ALBERT

Beschreibung: SUMMARYData sets were accumulated of annual average maximum, minimum and mean air temperature from a range of sites worldwide, specifically from non-urban locations such as agricultural research institutes, universities and other rural or island locations for the period 1975–2011 or longer where data were available. The data sets were then analysed using linear regression to determine the rate and direction of change in temperature over the reference periods. This analysis was performed to provide vegetable scientists with likely future temperature change scenarios up to 2025 and 2050 (on the assumption that recent trends are maintained) so that breeding, agronomic and other related research programmes may better respond to potential challenges from abiotic and biotic stresses to vegetable production. Substantial variation was evident between sites and between time runs at specific sites. At some locations rapid increases in air temperature are projected, such as for sites in East Asia, but at other locations little change is evident; in rare cases, local cooling is shown. The implications of variability and change in air temperature in the context of constraints to vegetable production and the opportunities to exploit the range of genetic diversity available in climatically uncertain environments are discussed. It is believed that modern agricultural science can address successfully the problems raised by climate uncertainty, yet the lack of sufficient, immediate investment in horticultural disciplines worldwide places the world at severe risk of failing to attain effective food and nutritional security.

Beschreibung: Increased expression levels of miR-181 family members have been shown to be associated with favorable outcome in patients with cytogenetically normal acute myeloid leukemia. Here we show that increased expression of miR-181a and miR-181b is also significantly (P 〈 .05; Cox regression) associated with favorable overall survival in cytogenetically abnormal AML (CA-AML) patients. We further show that up-regulation of a gene signature composed of 4 potential miR-181 targets (including HOXA7, HOXA9, HOXA11, and PBX3), associated with down-regulation of miR-181 family members, is an independent predictor of adverse overall survival on multivariable testing in analysis of 183 CA-AML patients. The independent prognostic impact of this 4-homeobox-gene signature was confirmed in a validation set of 271 CA-AML patients. Furthermore, our in vitro and in vivo studies indicated that ectopic expression of miR-181b significantly promoted apoptosis and inhibited viability/proliferation of leukemic cells and delayed leukemogenesis; such effects could be reversed by forced expression of PBX3. Thus, the up-regulation of the 4 homeobox genes resulting from the down-regulation of miR-181 family members probably contribute to the poor prognosis of patients with nonfavorable CA-AML. Restoring expression of miR-181b and/or targeting the HOXA/PBX3 pathways may provide new strategies to improve survival substantially.

Beschreibung: FoxP3+ confers suppressive properties and is confined to regulatory T cells (Treg) that potently inhibit autoreactive immune responses. In the transplant setting, natural CD4+ Treg are critical in controlling alloreactivity and the establishment of tolerance. We now identify an important CD8+ population of FoxP3+ Treg that convert from CD8+ conventional donor T cells after allogeneic but not syngeneic bone marrow transplantation. These CD8+ Treg undergo conversion in the mesenteric lymph nodes under the influence of recipient dendritic cells and TGF-β. Importantly, this population is as important for protection from GVHD as the well-studied natural CD4+FoxP3+ population and is more potent in exerting class I–restricted and antigen-specific suppression in vitro and in vivo. Critically, CD8+FoxP3+ Treg are exquisitely sensitive to inhibition by cyclosporine but can be massively and specifically expanded in vivo to prevent GVHD by coadministering rapamycin and IL-2 antibody complexes. CD8+FoxP3+ Treg thus represent a new regulatory population with considerable potential to preferentially subvert MHC class I–restricted T-cell responses after bone marrow transplantation.

Beschreibung: Abstract 2863 Background: JAK2 V617F is a recurrent, activating mutation in patients (pts) with BCR-ABL1-negative MPNs. Mutations in codon 515 of MPL occur in 1–5% and 5–10% of ET and PMF pts, respectively, and similar to JAK2 V617F, lead to constitutive JAK-STAT signaling. The acquisition of multiple mutational events affecting the JAK-STAT axis or components of the epigenetic machinery is common in MPNs and likely contributes to phenotypic diversity, including progression to acute myeloid leukemia. Mutated genes thus far implicated in MPN initiation and/or progression include TET2, CBL, SH2B3, ASXL1, DNMT3A, IDH1/2, IKZF1, EZH2, SRSF2, and TP53. In order to identify novel somatic mutations associated with classic BCR-ABL1-negative MPNs, we performed whole genome sequencing of DNA extracted from peripheral blood granulocytes and cultured skin fibroblasts from a patient with PMF and a known MPL W515K mutation. Methods: Whole genome sequencing (WGS) was undertaken in a 55 year-old man with untreated PMF four years after initial diagnosis. His DIPSS Plus risk group was low (score 0). His karyotype was normal, and molecular testing revealed wild-type JAK2 in addition to the MPL W515K mutation. WGS of purified granulocytes and paired cultured skin fibroblasts was performed using both Illumina HiSeq and Complete Genomics (CGI) platforms. The resulting data were analyzed using multiple independent aligners and variant callers. Amplicon-based targeted resequencing with the Illumina MiSeq platform was used to evaluate additional patient samples for recurrent mutations. Stanford institutional review board approval and informed pt consent was obtained for these analyses. Results: The PMF genome was sequenced to 88X (Illumina) and 128X (CGI) average fold coverage, and the cultured skin fibroblast genome was sequenced to 47X (Illumina) and 126X (CGI). The PMF genome had a low somatic mutation rate, consistent with that observed for other sequenced hematopoietic tumor genomes, with a low number of true somatic mutation calls. To definitively identify true mutations among various sequencing artefacts and germline variants, we use cultured skin fibroblasts which can be prepared with no contamination by neoplastic cells. In addition to re-identification of the MPL W515K mutation, this approach identified six additional somatic mutations that alter gene coding regions, splice sites, or known regulatory regions: a nonsense mutation in CARD6, implicated in modulation of NF-kappaB activation; a splice-site mutation in CAP2; three nonsynonymous point mutations in KIAA0355, SOX30, MFRP; and a 19-base pair (bp) deletion involving a regulatory region in the 5'-untranslated region (5'-UTR) of BRD2, a bromodomain-containing protein implicated in transcriptional regulation (Table). CARD6, BRD2, and KIAA0355, an uncharacterized protein, are expressed by the granulocytes derived from this patient, supporting a potential role in the development of PMF in this pt. Using massively parallel sequencing, we are currently examining the transcribed region of BRD2 and the coding region of the other five genes in additional pt samples. Analysis of the first 87 samples (MF=47, PV=20, ET=20) out of a cohort of 180 MPN pts has thus far not identified recurrent somatic mutations in these six genes. Conclusion: High-coverage genome sequencing of neoplastic and germline cells from a patient with MPL-mutated PMF identified six additional somatic mutations of potential functional significance. Work is ongoing to determine if somatic mutations in these genes are found in other patients with BCR-ABL1-negative MPNs, and their pathogenetic relevance to MPN biology. Disclosures: Snyder: Illumina: Consultancy; GenapSys: Membership on an entity's Board of Directors or advisory committees; Personalis: Consultancy, Founder Other.

Beschreibung: Abstract 1602 Introduction: MCL presents a therapeutic challenge remaining incurable with standard therapy. Most pts exhibit aggressive behaving advanced (adv) stage disease at diagnosis and require multi-agent chemotherapy, while others may have an indolent course. Improved outcomes with rituximab (R) and autologous stem cell transplant (ASCT) have been reported for select pts within the context of small clinical trials. In the province of BC, a new policy was introduced in 2003 recommending upfront ASCT for all eligible pts with adv stage MCL. This largely coincided with the availability of R which was included in the protocol: 6 cycles R-CHOP induction followed by ASCT and 2 cycles R maintenance (R weekly × 4 at 2 and 6 mo). The aim of this study was to review the clinical profile of MCL within a non-selected population of pts and to evaluate outcomes before and after this policy change. Methods: Using the BC Cancer Agency Centre for Lymphoid Cancer database, we identified all pts diagnosed with MCL between Jan 1990 and Dec 2010. Pathology was centrally reviewed and clinical data was retrieved from the database and medical records. Within this grp, we identified a SCT eligible cohort (

Beschreibung: Abstract 301 Background: While whole brain radiotherapy (WBRT) has long been considered the standard consolidative therapy in primary CNS lymphoma (PCNSL), concerns regarding irreversible neurocognitive effects of brain irradiation have prompted development of dose-intensive induction and consolidative chemotherapeutic approaches, with the aim to eliminate brain irradiation. We present the updated results of the first Phase II multicenter trial, conducted by a major cooperative group within the United States, to evaluate an intensive chemotherapy-alone strategy in newly diagnosed patients with PCNSL. Methods: Induction chemotherapy consisted of methotrexate (MTX), temozolomide, rituximab (MT-R) with HD-MTX (8 gm/m2) administered every 2 weeks × 8, weekly rituximab × 6 and temozolomide (150 mg/m2) starting day +7 and continued monthly × 5. Patients who achieved a CR received intensive consolidation cytarabine 2 gm/m2 BID on days 1–4 with etoposide 40 mg/kg over 96 h (EA). Assessment of BCL6 and MYC expression by lymphoma cells was performed by immunohistochemical analysis of diagnostic specimens and scored (10% increments) by a pathologist who was blinded to clinical outcome. Results: 44 newly diagnosed, immunocompetent patients with PCNSL were treated at 12 CALGB centers between 2005 and 2009. Patient characteristics were as follows: median age was 61 yr (range 12–76), median ECOG PS was 1, 58% were IELSG risk group 2–3. 98% of tumors were large B-cell lymphoma. 66% of patients exhibited CR to induction MT-R. With median overall follow-up of 5.3 years, 21 out of 46 patients exhibited disease progression and 17 have died. There was one treatment-related death (sepsis) during consolidation. There has been no evidence for significant treatment-related neurotoxicity. The median progression-free survival (PFS) is 4.0 years and estimated PFS rates with 95% confidence limits at 1, 2, 3 and 4 years are 0.66 (0.50, 0.76), 0.59 (0.43, 0.72), 0.52 (0.36, 0.64) and 0.47 (0.32, 0.61). The probability of 2-year PFS for patients who completed the entire regimen is 0.69 (0.47, 0.94). The estimated overall survival (OS) rate with 95% confidence limits at 4 years is 0.65 (0.49,0.77). Remarkably, event-free survival (EFS) was similar in patients older and younger than 60 (p〈 0.47). While ECOG PS〉1 and high IELSG score showed a trend toward inferior EFS, the most significant clinical prognostic variable was treatment delay: those patients who started remission induction therapy more than 30 days after diagnosis exhibited shorter EFS than patients who received MT-R within a month (2-sided p-value = 0.05). There was no relationship between treatment delay and IELSG prognostic score. While high MYC expression (〉50% lymphoma nuclei) was detected in 54% of cases, MYC was not prognostic. By contrast, high BCL6 expression (≥30% of lymphoma nuclei) was detected in 59% of cases and correlated as a continuous variable with inferior progression-free survival, event-free survival and overall survival. The 2-sided p-values for these models were p=0.045, p=0.019 and p=0.045 (log-rank test). Conclusions: CALGB 50202 (Alliance) demonstrates that induction MT-R followed by EA consolidation is feasible in the multicenter setting and yields rates of PFS and OS in newly diagnosed PCNSL patients that are at least comparable to combined modality treatment involving reduced dose whole brain irradiation. The MT-R-EA regimen is well-tolerated in patients age 〉60 and has similar efficacy in this population as in younger patients. Based upon these encouraging results, a successor, intergroup, randomized phase II trial, CALGB 51101 (Alliance) has been activated that compares dose-intensive consolidation (EA) with myeloablative chemotherapy and autologous stem cell transplant in this first randomized trial in PCNSL in which neither arm involves WBRT. Our observations regarding the association of treatment delay with adverse prognosis suggest that prompt initiation of therapy for PCNSL patients may translate into improved outcomes. In this first prospective analysis of molecular biomarkers in PCNSL in the setting of a clinical trial, high BCL6 expression was found to correlate with inferior outcome. This observation raises the possibility that in future studies BCL6 could be used as a biomarker in risk-adapted therapy and supports the investigation of BCL6 antagonists in the treatment of this disease. Supported by LLS and by NCI CA13908301. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Abstract 3269 Neutrophil elastase (NE) is an abundantly expressed enzyme with broad substrate specificity and the ability to regulate many components of the tumor microenvironment. Our laboratory has previously demonstrated that NE can gain entry into A549 lung tumor epithelial cells and increase their proliferation via degradation of insulin receptor substrate-1 (IRS-1). We have employed a variety of biochemical and in vitro assays to better define the mechanisms governing the internalization and trafficking of NE within tumor cells. Using radiolabeled 125I-NE binding assays, we have determined that NE gains entry into three unique lung tumor epithelial cell lines via a low-affinity/high abundance receptor binding interaction with a dissociation constant (Kd) of 284 nM. NE enters cells via an endocytic pathway that is both clathrin-and dynamin-dependent and caveolin-1- and flotillin-1-independent. The interaction of NE with this putative receptor is also dependent upon an intact tertiary structure, as heat-denatured as well as chemically-denatured NE is not internalized into lung tumor epithelial cells. Uptake is competitively inhibited by the highly homologous proteinase cathepsin G (CG) which, like NE, is internalized into lung tumor epithelial cells via a clathrin- and dynamin-dependent endocytic pathway. Affinity purification studies are currently underway to identify the receptor which mediates uptake of NE into tumor cells. Identification of this receptor may unveil an attractive therapeutic target which would inhibit the uptake of NE into tumor cells while leaving the anti-microbial activities of this proteinase intact. As NE exhibits broad substrate specificity, we performed an unbiased proteomic screen to uncover additional intracellular targets of NE. Because NE enters the cell via EEA1+ early endosomes, we concentrated on this subcellular compartment to restrict the number of potential targets to those that NE is known to access within the cell. Early endosomal protein was purified from total cell lysates using sucrose gradient fractionation and then subjected to an unbiased mass spectrometry-based proteomic analysis. Early endosomal lysates from NE-treated A549 cells exhibited marked reductions in 8 ubiquitin pathway-associated proteins, 3 prominent splicing factors, and 3 notable proteoglycans. Loss of these targets upon NE treatment was validated by western blot. Additional western blot analyses of total cell lysates from A549 cells (+/−) NE demonstrated changes in gene expression associated with an epithelial-to-mesenchymal transistion (EMT). Specifically, NE-treated cells upregulated alpha-smooth muscle actin, N-cadherin, and fibronectin-EDA, and downregulated E-cadherin in a dose-dependent fashion. These data expand the role of NE in regulating many aspects of solid tumor development, and characterization of the effect of NE on these pathways is currently under investigation. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Abstract 4576 Background: Chronic lymphocytic leukemia (CLL) has a highly variable clinical course. Some patients require treatment early while others can be monitored without therapy. CD38 expression has been shown in multiple cohorts to have prognostic significance. An elevated percentage of CD38 positive CLL lymphocytes at the time of diagnosis is correlated with a more rapid need for therapy and a shorter overall survival. The extent to which CD38 varies during the course of CLL, including after therapy, has only been evaluated in a limited fashion. Methods: From a cohort of over 500 CLL patients at the Duke University and Durham VA Medical Centers, we selected 136 patients in whom we had measured CD38 expression by flow cytometry on two or more occasions. We determined the first, maximum, minimum, and range (maximum – minimum) CD38 values. We compared these values to other molecular prognostic markers using Wilcoxon tests and assessed the prognostic significance of these values using Cox proportional hazard models and Kaplan-Meier analyses. Results: Of the 136 patients, 70% were male and 88% Caucasian, with a median age of 60. The majority had low clinical stage at diagnosis—either Rai stage 0 (68%) or 1 (19%). Molecular prognostic markers were also generally favorable. Eighty-two (67%) patients had mutated IGHV status, 69 (51%) were ZAP70 negative, and 76 (63%) had either 13q deletion or normal cytogenetics, determined by fluorescent in situ hybridization. CD38 expression was measured a median of 5.5 times (2 – 19). The median time between the first and last CD38 measurements was 1206 days (81 – 4109). The median values were 6% (0.6 – 99) for maximum CD38, 1.5% (0 to 84.5) for minimum CD38, and 4.9% (0.2 to 95.3) for CD38 range. Maximum, minimum, and CD38 range were significantly lower in patients with mutated compared to unmutated IGHV status (p 〈 0.005 for all parameters, Wilcoxon rank sum test). Elevated maximum and CD38 range were significantly associated with a more rapid time to therapy (TTT) and shorter overall survival (OS) in a univariate Cox proportional hazards model (p 〈 0.03 for all, Wald test). In a multivariate Cox proportional hazards model including first CD38 and maximum CD38 values, only maximum CD38 remained statistically significant. We found that patients with high CD38 variation (CD38 range greater than the median) had significantly shorter TTT and OS than patients with low CD38 variation (p = 0.002 for both, log rank test). Using receiver operator characteristic analyses, we determined that the best cut-off for dichotomizing the first CD38 according to TTT and OS in the entire Duke/Durham VA CLL cohort was 11%. Using this cut-off, 15 patients (11%) converted from CD38 negative to CD38 positive. Using the standard 30% cut-off, 14 patients (10%) converted from CD38 negative to CD38 positive. Patients with a first CD38 measurement less than 11% and subsequent measurements above 11% had a favorable OS, similar to patients with low CD38 for all measurements (p = 0.002, log rank test). However, patients with a first CD38 measurement less than 30% who had subsequent measurements above 30% had an inferior OS, similar to patients with high CD38 for all measurements (p = 0.006, log rank test). Lastly, among 24 patients with CD38 measurements before and after first therapy, the percentage of CD38 positive cells increased in 19 patients (79%), with a median value of 3.2% before to 6.9% after therapy (p = 0.005, Wilcoxon signed rank test). Conclusions: CD38 values vary as patients transition across the disease trajectory. This variation appears to have prognostic significance, with high variation associated with faster time to first therapy and shorter overall survival. Additionally, in our cohort, a patient's maximum CD38 value had more prognostic significance than a single initial measurement. Thus, longitudinally measuring CD38 throughout the clinical course of CLL could aid in the management of CLL patients, refining the initial prognostic assessment, and improving patient counseling and decision making. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Abstract 1364 Recently, we demonstrated that the anti-bacterial agent tigecycline preferentially induces death in AML and AML stem cells over normal hematopoietic cells through the inhibition of mitochondrial translation. This heightened sensitivity was due to increased mitochondrial mass and reliance on oxidative metabolism in the AML cells compared to normal hematopoietic cells. Here, we sought to better understand the mechanisms of sensitivity and resistance to inhibitors of mitochondrial translation. To establish cells resistant to tigecycline, we exposed TEX leukemia cells to increasing concentrations of tigecycline over 4 months and selected a population of TEX cells resistant to tigecycline (RTEX+TIG) with an IC50 〉 24 μM (versus an IC50 of 2.8 + 0.4 μM in wild type cells). We then profiled oxidative metabolism in the resistant cells. In RTEX+TIG cells, levels of Cox-1 and Cox-2, subunits of respiratory complex IV in the electron transport chain that are translated by mitochondrial ribosomes, were undetectable. In contrast, Cox-4 that is part of the same respiratory chain, but translated in the cytoplasm, was only slightly reduced. RTEX+TIG cells also had undetectable levels of oxygen consumption and increased rates of glycolysis compared to wild type cells. Moreover, RTEX+TIG cells were more sensitive to inhibitors of glycolysis and more resistant to hypoxia, thus demonstrating the functional importance to the change in their metabolic status. RTEX+TIG cells also had reduced mitochondrial membrane potential by 44.4 + 7.2% and reduced mitochondrial mass compared to wild type cells. Morphologically, RTEX+TIG cells had abnormally swollen mitochondria with irregular cristae structures. To understand the molecular basis for the metabolic changes in the RTEX+TIG cells, we performed RNA sequencing of the RTEX+TIG cells and wild type TEX cells. Unbiased analysis, by two independent approaches, of the promoter sequences of transcripts upregulated 1.5-fold or greater in RTEX+TIG cells demonstrated a significant over-representation of binding sites for the hypoxia-inducible factor 1 alpha HIF1α :HIF1β transcription factor complex. Specifically, a subset of HIF1α target genes involved in energy balance and cellular metabolism were coordinately upregulated in RTEX+TIG cells, corresponding with our phenotypic observations of the metabolic state of these cells. We validated the upregulation of HIF1α mRNA and protein by Q-RTPCR and immunoblotting. Strikingly, upon removal of tigecycline from RTEX+TIG cells, the cells re-established aerobic metabolism and oxidative phosphorylation. Levels of Cox-1 and Cox-2, oxygen consumption, glycolysis, mitochondrial mass and mitochondrial membrane potential returned to wild type levels. However, HIF1α remained elevated. Upon re-treatment with tigecycline, the cells remained resistant and the glycolytic phenotype was re-established. TEX cells display features of leukemia stem cells, including differentiation, self-renewal and hierarchical organization. Interestingly, RTEX+TIG cells were more differentiated and had reduced stemness compared to the wild type TEX cells. By immunohistochemistry, RTEX+TIG had increased non-specific esterase activity (NSE). In addition, RTEX+TIG cells had reduced clonogenic growth and ability to engraft immune deficient mice compared to wild type cells. Moreover, RNA sequencing data showed reduced expression of stem cell maintenance genes in RTEX+TIG cells. Depletion of mitochondrial DNA via prolonged exposure of parental cell lines to cationic lipophilic agents such as ethidium bromide produces rho-zero cells that have irreversibly lost mitochondrially translated proteins. These cells lack a functional respiratory chain and cannot derive energy from oxidative phosphorylation. Instead, these cells rely on glycolysis for their energy supply. Here, we have produced a reversible rho-zero like metabolic phenotype by sustained inhibition of mitochondrial translation. This work, therefore, highlights mechanisms of metabolic adaption to inhibition of oxidative phosphorylation. Finally, these data suggest a unique role for metabolism in differentiation and stemness in AML. Disclosures: No relevant conflicts of interest to declare.