ALBERT

Description: Triggering of the T-cell receptor (TCR) can produce very different responses, depending on the nature of the major histocompatibility complex/antigen peptide (MHCp) ligand. The molecular mechanisms that permit such fine discrimination are still unknown. We show here that an epitope in the cytoplasmic tail of the TCR CD3ϵ subunit, recognized by antibody APA1/1, is only detected when the TCR is fully activated. Exposure of the APA1/1 epitope is shown to be fast and independent of tyrosine kinase activity and that it takes place even when T cells are stimulated at 0°C. These results suggest that APA1/1 detects a conformational change in the TCR. APA1/1 staining concentrates in a restricted area of the immunologic synapse. Most important, we show that full agonist, but not partial agonist, peptides induce exposure of the APA1/1 epitope, indicating a correlation between the induction of the conformational change in the TCR and full T-cell activation. Finally, the conformational change is shown to occur in T cells that are being stimulated by antigen in vivo. Therefore, these results demonstrate that the TCR undergoes a conformational change on MHCp binding in vitro and in vivo, and they establish a molecular correlate for productive TCR engagement. (Blood. 2005;106:601-608)

Description: Xenotransplantation of acute myeloid leukemia (AML) into immunodeficient mice has been critical for understanding leukemogenesis in vivo and defining self-renewing leukemia-initiating cell subfractions (LICs). Although AML-engraftment capacity is considered an inherent property of LICs, substrains of NOD/SCID mice that possess additional deletions such as the IL2Rγcnull (NSG) have been described as a more sensitive recipient to assay human LIC function. Using 23 AML-patient samples, 39% demonstrated no detectable engraftment in NOD/SCID and were categorized as AMLs devoid of LICs. However, 33% of AML patients lacking AML-LICs were capable of engrafting NSG recipients, but produced a monoclonal T-cell proliferative disorder similar to T-ALL. These grafts demonstrated self-renewal capacity as measured by in vivo serial passage and were restricted to CD34-positive fraction, and were defined as LICs. Molecular analysis for translocations in MLL genes indicated that these AML patient-derived LICs all expressed the MLL-AFX1 fusion product. Our results reveal that the in vivo human versus xenograft host microenvironment dictates the developmental capacity of human LICs residing in a small subset of patients diagnosed with AML harboring MLL mutations. These findings have implications both for the basic biology of CSC function, and for the use of in vivo models of the leukemogenic process in preclinical or diagnostic studies.

Description: Prognostic heterogeneity of AML with intermediate-risk cytogenetics (AML-IR) is mostly clarified by determination of mutations of NPM1 gene (NPM1mut), internal tandem duplication of FLT3 gene (FLT3-ITD), and biallelic mutation of CEBPA (CEBPAmut). Moreover, intrinsic characteristics of these mutations such as allelic burden of FLT3-ITD and additional gene lesions described in this population might provide a refined prognostic categorization and guide postremission strategy. In this context, we analyzed the associated features of DNMT3A mutations (DNMT3Amut) and their prognostic impact and interaction with major molecular categories in patients with AML-IR. Overall, 120 patients (52% male; median age: 51, range: 18-71) diagnosed with de novo AML-IR (MRC definition) in the cooperative group CETLAM between 1994 and 2013 who received intensive AML chemotherapy were included in the analysis. DNMT3Amut were analyzed by RT-PCR and Sanger sequencing as previously described, and were found in 44 patients (37%), 25 (57%) with a mutation in the R882 position. NPM1 and FLT3-ITD were analyzed in all patients, and CEBPA in 53. Patients with DNMT3Amut were older (53 vs. 48.5 years, p=0.019), had a higher leucocyte count at diagnosis (median value: 32.85 vs. 16.3x109/L, p=0.019), and showed a trend to an association with FLT3-ITD (p=0.081) compared to patients with wild-type DNMT3A (DNMT3Awt). For survival analysis, only patients up to 60 years old were analyzed. For a deeper insight on the interaction of DNMT3A with other mutations, we grouped patients in two different molecular categories according to presence of NPM1mut, FLT3-ITD, and CEBPA, as well as FLT3-ITD/wt ratio (based on a previous analysis detailed in Pratcorona M, Blood 2013, 121(14):2734-8). Thus, we distinguished two molecular categories: 1) a favorable molecular subgroup (n=39): NPM1mut without FLT3-ITD or a low ratio ITD/wt ratio, 0.5 for concomitant NPM1mut). In the overall series, complete response rate (CR), survival (OS), and leukemia free survival (LFS) were 84%, 43±5% (5-yr), and 38±5% (5-yr), respectively. There was no effect of DNMT3A in the global series. Nonetheless, outcome of patients with DNMT3A from the favorable subgroup was significantly worse, with an inferior 5-year OS and LFS (40±14% vs 76±8%, p=0.029; 35±14% vs 71±9%, p=0.031). On the contrary, DNMT3A mutations did not confer a different outcome in other molecular subgroups. Of note, patients with DNMT3Amut showed a trend for a better OS after receiving an allogeneic stem cell transplantation after first CR (OS at 5 years 37±12 vs 78±14, p=0.122) (Figure 2). Identification of the prognostic value of DNMT3A in the favorable molecular group might be of relevance, since general guidelines do not recommend allogeneic stem cell transplantation in first CR in this group. Based on this finding, this recommendation may be reconsidered in cases with DNMT3Amut, but larger studies are warranted to confirm it. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction Younger patients with acute myeloid leukemia and intermediate cytogenetic risk (AML-IR) harboring NPM1 mutation (NPM1mut) without FLT3 internal tandem duplication (FLT3-ITD) have a relatively favorable prognosis, and are not deemed as candidates to receive an allogeneic hematopoietic stem cell transplantation in first complete remission (CR1). However, it remains uncertain if this favorable prognosis is also maintained in older patients within the same molecular features with some conflicting results. Patients and methods We analyzed the cohort of patients ≥60 year-old considered fit for intensive chemotherapy and included in the CETLAM protocols LMA-2003 and LMA-2012 for patients up to 70, consisting of a standard induction chemotherapy, HiDAC post-remission therapy, followed by alloHSCT in selected patients with high-risk features. Overall we identified 192 patients between 60 and 71 year-old diagnosed with AML-IR with known NPM1 and FLT3 mutational status. Results We identified 192 AML-IR patients (93♀, 99♂) aged 〉=60 (median age was 65 years old (range: 60-71)), with a median WBC count 10.6 x 109/L (range: 0.23-400 x 109/L), median bone marrow blasts 65% (range: 20-100%). Overall, CR rate was 78%, five-year overall survival (OS) was 30±4%, and leukemia-free survival (LFS) was 31±4%. Patients were classified in three molecular groups depending on NPM1mut and FLT3-ITD: 40 patients harbored NPM1mut/FLT3 without ITD (FAV group), 98 patients had NPM1wt/FLT3wt, and 54 had a FLT3-ITD. Five-year OS of these 3 groups were: 64±9%, 25±6%, and 19±6%, respectively (p60 who received intensive chemotherapy, with a high proportion of long-standing responses after HiDAC-based post-remission therapy. Disclosures No relevant conflicts of interest to declare.

Description: Background The simultaneous administration of G-CSF and chemotherapy as a priming strategy has resulted in a clinical benefit in determined subsets of patients diagnosed with acute myeloid leukemia (AML) (Löwenberg et al, NEJM 2003; Pabst T, et al, Blood 2012). However, the mechanism responsible for this anti-leukemic effect is not fully characterized. We hypothesize that the clinical benefit may occur at least partially by the effect of G-CSF on leukemic stem cells (LSC). Objective The main goal of this project was to determine the effect of G-CSF on primary AML samples in vitro, especially on LSCs. Methods and patients Peripheral blood mononuclear cells (PBMC) from 10 AML patients were treated with G-CSF at increasing doses, alone or in co-culture with HS-5 stroma cells. Cell viability (7-AAD -eBioscience- cell death exclusion and volumetric cell counting) and surface phenotype was determined by flow cytometry (FACSVerse, BD) 72 hours after treatment. Data were analyzed using the FlowJo (Trastar) software. For clonogenicity assays, AML primary samples were treated for 18 hours with G-CSF at increasing concentrations and cultured in H4034 Optimum MethoCult (StemCell Technologies) for 14 days. Colonies were counted based on cellularity and morphology criteria. Results G-CSF treatment showed no effect on cell viability of the bulk leukemic population or on the CD34 + immature subpopulation. A dose-dependent increase in CXCR4 surface expression was observed, reaching a 1.4-fold of change at the highest concentration of G-CSF (100 μg/mL). In contrast, treatment of leukemia cells with G-CSF in the presence of stroma cells reduced the overall cell viability. Thus, a 32% decrease of cell viability was measured at the highest concentration used (p = 0.0006), while no significant changes in the frequency of each leukemic subpopulations were observed. Clonogenic capacity was significantly reduced in a dose-dependent manner upon treatment with G-CSF, achieving a 41% reduction at the highest G-CSF concentration (100 μg/mL). Conclusions G-CSF reduces the viability of leukemic cells when these cells are in co-culture with the HS-5 stroma cell line, suggesting that the presence of stroma cells is required for the cytotoxical effect of G-CSF on the blast population. Interestingly, G-CSF treatment decreased the clonogenic capacity of AML samples, therefore suggesting that G-CSF exerts its effect at least partially on LSCs. Our findings support the design of studies to explore new strategies of chemotherapy priming in AML patients. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3278 Specific targets of signaling pathways that control self-renewal and survival of acute myeloid leukemic stem cells (LSCs) vs. normal hematopoietic stem cells (HSCs) are largely unknown. Here, using a small molecule (CWP232228) derived from a parent compound that inhibits Wnt/TCF targets, we demonstrate reduction of primary human AML-blast growth and clonogenic capacity ex vivo, without effects on normal hematopoietic progenitors. Upon establishment of AML or normal hematopoiesis in immune-deficient recipients, in vivo administration of CWP232228 reduced leukemic disease and abolished LSC self-renewal, with no effect on normal HSC function. In vivo gene profiling and ex vivo molecular studies revealed that CWP232228 induces apoptosis and differentiation of AML-blasts via inhibition of Wnt/b-catenin signaling and activation of non-canonical Wnt signaling which phenocopies the effects of this small molecule. Our study reveals an in vivo differential dependence of AML on canonical vs. non-canonical Wnt signaling that allows therapeutic targeting of LSCs whilst sparing normal HSCs. Disclosures: Chung: Choongwae Pharma Corporation: Employment.

Description: Introduction Multiple myeloma (MM)remains an incurable disease because most of the available drugs do not destroy clonogenic tumor cell (CTC). Natural Killer (NK) cells exert cytotoxicity against MM cells; improving NK cell cytotoxicity might be part of the mechanism of action of effective anti-myeloma drugs such as lenalidomide or bortezomib. By coculture with the genetically-modified K562-mb15-41BBL cell line it is possible to expand ex vivo large numbers of activated NK cells from MM patients. We are conducting a phase I clinical trial to evaluate feasibility, safety and tolerability of these NK cells (termed NKAEs) infused in MM patients an autologous setting (EudraCT 2012-000514-11). Because the activity of NKAEs against MM CTCs is unknown, we addressed this issue and analyzed NK cell ligands and receptors pathways mediating CTCs destruction. Methods and Patients Peripheral blood (PB) was collected from MM patients (n=36) or healthy donors (n=14).To activate and expand NK cell from MM patients, peripheral blood mononuclear cell were co-cultured with K562-mb15-41BBL cells and 100 IU/ml IL-2. We used time-resolved fluorescence to detect activity of NK cells on bulk MM cells and methylcellulose clonogenic assays to determine NK cell specific activity on MM CTCs. We analyzed NK and MM receptor expression profile by flow cytometry and Real Time PCR, and identified the side population (SP) by DyeCycle Violet efflux. Three MM patients on 2nd or later relapse have been enrolled in the phase I clinical trial to date. We collected 200 ml of PB from patients to produce autologous NKAEs under GMP conditions and cells were harvested on day 14 and 21 for infusions. Four cycles of pharmacological treatment with 2 infusions of 7.5 x 106 autologous NKAEs/kg on day 1 and 8 of each cycle were performed. Results NK cells from patients (n=20) produced 26.6±12.7% lysis of bulk MM cells, similar to NK cells from healthy donors (17±7.8%), while cytotoxicity by NKAEs from MM patients was 68± 0.7% (n=3) at 8:1 ratio. In methylcellulose assays, MM cell killing was higher on CTCs (47±16.8% in MM patients and 57±8% in healthy donors) than on corresponding bulk MM cells (p

Description: Introduction Patients with intermediate-risk cytogenetic AML (IR-AML) have a heterogeneous prognosis, and are currently further stratified based on determined gene mutations. However, the optimal post-remission therapy, especially in younger patients, is unclear. Recently, miR-3151, a novel microRNA (miRNA) located in intron 1 of the BAALC gene, has been identified. High miR-3151 expression –either alone or in combination with high BAALClevels– independently correlates with poor prognosis in patients ≥ 60 years with normal cytogenetics AML (CN-AML) (Eisfeld AK, et al. Blood 2012). However, the prognostic value of miR-3151 in younger patients (≤60 years) with IR-AML has not been examined. We hypothesized that miR-3151 expression could also be a prognostic marker in younger patients. Aim To analyze whether miR-3151 expression – either alone or in combination with BAALC– improved prognostic assessment in younger patients (up to 60 years) with IR-AML and to characterize in this subset of patients the miRNA signature associated with high miR-3151 expression. Methods Samples were available from two separate cohorts of patients with IR-AML who had received intensive therapy: a training set of 76 patients from a single institution and a validation set of 108 patients from several centers who had been treated within the CETLAM AML-2003 protocol. Information on NPM1, FLT3-ITD and CEBPA was available for both patient cohorts. The expression levels of 670 miRNAs had previously been analyzed in the 76 patients in the training set. In the present study, the expression of miR-3151 and BAALC was analyzed using TaqMan® MicroRNA Assay and TaqMan® Gene Expression Assay (Applied Biosystems), respectively. Expression levels of miR-3151 and BAALC –both alone and in combination – were correlated with patient outcome. Statistical analyses were performed with SPSS v.15.0.1, R software v.2.9.0 and TIGR MultiExperiment Viewerv4.0. Results In the training set, higher expression of miR-3151(dichotomized by its median value of normalized expression) correlated with a shorter 5-year overall survival (OS) (32%±17% vs. 61±17%, p=0.029) and 5-year leukemia-free survival (LFS) (29%±17% vs. 58±17%, p=0.036) in patients ≤ 60 yrs. When the analysis was restricted to patients with CN-AML, miR-3151 expression retained its prognostic significance (p= 0.016). In addition, increased BAALCexpression was associated with shorter OS (28%±20% vs. 58±14%, p=0.054) and LFS (17%±18% vs. 55±14%, p=0.039). In the multivariate analysis for OS and LFS, including age, WBC, NPM1mut, FLT3-ITD, BAALC and miR-3151 expression levels as covariates, miR-3151 showed independent prognostic significance for OS (p=0.016; HR=2.52, 95% CI: 1.2-5.4),and a statistical trend for LFS (p=0.09). Patients with low expression of both miR-3151 and BAALC had better prognosis (OS: 66%±18% vs. 34±16%, p=0.027; LFS: 67%±20% vs. 27±16%, p=0.009).Interestingly, the combination of both miR-3151 and BAALC retained a significant prognostic value for LFS within the favorable molecular subgroup/ ELN favorable subgroup (patients harboring NPM1mut without FLT3-ITD or biallelic CEBPAmut; LFS: 44%±30% vs. 100%, p=0.017) and showed a trend in the unfavorable molecular subgroup/ELN Intermediate I&II subgroups (patients lacking both NPM1 and CEBPA mutations and/or harboring FLT3-ITD; OS: p=0.064 and LFS: p=0.072). In the validation set, miR-3151 overexpression confirmed its prognostic impact in patients in the univariate analysis for OS (45%±12% vs. 26±19%, p=0.039) and LFS (51%±14% vs. 30±24%, p=0.034) and in the multivariate analysis for OS (OS: p=0.038; HR 1.88, IC 95%: 1.06-3.34) and LFS (p= 0.014; HR 2.411 (1.198-4.855) Finally, a supervised analysis revealed that samples exhibiting high levels of miR-3151 expression had a distinctive miRNA signature including miR-509, miR-135a, miR-100*, miR-186*, let-7a* and miR-501. Conclusion miR-3151 is an independent prognostic marker in patients with IR-AML. The study of miR-3151 refines the molecular prognostic stratification of these patients and hence could be of help to guide therapy. Acknowledgments Marina Díaz-Beyá is supported by Fundación Española de Hematologia y Hemoterapia. This research is supported by Sociedad Española de Hematologia y Hemoterapia and by grants from Fondo de Investigaciones Sanitarias FIS-PI080158. Disclosures: No relevant conflicts of interest to declare.