyzed in 961 AML patients by a LightCycler-based melting curve assay. Female/male ratio was 433/528 and age ranged from 13.1–100.4 years (median, 66.7 years). The results were compared to a healthy control cohort from the KORA (Cooperative Health Research in the Region of Augsburg) survey S4, which consists of 475 cases who where matched to the leukemia samples with respect to sex (193f/282m) and age (median: 67, range 32–81 years). Informed consent for participation in anonymized genetic studies was obtained from all individuals. IDH1G105 in the healthy cohort was analyzed by Sanger sequencing. Further mutation analyses were available in subsets of the AML patients, respectively, as follows: (IDH1R132 n=625, IDH2R140 n=587, IDH2R172 n=590, FLT3-ITD n=629, FLT3-TKD n=503, NPM1 n=628, CEBPA n=587, RUNX1 n=231, MLL-PTD n=629, NRAS n=273, KRAS n=133, ASXL1 n=470) and were analyzed as described previously. A subcohort of 634 AML with intermediate risk karyotype was analyzed for survival. Female/male ratio of this subcohort was 280/354 and age ranged from 15.7–86.6 years (median, 66.9 years). The adverse impact of IDH1R132, IDH2R140 and IDH2R172 on the NPM1+/FLT3-ITD- group has been shown previously for this group (Blood 2010 116: Abstract 102). Results: The IDH1105GGT minor allele was detected in 11.2% (108/961) in AML and in 8.8% (42/475) of the KORA control. This slight difference does not reach statistical significance (p=0.17) and thus there is no indication that this variance is a predisposing factor for leukemia. Also the frequency of a homozygous IDH1105GGT minor allele was not different between the AML cohort (3/108, 2.8%) and the KORA cohort (2/42, 4.8%, n.s.). In the AML cohort there was no association of the IDH1105GGT minor allele with age, WBC, platelet count or any of the above mentioned molecular mutations. In contrast, some differences in survival were observed: patients with the IDH1105GGT minor allele had a longer event free survival (EFS) than those with the IDH1105GGC major allele (median: 30.1 vs. 21.6 months, p=0.052) in the intermediate risk cohort. This prognostically favourable effect of the IDH1105GGT minor allele was most prominent in the NPM1+/FLT3- group with a median EFS of 45.1 vs 23.5 months as compared to those with the IDH1105GGC major allele (p=0.015). Conclusions: 1) The polymorphic IDH1105GGT minor allele was not found to be a marker predisposing for AML. 2) No association of the IDH1105GGT minor allele to any mutation or other biological parameters was detected. 3) We were not able to reproduce the previously published adverse impact of the IDH1105GGT minor allele on survival in AML. In contrast, in our cohort of 475 patients with intermediate risk AML the EFS was even better in patients carrying the IDH1105GGT minor allele, especially in the subcohort with NPM1+/FLT3−ITD−. Thus, we would suppose that the IDH1105GGT minor allele is a favorable molecular marker in intermediate risk AML. However, as these findings are in contrast to previously published data, further confirmation in additional studies is necessary to draw firm conclusions on the utility of the IDH1105 polymorphism as a marker for diagnostics and prognosis in AML. Disclosures:Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Eder:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Low Expression of the Novel Tumor Suppressor Trim62 Is An Independent Adverse Prognostic Factor for Survival In Acute Myeloid

Abstract 563▪FN2▪This icon denotes a clinically relevant abstract Background:The role of TRIM (tripartite motif) proteins in human cancer is unknown except for TRIM19 (PML, promyelocytic leukemia gene), which acquires oncogenic activity when fused to RARα (retinoic acid receptor alpha) and causes acute promyelocytic leukemia. We have confirmed a putative role of TRIM62 as a tumor suppressor by genetically deleting the TRIM62 locus in a mouse model. TRIM62 maps to chromosome 1p35.1, a genomic region frequently associated with loss of heterozygosity in human cancer, including leukemia. Methodology:We assessed TRIM62 expression by high-throughput reverse phase protein array (RPPA) technology in 511 pts. Eleven CD34+ bone marrow (BM) and 10 normal peripheral blood (PB) lymphocyte samples were used as controls. Samples were printed as 5 serial 1:2 dilutions in duplicate using an Aushon 2470 Arrayer. Slides were probed with 195 antibodies against apoptosis, cell cycle, signaling, integrins, and phosphatases among other functional protein groups. Patients were segregated according to TRIM62 levels into TRIM62LOW (lower two tertiles) and TRIM62HIGH (highest tertile). Results:The level of TRIM62 protein in AML samples was significantly below (p=0.0048) that of CD34+ cells obtained from healthy volunteers., The level of expression was similar in the paired PB and BM derived AML samples (p=0.2433). Cases of AML-M4 and –M5 exhibited lower expression of TRIM62 than other FAB subtypes.,Stratification according to specific cytogenetic abnormalities (p=0.9286), standard cytogenetic risk criteria (favorable, intermediate, poor; p=0.6824), or mutational status (FLT3, NPM1, RAS, p53, or IDH1/2) failed to show statistically significant differences in TRIM62 expression.Overall survival (OS) was significantly different in TRIM62LOW and TRIM62HIGH for all patients (40.4 vs. 75.6 weeks, p=0.00002), and this difference was greater among patients with cytogenetically normal AML (CN-AML; 47 vs. 125.3 weeks, p=0.00001; Figure 1‘A). TRIM62HIGH patients had significantly longer OS compared with TRIM62LOW patients, regardless of whether they had received standard cytarabine-based chemotherapy (p=0.00003) or epigenetic therapy (i.e. hypomethylating agents with or without histone deacetylase inhibitors; p=0.02). TRIM62LOW was associated with a trend toward shorter remission duration compared with TRIM62HIGH for all cases (38 vs. 63 weeks, p=0.06) and in CN-AML patients (47.7 vs. 58.1 weeks, p=0.09). By multivariate analysis, TRIM62HIGH was identified as an independent favorable risk factor for survival both in the whole cohort (p=0.0085) and in patients with CN-AML (p=0.0014). Notably, TRIM62LOW discriminated a subset of patients with a distinct poor prognosis among those with FLT3MUT (36 vs. 128.5 weeks, p=0.006), FLT3WT (66 vs. 129 weeks, p=0.001), NPM1MUT (34.6 vs. 253.1 weeks, p=0.0026), NPM1WT (51.7 vs. 102.6 weeks, p=0.017), and RASWT (46.3 vs. 110, p=0.0002). Among patients with the most favorable prognosis (FLT3WTNPM1MUT CN-AML), TRIM62HIGH identified a subset of patients for whom stem cell transplant is not recommended: OS for FLT3WTNPM1MUTTRIM62HIGH vs. FLT3WTNPM1MUTTRIM62LOW patients was 100% vs. 35%, after 6 years of follow-up (Figure 1B). When patients with FLT3WTNPM1mutant were excluded from the analysis, TRIM62 still stratified two distinct groups of patients with AML regarding OS, both for the whole cohort (p=0.03) and for those with CN-AML (p=0.0006). By assessing the level of 195 other proteins on the same RPPA chip, we showed that TRIM62 levels correlated significantly and positively (R>0.2) with proteins involved in leukemia stem cell pathways (β-catenin and Notch), migration/homing (fibronectin, integrin β3), hypoxia (VHL, HIF1α), and the p53 pathway (MDM2, p21). [Display omitted] Conclusions:TRIM62 loss, consistent with a role as a tumor suppressor, represents a powerful independent adverse prognostic factor in AML. TRIM62HIGH identifies a population of CN-AML patients with superior outcomes compared with TRIM62LOW amongst those with CN-AML even after stratification by NPM1 and FLT3 mutational status. Disclosures:No relevant conflicts of interest to declare.

ID1 Expression Correlates with CEBPA Mutational Status and Is Not An Independent Risk Factor in Cytogenetically Normal AML,

Abstract 3554 Background:Acute myeloid leukemia with normal karyotype (CN-AML) is a heterogenous disease. During the last years, mutations in several genes (e.g. NPM1, FLT3, CEBPA, WT1, IDH1, IDH2) have been identified which are involved in the pathogenesis of AML and affect the prognosis of these patients. Moreover, deregulated expression of genes such as MN1, BAALC, ERG and WT1 was demonstrated to be predictive of outcome in CN-AML. Recently, high expression of the ID1 gene was described as a negative prognostic factor in AML (Tang et al. Blood 2009, 114:2993–3000). Aims:We have shown that C/EBPα, a transcription factor encoded by the CEBPA gene, binds to a regulatory element in the promoter region of the ID1 gene and regulates ID1 expression in leukemic cells (Wagner et al. Proc Natl Acad Sci USA 2006, 103:6338–6343). Therefore, we wanted to analyze the prognostic impact of ID1 expression in CN-AML in the context of other molecular markers, in particular CEBPA mutations. Methods:ID1 expression was quantified normalized to ABL by real time RT-PCR in 269 patients (age 16–60 years) with CN-AML treated with intensive double induction and consolidation therapy within the AMLSG 295 and 0199 trials (NCT00209833). The patients were also analyzed for mutations in the genes NPM1, FLT3, CEBPA, WT1, IDH1 and IDH2. Median follow up was 79 months. Results:Expression of ID1 varied over a 3-log range. High expression of ID1 (ID1high, defined as > median expression level) was significantly associated with the presence of a FLT3 -ITD or an IDH2 mutation and WT1 wildtype. Moreover, ID1 expression was closely associated with CEBPA mutational status. Altogether, 41 patients (15%) harboured a CEBPA mutation (24 monoallelic and 17 biallelic mutations). ID1 expression in the CEBPA wildtype patients was significantly higher than in patients with monoallelic CEBPA mutations and these patients had a significantly higher ID1 expression compared to patients with biallelic CEBPA mutations (p = 0.001). ID1high patients had a trend to a lower complete remission (CR) rate (74% vs. 84%; p = 0.07), but in multivariate analysis only blast clearance on day 15 after induction 1, age and WT1 SNP rs16754 were independent predictors for the achievement of CR. In univariate analysis, ID1high patients had an inferior overall survival (OS) compared to patients with low expression (median OS 29 vs. 78 months, 5 year OS 39% vs. 53%, p = 0.026). ID1high status was an independent negative prognostic factor in multivariate analysis when analyzed together with NPM1, FLT3 -ITD, WT1, IDH1, IDH2, extramedullary disease and platelet counts (HR 1.51; 95% CI 1.06–2.19). However, when also CEBPA mutational status was entered into the model, ID1 expression lost its prognostic impact and the only independent prognostic factors were age, platelets, CEBPA mutations, NPM1 /FLT3 -ITD risk group and WT1 SNP rs16754. Likewise, ID1high patients had a trend to an inferior relapse-free survival (RFS; HR 1.36, 95% CI 0.96–1.93, p = 0.086) in univariate analysis. However, in multivariate analysis including CEBPA mutational status, ID1 expression had no impact on RFS and the only prognostic factors for RFS were NPM1 and CEBPA mutations and WT1 SNP rs16754. In CEBPA wildtype patients, ID1 expression had no impact on CR-rate, OS or RFS in univariate or multivariate analysis. Conclusions:CEBPA mutations seem to deregulate ID1 expression in CN-AML. Therefore, ID1 expression is not an independent prognostic factor in CN-AML. Disclosures:No relevant conflicts of interest to declare.

Hsa-Mir-199b-5p Targets PODXL and DDR1: Decreased Levels of Mir-199b-5p Correlate with Elevated Expressions of PODXL and DDR1 in Acute Myeloid Leukemia

Abstract 1463Discovery of new prognostic factors in AML is driving our understanding of disease biology and development of new therapeutic targets. Aberrant expression of Podocalyxin (PODXL), a CD34 orthologue, has recently been associated with a subset of aggressive cancers including acute myeloid and lymphoid leukemia, myeloid sarcomas, and certain breast, liver, pancreatic and kidney tumors. Herein, via tissue microarray (TMA) approach, we discovered 50% of M1 and 100% of M2 and M4 FAB-subtype patients showed significantly elevated PODXL levels in bone marrow compared to normal subjects. Importantly, several recent investigations have linked aberrant miRNA expression with AML. A recent study evaluated miRNA expression in 122 untreated adult AML cases using a microarray platform. Interestingly, one of the significantly down regulated miRNA in Trisomy 8 associated AML was miR-199b. Via applying TargetScan prediction algorithm, we discovered that PODXL is a highly conserved target of miR-199b-5p. To test the silencing potential of miR-199b-5p on PODXL, miRIDIAN Mimic hsa-miR-199b-5p (and negative control) were transfected in THP-1 and HeLa cells. Analysis of PODXL transcript levels post transfection confirmed that PODXL is indeed a strong target of miR-199b-5p. This was further confirmed via generating stable cell lines expressing miR-199b-5p. Further prediction analyses of miR-199b- 5p targets identified Discoidin domain receptor 1 (DDR1) as another highly conserved target (4x-3'UTR sites). DDR1 is a class of collagen receptor, expressed on human leukocytes, including neutrophils, monocytes, lymphocytes and podocytes. In addition, DDR1 is significantly overexpressed in several human tumors of breast, ovarian, esophageal, and brain origin. Experimental validation via a similar approach outlined for PODXL confirmed that DDR1 like PODXL is also a specific target of miR-199b-5p. In addition, this was further confirmed via employing 3'UTR-luciferase assays for both PODXL and DDR1 where seed regions in the respective 3'UTRs were mutated for miR-199b-5p binding. Experimental results showed that mutation of seed regions significantly inhibited the ability of miR-199b-5p to target PODXL and DDR1. Interestingly, previous studies have demonstrated both PODXL and DDR1 play very similar functional roles in kidney by regulating slit diaphragms and foot processes. Therefore, we next evaluated the expression of DDR1 in the same AML patient set via TMA approach. For the first time, IHC analyses showed DDR1 levels were highly up regulated in AML. Interestingly, DDR1 expression was significantly up regulated in 100% of M1 AML cases and in 25% of M2 and M4 AML patients. Significantly, DDR1 expression was highly elevated in the same AML cases where PODXL levels were increased. Most importantly, via extracting miRNA from paraffin embedded bone marrow tissues of these AML patients and quantitative RT-PCR based assay, we found that miR-199b-5p levels were significantly decreased in most of the AML patients with elevated PODXL and DDR1 expressions. The miRNA qRT-PCR data was normalized using RNU6 and SNORD48 as internal controls. Furthermore, 55% of these AML cases were patients with persistent AML. A recent investigation revealed that overexpression of miR-199b-5p in Medulloblastoma (MB) cancer stem cells results in down regulation of CD133+ tumor initiating cells and causes depletion of the side population compartment of MB tumor stem cell population via negatively regulating Hes1 mediated Notch signaling pathway. Therefore, it is tempting to hypothesize that down regulation of hsa-miR-199b-5p in AML may provide proliferative advantage for leukemic stem cells in addition to accelerating migration and invasion functions via up regulation of PODXL and DDR1. Current investigations are focused on employing retroviral transduction of miR-199b-5p antagomir into human bone marrow derived CD34 cells to test the leukemogenic potential of miR-199b-5p silencing via colony forming assays. Taken together, our studies have identified that miR-199b-5p targets PODXL and DDR1 and demonstrate that concurrent increased expressions of PODXL and DDR1 in AML correlates with decreased expression of miR-199b-5p in AML. Disclosures:No relevant conflicts of interest to declare.

Coexpression of Bmi-1 and Interleukin-3 Receptor Alpha Chain (CD123) Correlate with Poor Prognosis In Acute Myeloid Leukemia

AbstractAbstract 4324 IntroductionAcute myeloid leukemia (AML) is characterized by the accumulation of immature cells due to disturbed differentiation and proliferation of the myeloid lineage. The traditional AML classification based on morphology, immunophenotyping, and cytogenetic abnormalities is not perfect, in part, because the leukemic population is functionally heterogeneous. AML cells comprise leukemic stem cells (LSCs) and mature leukemia cells that have differentiated abnormally. There has been a recent effort to identify new markers underlying this functional heterogeneity. Bmi-1 is a member of the Polycomb-group (PcG) proteins and is thought to inhibit inhibits cellular senescence and apoptosis. Bmi-1 was also recognized as an essential regulator of the self-renewal capabilities of both hematopoietic stem cells (HSCs) and leukemic stem cells (LSCs). Additional markers of LSCs have been described recently. One of these, interleukin-3 receptor alpha chain (CD123) expressed on AML cells caused higher cycling activity and resistance to apoptosis induced by the lack of growth factor. Coexpression of Bmi-1 and CD123 might be indicative of poor prognosis and might identify patients requiring aggressive therapeutic schedules. Patients and methodsCells lines used included two AML lines (HL-60 and KG-1a), one chronic myeloid leukemia in blast crisis (K562), and one pre-B acute lymphoblastic leukemia (SD-1). A cohort of 58 patients newly diagnosed with AML during the previous five years at our center. Patient BM samples were obtained during routine diagnostic procedures before antineoplastic chemotherapy was started. Diagnosis was based on morphology, cytochemistry, and expression of leukocyte differentiation antigens. All patients were characterized by cytogenetics. Flow-cytometry was used to analyze bone marrow samples to identify Bmi-1+CD123+ population. ResultsPatient samples were divided into 2 groups. Patients in FAB subtypes M0 and M1 comprised group A (N = 20), and patients in all other subtypes comprised group B (N = 38). The median percentages of Bmi-1 positive cells and CD123 positive cells were higher in group A than in group B and healthy controls. There were significant differences in Bmi-1 expression between groups A and B (p = 0.002) and between healthy controls and all AML patients (p = 0.0007). There were no significance differences in CD123 expression between groups A and B but the percentage of CD123 positive cells in healthy controls was significantly lower than in all AML patients (p = 0.0001). Among all AML patients Bmi-1 expression and CD123 expression in CD34+ cells were highly correlated (R = 0.76, p = 0.001). ConclusionsWe thought it important to test coexpression of Bmi-1 and CD123 in the CD34 BM population of AML patients as these markers were both prognostic factors in AML. Our results showed that expression of these markers correlated and together provided an important predictor of prognosis in AML. The criteria of AML stratification are commonly known. However, CD123 and Bmi-1 could become “new” prognostic factors in AML as well as targets for therapeutic approaches. The facts that Bmi-1 is localized in the nucleus (Gil, et al 2005) and is expressed in both hematopoietic and leukemic stem cells (Lessard, et al 2003) makes the hypothetical use of Bmi-1-targeted inhibitors more complex. Thus, further investigation to refine prognosis and to optimize therapy of AML based on Bmi-1 expression in combination with other markers is needed. Disclosures:No relevant conflicts of interest to declare.

The Prognostic Impact of High EVI1 expression In AML Is Due to Its Close Correlation to Rearrangements Involving EVI1 or MLL, which Are Cytogenetically Cryptic In a Subset of Patients: a Study on 332 Cases.

AbstractAbstract 1676 Introduction:High EVI1 expression has been proposed as a negative prognostic factor in AML. An association between high EVI1 expression and distinct cytogenetic subgroups, such as 3q26-rearrangements, MLL-rearrangements and -7/7q- have been reported. Both 3q26- and MLL-rearrangements can be difficult to detect by chromosome banding analyses or may even be cytogenetically cryptic in a subset of patients due to limited resolution. Therefore, only studies using FISH for the detection of cryptic EVI1- or MLL-rearrangements can clarify their frequencies in AML with elevated EVI1 expression. Methods/Patients::The study cohort was composed of 332 AML cases with a) normal karyotype (NK) (n=211), b) -7/7q- (n=77), and for comparison c) 3q26-rearrangements (n=38), and d) MLL-rearrangement (n=6). In all cases EVI1 expression was investigated using quantitative PCR calculating a % EVI1/ABL1 expression. In all cases FISH for EVI1 rearrangement was performed in addition to chromosome banding analysis. Cases with high EVI1 expression were also analyzed for MLL rearrangements by FISH. Results:In the total cohort, EVI1 expression varied between 0 and 1614 (median: 21.1). The highest EVI1 expression was measured in cases with cytogenetically identified 3q26-rearrangements (range: 6.1–566.4; median: 81.9) and in AML with MLL-rearrangements (range: 46.7–831; median: 239). The EVI1 expression was significantly lower in AML with NK (range: 0–1614; median: 0.5, p<0.001) and AML with -7/7q- (range: 0.03–199; mean: 34.5; median: 10.7, p<0.001). In the subgroup of cases with NK 4 MLL-rearrangements (1.9%) were detected by FISH and subsequently verified by fusion gene specific PCR. In addition, 4 cases with cryptic EVI1-rearrangements (1.9%) were identified by FISH analysis. Further genetic analysis revealed that these were due to t(3;8)(q26;q24) (n=2) and t(3;21)(q26;q11) (n=1). In one case, the EVI1-rearrangement could not be further analyzed due to lack of material. In the -7/7q- cohort 14/77 cases (18.2%) with cytogenetically cryptic EVI1 rearrangement including 3 novel recurrent abnormalities were detected: t(3;21)(q26;q11) (n=3), inv(3)(p24q26) (n=4) and t(3;8)(q26;q24) (n=2). In 5 cases FISH analysis revealed that the 7q- was not caused by an interstitial deletion but due to an unbalanced rearrangement between chromosomes 7 and 3: der(7)t(3;7)(q26;q21). In these 5 cases high-resolution SNP microarray were performed and revealed breakpoints in the CDK6 gene and centromeric of the EVI1 gene. Further mutation screening revealed that none of the cases with EVI1- or MLL-rearrangement harboured mutations in NPM1 or CEPBA. In 254 cases clinical follow-up data was available. Different cut-off levels of EVI1 expression were tested, and a cut-off at 30% EVI1/ABL1 expression was the lowest level that had a significant impact on outcome. Separating the cohort at this cut-off into high EVI1 (n=67) and low EVI1 expressors (n=187) showed a shorter EFS in patients with high EVI1-expression (p=0.001; relative risk (RR)=1.87, median EFS 6.2 vs 15.0 months (mo)), while no impact on OS was observed. When the same analyses were performed with respect to EVI1-rearrangements we observed both a significantly shorter EFS in cases with EVI1-rearrangement (n=39) vs all others (n=215) (p=0.001; RR=2.03, median EFS 4.6 vs 15.0 mo) and a significantly shorter OS (p=0.026; RR=1.73, median OS 10.1 vs 26.3 mo). Analyzing the impact of high EVI1 expression separately in the cohort without EVI1 rearrangement revealed no impact of EVI1 expression on EFS. Conclusions:The negative prognostic impact of high EVI1 expression is strongly associated with EVI1- or MLL-rearrangements and is absent in AML without EVI1- and MLL-rearrangement. Applying FISH in addition to chromosome banding analysis we identified cryptic rearrangements in 3.8% of AML with normal karyotype and in 18.2% of AML with -7/7q-, including 3 novel recurrent cytogenetically cryptic EVI1-rearrangements. This data supports the routine performance of FISH screening for EVI1- and MLL-rearrangements in patients with normal karyotype or 7q-/-7 and without NPM1 mutation and CEPBA mutation to assign patients to the correct biologic entity. The postulated independent prognostic impact of EVI1 expression should be tested further including this laboratory workflow as these parameters may have important impact on prognosis and future treatment strategies. Disclosures:Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Grossmann:MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

A Retrospective Multi-Center Analysis of Prognostic Factors Determining Outcomes In Patients with Secondary AML

AbstractAbstract 2139 Introduction:Secondary AML (sAML), a well recognized poor prognostic factor in AML, is a heterogeneous group of diseases, including both AML evolving from myelodysplastic syndromes (MDS, MDS→AML) and AML following chemotherapy for another malignancy, i.e. treatment-related AML (tAML). These two subtypes of poor risk AML have similar outcomes when treated with the same cytarabine-based chemotherapy regimen, supporting their inclusion into the same clinical category. A combined data set of patients from a single centre and a phase II study was analyzed to determine which patient and disease factors were of prognostic significance in predicting response to treatment. This is a large data set of sAML patients including patients treated with a variety of cytarabine-based induction regimens. Materials and Methods:Data from 77 newly diagnosed sAML patients treated with cytarabine-based induction chemotherapy at the Cleveland Clinic between 1997 and 2007 and 88 newly diagnosed sAML patients treated with amonafide + cytarabine in a phase II study were combined. The focus of the analysis was to ascertain the prognostic factors affecting overall survival (OS) from time of diagnosis. Initially, univariate Cox proportional hazard models for each prognostic factor were fitted. Hazard ratios and their associated 95% confidence intervals and Wald test p-values were produced. Prognostic factors were: age at diagnosis (<60 years, ≥60 years), gender (male, female), WBC (<20 x109/L, ≥20 x109/L), peripheral blasts (<20%, ≥20%), bone marrow blasts (<40%, ≥40%), cytogenetics CALGB class (unfavorable, intermediate, favorable, unknown), secondary disorder class (MDS, tAML), prior chemotherapy (yes, no), prior radiotherapy (yes, no). Subsequently a multivariate model was produced using the stepwise selection method and a significance level of 5%, having imputed missing data with mean/median values as appropriate. Results:In the combined data set of 165 patients with newly diagnosed sAML, the results for each significant prognostic factor are outlined in the table below.The final multivariate model (displayed in the table below) determined unfavorable cytogenetics, male gender and advanced age as prognostic factors predictive of worse OS. Conclusions:In a combined data set of 178 patients with sAML from 22 US centers, cytogenetics, age and gender were identified as prognostic factors of significance in determining the outcome when treating sAML patients with induction chemotherapy. For patients with these risk factors, additional therapeutic strategies, including early stem cell transplantation, should be considered. Disclosures:Tejura: Antisoma: Employment. Sekeres: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Hypomethylating Agents Correct Subcellular Localization of FOXO3a and (re)Establish Expression of Its Transcriptional Targets In AML and MDS- Cells

AbstractAbstract 2173 Background:We previously found deregulation of DNA-damage response (DDR) signaling in MDS and AML, notably the “uncoupling” between upstream proteins (ATM) and downstream effectors (Chk-1/2) in high-risk MDS and AML (Boehrer et al., Oncogene 2009). Here, we assessed if and to which degree the hypomethylating agents azacitidine (AZA) and decitabine (DAC), widely used agents in the treatment of high-risk MDS and AML, can overcome these defects. Methods:MDS (MOLM-13)- and AML- (HL-60, KG-1)-derived cell lines were incubated with AZA (1-2μM) and DAC (1-2μM). The drugs capacity to induce apoptosis (DiOC6(3)/PI), cell cycle arrest (PI) and their impact on the expression level (immunoblot) and subcellular localization (immunoflourescence) of DDR-related proteins were concomitantly assessed. Functional relevance was determined by co-incubation with biochemical inhibitors of ATM (KU-55933), ATM/ATR (caffeine), Chk-1 (UCN-01), and Chk-2 (NSC-109555). Most important findings were recapitulated on MDS- and AML- patient cells (n=5) after selection for CD34-positvity by immunomagnetic beads. Results:As previously described, both drugs induced dose- and time-dependent apoptosis and cell cycle arrest in G2/M. Noteworthy, whereas both drugs completely abrogated expression of DNA methyltransferases 1 and 3a as early as 4h of incubation with 2μM (abrogation sustained at 24 and 48h), they differentially affected DDR-related signaling. Indeed, the increased capacity of DAC (as compared to AZA) to induce G2/M-arrest (G2/M-increase at 48h DEC: 20%, AZA: +6%) was accompanied by an earlier and more efficient activation of the checkpoint-kinases-1 and -2 (phosphorylation of Chk-1-Ser317 and Chk-2-Ser68) as well as their downstream target y-H2AX (phosphorylation on Ser139). Noteworthy, DAC (and to a lesser extent AZA) modified activation of FOXO3a (diminished phosphorylation on Ser253) and concomitantly enabled nuclear translocation of non-phosphorylated FOXO3a. In addition, nuclear translocation of FOXO3a was accompanied by upregulation of its transcriptional targets known to confer G2/M- arrest (p21, p27) and apoptosis (BH3-only protein BIM). Confirming the functional relevance of our findings, we found that inhibition of ATM (KU-55933) or ATM/ATR (caffeine) had little to no impact on AZA /DAC-induced apoptosis or inhibition of cell cycle progression, whereas inhibition of Chk-1 by UCN-01 (and to a lesser degree inhibition of Chk-2 by NSC-1095555) abrogated G2/M-arrest induced by hypomethylating agents. Finally we showed that both DAC and AZA (2μM) can induce the cytoplasmic-nuclear shift of FOXO3a in MDS- and AML-derived cell lines and to a lesser extent in patient blasts, an effect observed as early as 4h of the incubation period. Conclusions:Hypomethylating agents can correct aberrant DDR-signaling in AML-and MDS blasts, and can abrogate constitutive phosphorylation of FOXO3a thereby “correcting” its aberrant cytoplasmic localization and (re-)establishing its function as a transcriptional regulator. This possible mode of action of hypomethylating agents may be important considering that aberrant phosphorylation and localization of FOXO3a is an adverse prognostic factor in AML, and that correction of its deregulation has been described as an attractive therapeutic aim. Disclosures:Fenaux: CELGENE, JANSSEN CILAG, AMGEN, ROCHE, GSK, NOVARTIS, MERCK, CEPHALON: Consultancy.