ALBERT

Description: Key PointsStaphylococcal enterotoxins stimulate benign T cells to induce activation of the immunoregulatory Stat3/IL-10 axis in malignant T cells. Colonization with enterotoxin-producing Staphylococcus aureus may promote immune dysregulation in cutaneous T-cell lymphoma.

Description: Introduction Growth differentiation factor 15 (GDF15) is a multifunctional growth factor of the transforming growth factor beta (TGFbeta) family that plays a complex role in several types of cancers. In multiple myeloma, GDF15 was recently shown to enhance the tumor-initiating and self-renewal potential of the cancer cells (Tanno et al, Blood 2014). Moreover, blood and bone marrow plasma levels of GDF15 are elevated in myeloma patients compared with healthy persons, and high serum levels are associated with a poor prognosis (Corre et al, Cancer Research 2012). GDF15 seems important for bone remodeling during hypoxia (Hino et al, JBMR 2012), and one study proposed GDF15 to increase osteoclast activation in prostate cancer metastasizing to bone (Wakchoure et al, Prostate 2009). Whether GDF15 plays a role in the bone disease of multiple myeloma is not well characterized. Aim Our aim was to investigate whether high GDF15 serum levels are associated with multiple myeloma bone disease and to characterize the effect of GDF15 on osteoclast differentiation in vitro. Methods GDF15 was measured in serum samples obtained at diagnosis from 138 myeloma patients and 58 age and sex-matched healthy controls. The patient serum samples were collected for the Nordic Myeloma Study Group during a randomized phase 3 clinical trial which compared the effect of two different doses of pamidronate on bone. The bone disease was therefore particularly well-characterized in this study (Gimsing et al, Lancet Oncol 2010). Peripheral blood mononuclear cells (PBMC) isolated from buffy coats were cultured in osteoclast medium (a-MEM with human serum (20%), M-CSF (30ng/ml) and RANKL (50ng/ml)) for up to 14 days with or without GDF15. Purchased pre-osteoclasts (Lonza Inc.) were cultured in purchased bullet kit (OC medium with M-CSF (33ng/ml) and RANKL (66ng/ml)) for 7 days with or without GDF15. Cells positive for tartrate resistant acidic phosphatase (TRAP) staining and with more than two nuclei were counted as osteoclasts. Results GDF15 was significantly higher in serum obtained from myeloma patients (median 1.08 ng/ml, range 27.91) compared with healthy controls (median 0.46 ng/ml, range 1.66, Independent samples Kruskal-Wallis test p〈 0.0001). Moreover, serum GDF15 was elevated in patients with a more advanced osteolytic bone disease (n= 51, median 1.44 ng/ml, range 6.48) as compared to patients without osteolytic lesions (n= 16, median 0.84 ng/ml, range 10.62) at inclusion (p

Description: Background Recombinant FVIIa (NovoSeven®) is used for the treatment of spontaneous bleeds in haemophilia patients with inhibitory antibodies against FVIII or FIX. Two theories have been proposed to describe the mechanism of action of FVIIa (van't Veer C et. al., (2000) Blood 95:1330-5, Monroe DM et. al., (1998) Blood Coagulation Fibrinolysis 9(S1):S15-S20); a) competition with zymogen FVII to bind to surface exposed TF at site of injury, and b) activation of FX by FVIIa bound to phospholipids exposed on activated platelets. This study aims at generation and characterization of a novel reagent that will provide new insights into the extent of TF influence in vascular injury. Aim To engineer and characterize FVIIa variant with abrogated TF binding ability and to address influence of TF on FVIIa activity in bleeding models (in whole blood clotting). Methods Based on the x-ray crystal structure (PDB: 1DAN), the binding interface between soluble TF (sTF) and FVIIa on the FVIIa light chain was targeted for engineering a new N-glycan. FVIIa variant I69N/F71T was expressed in BHK cells. Majority of the FVIIa variant was additionally glycosylated as assessed by PNGase treatment and SDS-PAGE analysis. Low-levels of non-glycosylated FVIIa variant were removed by passing the FVIIa variant through a sTF column. The resulting FVIIa preparation was homogenous as assessed by HPLC analysis and was characterized as reported earlier (Persson E et al., (2009) FEBS J. 276:3099-109). Results We have successfully introduced a new N-glycan in FVIIa by engineering an N-glycosylation site Asn-X-Ser/Thr in the FVIIa light chain that provides steric hindrance to sTF. WT-FVIIa binds to sTF with Kd of 6 nM, I69N/F71T-FVIIa displays abrogated sTF binding ability up to 10 µM sTF. At higher concentrations, sTF may interact with FVIIa protease domain directly. Proteolytic and amidolytic activity of WT and FVIIa variant are comparable in the absence of sTF, suggesting that the additional N-glycan does not perturb the protease domain. Data from FX activation and ATIII inhibition assays show virtually no effect of sTF on the functional properties of I69N/F71T-FVIIa in presence or absence of phospholipids. Thrombelastography (TEG) assay initiated by kaolin in haemophilia like human whole blood, in the presence of a TF specific inhibitory antibody, revealed no significant differences in clotting time (R-time) and maximum thrombus generation (MTG) for WT and I69N/F71T FVIIa variants. Similar data when TEG assay was initiated by re-lipidated TF (Innovin®) showed marked differences between WT and I69N/F71T FVIIa variants suggesting loss in the TF binding ability of the FVIIa variant. Conclusions Present work demonstrates that, using exclusive mutagenesis strategy, it is possible to engineer FVIIa variant with minimal changes in the primary sequence that displays abrogated TF binding ability but intact amidolytic and proteolytic activity. Functional properties of this variant were characterized by in vitro biochemical and TEG assays. Such a variant should prove useful to delineate involvement of TF in FVIIa activity in bleeding models. Disclosures Gandhi: Novo Nordisk A/S: Employment. Grøn:Novo Nordisk A/S: Employment. Petersen:Novo Nordisk A/S: Employment. Reedtz-Runge:Novo Nordisk A/S: Employment. Olsen:Novo Nordisk A/S: Employment. Østergaard:Novo Nordisk A/S: Employment.

Description: Pediatric acute lymphoblastic leukemia (ALL) is characterized by recurrent chromosomal translocations that frequently occur in utero in preleukemic cells. The translocation t(12;21) resulting in the formation of the chimeric transcription factor ETV6-RUNX1 is the most frequent structural aberration occurring in 25% of B-cell ALL. A previous study suggested that ETV6-RUNX1 positive preleukemic cells are present in every hundredth human newborn, thus exceeding the actually observed incidence of ETV6-RUNX1 positive ALL in children by a factor of 100. This finding indicated that secondary, but relatively rare cooperating oncogenic hits are necessary for the development of overt leukemia. However, later studies could not confirm this high incidence of preleukemic cells in newborns. To analyze the actual frequency of ETV6-RUNX1 preleukemic cells in newborns we developed a PCR based method termed genomic inverse PCR for exploration of ligated breakpoints (GIPFEL) and applied this technique to a population-based screening of ≈1000 cord blood samples from healthy newborns. The GIPFEL method is capable to detect the most common gene fusions associated with childhood leukemia without prior knowledge of the exact breakpoint. In the case of ETV6-RUNX1 positive leukemia, GIPFEL exploits the unique presence of a genomic fragment joining material from chromosomes 12 and 21 in the translocation-positive cells. These fragments can be digested and re-circularized by ligation creating a junction across the restriction site whose sequence can be predicted from published genome data. Importantly, the ligation site is independent of the translocation point within the individual DNA circle. The published breakpoint regions of the ETV6 and RUNX1 genes involved in the translocation were analyzed in silico for restriction sites that allow digestion of all possible translocation events to yield fragments smaller than approximately 50 kb. This condition was met for ETV6-RUNX1 breakpoints by digestion with SacI. Primer pairs were designed amplifying the complete set of theoretically predicted circularized fragments requiring 36 primers for the ETV6-RUNX1 translocation. Genomic DNA was prepared from cell lines, diagnostic specimens from ALL patients, peripheral blood from healthy donors and cord blood samples from newborns by column purification. The equivalent of approximately 4x105 cells (2.5 µg DNA) was subjected to the SacI restriction digest, ligated and remaining linear DNA was removed by exonuclease III. After ethanol precipitation the reaction products were subjected to a partially multiplexed, semi-nested PCR to quantify all possible ligation/junction products specific for the translocation. An internal RUNX1 genomic ligation product served as a quality control and allowed the relative quantification of the translocation product. In a first proof-of-principle study employing the ETV6-RUNX1 translocation positive cell line REH, process optimization close to the theoretical limits was carried out. Cell dilution and mixing studies revealed that under optimal conditions approximately 40 translocation positive cells (=10-4) present in the input DNA are sufficient to produce a reliable output signal. The method was next tested in a blinded study with 60 samples obtained from ETV6-RUNX1 diagnostic ALL samples. ETV6-RUNX1 samples positive at 10-4, being diluted from these diagnostic samples, still gave a reliable output signal. There was no false positive result. Detection coverage (=sensitivity) was 64%. This method was then applied to a retrospective sample set of cryopreserved anonymized cord blood samples of ≈1000 healthy newborns to determine frequency and levels of translocation-positive cells. First results will be presented. In conclusion this population-based study will allow an estimate of the actual incidence of ETV6-RUNX1 positive preleukemic cells in healthy newborns. The results will enable us to evaluate the penetrance and leukemia inducing potential of the chimeric transcription factor ETV6-RUNX1 in human newborns and will provide a basis for the assessment of potential secondary environmental or spontaneously occurring cooperating oncogenic lesions in ETV6-RUNX1 positive childhood leukemia. Disclosures No relevant conflicts of interest to declare.

Description: The short half-life of coagulation factor VIIa (FVIIa) in circulation is the result of elimination through multiple pathways of which inactivation by the plasma inhibitor antithrombin (AT) accounts for as much as 65% of the total clearance in humans. Remarkably, the rate of inhibition in vivo is about 30 times greater than the uncatalyzed rate of inhibition in vitro suggesting the presence of rate enhancing components in vivo (Agersø et al. (2011) J Thromb Haemost, 9:333-338). Prime candidates include endogenous heparin-like glycosaminoglycans (GAGs) potentiating the reactivity of antithrombin, or tissue factor (TF) which upon binding to FVIIa increases its susceptibility to inhibition. In the present study site-directed mutagenesis of FVIIa was undertaken to identify variants with altered AT reactivity in order to explore the relationship between the reactivity of FVIIa with AT in vitro and in vivo as well as the nature of endogenous rate enhancing components. The pharmacokinetic properties of FVIIa variants were determined in Sprague Dawley rats as this model recapitulates the aspects of AT-mediated FVIIa clearance observed in humans and allows for interaction of human FVIIa with endogenous rat TF. Similar to the human situation, inactivation of wild-type FVIIa in rat is evident as an accumulation of circulating FVIIa-AT complexes and a progressive divergence of the pharmacokinetic profiles representing FVIIa clot activity and total FVIIa antigen. Initially, the ability to modulate the in vivo complex formation with AT was investigated using two FVIIa variants exhibiting enhanced (〉200%) or reduced (

Description: Introduction Cutaneous T-cell lymphomas (CTCL) are rare malignancies, which present in the skin and presumably arise from malignant transformation of T-cells normally destined to home to the cutaneous environment. MicroRNAs (miRs) regulate gene expression at the post transcriptional level. Many studies have shown that altered miR expression is a central event in lymphomagenesis, and that miRs have potential as both diagnostic and predictive tumor markers. In CTCL we have previously identified and validated a 3 miR classifier that distinguishes CTCL from BID with 〉 95% accuracy, based upon the up-regulation of miR-155 combined with the down-regulation of miR-203 and miR-205. In normal adult tissues, miR-203 is mainly associated with keratinocyte differentiation, acting to repress stemness, and to induce cell cycle arrest and differentiation. In cancer, miR-203 has been shown to hold tumor suppressor properties, and may be down-regulated by promoter hyper-methylation. The function and implications of miR-203 for CTCL has not previously been described. In this study we have investigated the regulation and function of miR-203 in primary CTCL biopsies and cell lines. Materials and Methods Twenty-one fresh frozen primary CTCL biopsies, IL-2 independent CTCL cell lines (MyLa2059 and PDB2B), and the IL-2 dependent CTCL cell lines (SeAx and SeZ4) were analyzed in this study. Promoter methylation was analyzed by methylation specific melting curve analysis. Cell lines were transfected by electroporation of miR-203 mimic or non-template-control (mirVana, Ambion). Proliferation was measured by 3H-Thymidine and apoptosis by MMT assays. MiR-203 mimic and mock transfected cells were examined by Affymetrix RNA expression arrays (GeneChip Human Gene 2.0 ST). IL2Rβ mRNA expression was confirmed by qPCR and IL2Rβ protein levels by flow cytometry as measured by CD122 (IL2Rβ-chain), compared to CD25 (IL2Rα-chain) and CD132 (IL2Rγ-chain). Cloning was done according to the manufacturers’ recommendation (In-Fusion, Clontech) and luciferase reporter assays were performed using the Dual-Glo system (Promega). Results We show that miR-203 is epigenetically silenced by DNA methylation in both CTCL cell lines and in 9 of 21 (43%) of primary CTCL samples, and that miR-203 can be up-regulated by the hypo-methylating agents 5-azacytidine and 5-aza-2-deoxycytidine in vitro. We also show, that forced miR-203 expression in CTCL cells targets known oncogenes such as p63, Survivin and CREB. Furthermore, it is shown that induction of miR-203 reduces cell viability and decreases proliferation. mRNA array analysis of miR-203 mimic and mock transfected cells lead to the identification of 19 significantly de-regulated genes (P2), including the as yet unrecognized miR-203 target molecule IL2Rb, which is essential for IL-2 induced JAK/STAT signaling. qPCR and FACS analysis confirmed this up-regulation both at the mRNA and protein level. The IL-2 dependent cell line SeAx showed significantly more profound down-regulation of IL2Rβ upon in miR203 transfected cell lines. Preliminary luciferase reporter assays confirm that IL2Rβ expression is regulated by miR-203, providing novel evidence that miR-203 may act in concert with IL-2/STAT in CTCL pathogenesis. These experiments are currently being validated. Conclusion We provide the first evidence that miR-203 acts as a tumor suppressor in CTCL. Furthermore we show that down-regulation of miR-203 leads to increased expression of an as yet unidentified target gene, IL2Rβ, which is directly involved in JAK/STAT signaling, that plays an essential role in the regulation of T-cell proliferation. Thus, we suggest that epigenetic miR-203 down-regulation and IL2Rβ up-regulation are important early and driving events in CTCL pathogenesis. Disclosures No relevant conflicts of interest to declare.

Description: Hematopoietic cells and cell surface molecules have both been defined in the hundreds, and the cell-specific profiles arising from the presence of specific proteins on the surface of different cells or biological states (e.g. developmental stages, disease states, etc.) represent data of high combinatorial complexity. The dynamic surface marker profiles of cells have been extensively used for cell sorting and for therapeutics where specific surface markers are used to direct therapeutic agents to diseased cells, using either monoclonal antibodies or cell-based therapies. Immunophenotyping is commonly used to define and sort cells based on the proteins present on their surface. In order to efficiently separate similar cells, a large number of surface proteins are often used. Complete knowledge about unique, cell-specific profiles of surface protein expression are likely to reveal much simpler surface profiles than currently used, as well as the definition of surface profiles where non are currently defined. In cancer immunotherapy, adoptive transfer of chimeric antigen receptor (CAR) engineered T cells show promise as therapy modality. Currently, the main achievements utilizing this technique have been made targeting single malignancy-specific surface molecules, but progress is being made in requiring binding of several ligands before lymphocyte activation, which will increases the specificity of the therapy and thereby decrease off-target effects. Defining surface protein expression profiles for cell stratification and CAR therapy in silicorequires information about expression of a large number of surface proteins on a large number of cells. At present, no high-throughput technique for measuring surface protein expression exists, although efforts to increase throughput using mass spectrometry and computational prediction of protein expression from mRNA expression are being explored. However, surface molecule expression on individual cells has been characterized at low rates using immunohistochemistry or flow cytometry for decades, and vast amounts of cell-specific expression has been measured and published. This represents a rich, but unstructured source of data and information. To facilitate the definition of unique surface molecule profiles, we collected and organized large amounts of these data of human hematopoietic cells and the corresponding quantitative or qualitative presence (depending on availability) of known molecular surface molecules from the primary literature. To do so, we employed text mining techniques for article classification (as either containing information about surface protein expression or not) and subsequently extensive manual curation to assemble the data foundation for defining cell surface profiles for stratification and therapy. To analyze these data, we have developed algorithms for selection of cell surface protein for cell stratification and for target selection for CAR-based therapies. The resulting database contains expression of 305 surface proteins across 206 hematopoietic cells, totaling 6153 data points. We have applied our algorithm to define unique profiles for each of the 206 cells, thus characterizing the surface profiles of the majority of hematopoietic cells to increase efficiency and specificity of cell stratification and therapy targeting. Future efforts will include expanding the database to contain surface protein expression for cells in all human tissues, as well as experimental validation of discovered surface profiles. Disclosures No relevant conflicts of interest to declare.