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The process of angiogenesis contributes to leukemogenesis, indicating a place for angiotargeting therapy in the treatment of AML. In this issue of Blood, Madlambayan and colleagues demonstrate that combining antivascular agents that target endothelial cells and modulate the local cytokine network can be an effective therapeutic strategy in the fight against leukemia.1 The authors combined the VEGF-specific antibody bevacizumab with the vascular disrupting agent OXi4503. This combination may soon become a clinical reality: bevacizumab is already in clinical use and a clinical trial of OXi4503 in treatment of AML was recently registered (ClinicalTrials.gov identifier: NCT01085656).

A differentiation block with accumulation of immature myeloid cells characterizes acute myelogenous leukemia (AML). However, native AML cells often show some morphological signs of differentiation that allow a classification into different subsets, and further differentiation may be induced by exposure to various soluble mediators, e.g., all trans-retinoic acid (ATRA) and several cytokines. Combination therapy with ATRA and chemotherapy should now be regarded as the standard treatment for the acute promyelocytic leukemia variant of AML. Several agents can induce leukemic cell differentiation for other AML subtypes, although these effects differ between patients. Differentiation may then be associated with induction of apoptosis, and differentiation-inducing therapy may therefore become useful in combination with intensive chemotherapy to increase the susceptibility of AML blasts to drug-induced apoptosis. However, it should be emphasized that differentiation and apoptosis can occur as separate events with different regulation in AML cells, and future studies in AML should therefore focus on: A) the identification of new agents with more predictable effects on differentiation and apoptosis; B) the use of clinical and laboratory parameters to define new subsets of AML patients in which differentiation/apoptosis induction has a predictable and beneficial effect, and C) further characterization of how AML blast sensitivity to drug-induced apoptosis is modulated by differentiation induction.

The aim of the study was to characterize effects of exogenous cytokines on T lymphocytes derived from acute leukemia patients with chemotherapy-induced leukopenia. We investigated the cytokine responsiveness of long-term expanded CD4+ and CD8+ T cell clones and the effects of exogenous cytokines on anti-CD3-stimulated polyclonal T cell responses. After mitogenic activation in the presence of acute myelogenous leukemia (AML) blasts, most CD4+ and CD8+ clones proliferated in response to interleukin-2 (IL-2). Although a majority of the IL-2-responsive clones could also proliferate in the presence of exogenous IL-4, IL-7, IL-9, IL-10, IL-12, and IL-15, only IL-15 responses were equal to or exceeded the corresponding IL-2 responses. Exogenous cytokines were also added during T cell activation with phytohemagglutinin (PHA) + accessory leukemia cells derived from different AML patients, and all the cytokines then had divergent effects that depended on both differences between clones and differences between AML patients. However, for most of these T cell clone/AML blast combinations, IL-2 and IL-15 caused enhanced T cell proliferation. IL-2 and IL-15 also enhanced anti-CD3-stimulated polyclonal responses of nonexpanded T cells derived from cytopenic patients, whereas other cytokines had only minor effects. Our results demonstrate that cytokine-responsive T cells remain in the circulation during chemotherapy-induced cytopenia, and combination therapy including intensive chemotherapy and T cell-targeting cytokine therapy should, therefore, be possible in AML.

Both clinical and experimental evidence indicate that T lymphocytes can mediate antileukemic effects in acute myelogenous leukemia (AML). These antileukemic effects can be either nonspecific cytotoxicity (killer cell activity) or reactivity against leukemia-specific antigenic peptides presented by self-HLA molecules. The antigen-specific T cell activation requires recognition of specific peptides together with costimulatory signalling. For most patients the AML blasts express both HLA class I and class II molecules for antigenic presentation, but patients are heterogeneous with regard to: (1) expression of costimulatory binding molecules; (2) expression of receptors/counterreceptors involved in induction of apoptosis; (3) constitutive release of immunomodulatory soluble mediators. This heterogeneity suggests that the ability of AML blasts to initiate an antileukemic T cell response will differ between individual patients. Thus, clinical approaches for immunotherapy in AML have to overcome three major problems. First, the therapy should reduce the patient heterogeneity so that therapeutic effects become more predictable; or alternatively one should define patient subsets which are likely to benefit from immunotherapy. Second, immunotherapy should enhance antileukemic T cell reactivity or blast susceptibility to immune attacks. Third, the therapeutic procedures must be safe and suitable for routine use. All three problems probably have to be solved before immunotherapy can become a routine treatment.

Proliferative responses and cytokine secretion were compared when AML blasts were cultured in the three serum-free media, X-Vivo 10, X-Vivo 15, and defined serum-free medium (IMDM with mercaptoethanol, low-density lipoprotein, albumin, and transferrin) and in media containing 10% inactivated fetal bovine serum (FBS). The following AML blast functions were investigated: (a) constitutive cytokine secretion, (b) autonomous and cytokine-dependent proliferation, and (c) accessory cell function during T cell activation. Constitutive cytokine secretion and accessory cell function differed markedly when using different culture media. For the constitutive AML blast secretion of IL-1beta, IL-6, and tumor necrosis factor (TNF)-alpha, no qualitative differences were seen, but quantitative differences were observed with decreased cytokine levels for cells cultured in X-Vivo 10 and X-Vivo 15. The accessory cell function of AML blasts was also decreased in the X-Vivo media, whereas differences were less pronounced when comparing AML blast proliferation. Our results clearly demonstrate that a well-characterized culture system is essential for in vitro studies of AML blast functions.

Epigenetic events are important in carcinogenesis and for the susceptibility of malignant cells to chemotherapy, and this knowledge has identified a new generation of oncogenes and thereby also a new class of possible therapeutic targets for anticancer treatment. RNA reflects gene expression and can thereby be used for identification of therapeutic targets through global gene expression analysis or siRNA screening. However, molecular events at the RNA level are important regulatory mechanisms (i.e., the small regulatory RNAs) and a level for posttranscriptional modification of proteins through alternative RNA splicing. Recent publications have described RNA repair mechanisms that may be involved in carcinogenesis as well as chemosensitivity of human cancer. Most available clinical studies try to target gene expression and thereby RNA-associated regulatory mechanisms mainly through non-specific targeting of gene expression by histone deacetylase inhibition or demethylating agents, but the use of bcl-2 antisense represents an example of specific therapy. Future RNA-targeting studies have to focus on the development of new therapeutic strategies: (i) new therapeutic agents should possibly be more specifically directed towards the target molecules rather than towards more general molecular mechanisms with effects on a wide range of intracellular regulators; and (ii) drug delivery systems have to be developed so that the treatment can be directed towards the localization of the malignant disease to reduce the risk of serious side effects.

The effects of insulin-like growth factor-1 (IGF-1) on highly enriched human umbilical cord CD34+ cells were investigated in vitro. CD34+ cells were cultured in serum-free medium containing stem cell factor (SCF), GM-CSF, and interleukin-3 (IL-3). Culture of CD34+ cells for one week in the presence of these cytokines resulted in a dose-dependent increase in total cell number. Addition of G-CSF together with SCF+IL-3+GM-CSF increased the proliferation of myelopoietic cells as determined by the number of cells expressing the myelomonocytic marker CD64 and the granulocytic marker CD15 without significantly altering the number of CD34+ cells in the cultures. In the presence of G-CSF, IGF-1 induced a dose-dependent increase in the total cell number and a moderate but significant increase in the percentages of CD15+, CD64+ cells with sustained CD34+ cell proliferation. We conclude that IGF-1 can enhance the in vitro proliferation of committed progenitor cells derived from umbilical cord CD34+ cells.

T lymphocytes are important both for the host defence against infections and probably also as antileukaemic effector cells in patients with acute leukaemia. To investigate the T lymphocyte cytokine repertoire of clonogenic T lymphocytes, CD4+ and CD8+ T lymphocyte clones were prepared from acute leukaemia patients with chemotherapy-induced cytopenia (leucocytes0.5x 10(9)/l). A majority of both CD4+ and CD8+ clones secreted detectable interleukin-2 (IL-2), IL-10, IL-13, granulocyte/macrophage-colony-stimulating factor and interferon gamma (IFNgamma) in response to phytohaemagglutinin + accessory cells (Epstein-Barr-virus-transformed B cell line, 80-Gy-irradiated). The CD4+ clones showed significantly higher levels of IL-10 secretion than the CD8+ clones. Decreased levels of IL-2, IL-13 and IFNgamma were observed when acute myelogenous leukaemia (AML) blasts were used instead of cells from the B cell line as accessory cells during phytohaemagglutinin activation, but the differences in IL-13 and IFNgamma levels were reversed by addition of exogenous IL-2. On the basis of these results we conclude: (i) the remaining clonogenic T lymphocytes derived from acute leukaemia patients with therapy-induced leucopenia can respond to activation with a broad cytokine response, and T-cell-derived cytokines may then contribute to cytokine responses during complicating infections in these patients; (ii) although T cells can modulate AML blast functions and mediate antileukaemic effects, the leukaemia blasts will also modulate T cell functions and alter the cytokine profile of activated T lymphocytes.

Translation of mRNA is a prerequisite for cell proliferation, differentiation and viability. We have studied the effect of ribosome protein factors (GPRE) on acute myeloid leukemia (AML) blast cells. Ribosomes were isolated from MPC-11 cells using ultra-centrifugation. GPRE were extracted using a high KCl procedure. Blast cells from six AML patients were grown in suspension cultures for 24 and 96 h. GPRE or granulocyte macrophage-colony stimulating factor (GM-CSF) were added at the start of the incubation. GPRE, but not GM-CSF, prevented chromatin condensation and fragmentation of blast cell nuclei in AML-M2, -M4 and -M5 and the loss of nucleoli in AML-M2 and -M5. The fraction of phagocytosing blast cells in AML-M1, -M2, -M4 and -M5 was increased by GPRE. GPRE stimulated opsonin-dependent and -independent attachment and internalisation of N. meningitidis. GPRE increased the fraction of blasts expressing CD11b and CD32 in AML-M2 and -M5. GPRE diminished the fraction of AML-M5 cells bearing CD35 and CD32. GPRE also decreased the fraction of CD11c-bearing AML-M2 and -M5 cells. GM-CSF potentiated effects of GPRE in AML-M1, -M2, -M4 and -M5. GPRE and GM-CSF in combination affected phagocytosis and surface antigen expression in blast cells that were not influenced by either factor alone. Neither GPRE nor GM-CSF induced terminal differentiation or DNA-synthesis. We conclude that GPRE affects AML blast cell morphology, function and surface molecule expression, possibly by inhibiting apoptosis. The effects of GPRE may be mediated by ribosomal proteins that regulate translation and modulate the subcellular distribution of mRNA species.

T lymphocyte secretion of interleukin-13 (IL-13) in response to different activation signals was characterized in vitro. IL-13 release was investigated when virus transformed B lymphocytes or acute myelogenous leukaemia (AML) blasts were used as accessory cells during T cell activation. First, a majority of both CD4+ and CD8+ TCR alpha beta + T lymphocyte clones, derived from normal individuals and bone marrow transplant recipients, secreted IL-13 in response to a standardized mitogenic activation signal (phytohaemagglutinin + IL-2 + B lymphocyte accessory cells). The CD4+ cells showed significantly higher IL-13 levels than the CD8+ subsets. Second, when leukaemic accessory cells (more than 95% AML blasts) were used during T cell activation, IL-13 was released both during alloactivation of normal T lymphocytes and during mitogen activation of posttransplant T cells. Third, when normal T lymphocytes were stimulated with allogeneic AML blasts, addition of IL-13-neutralizing monoclonal antibodies decreased interferon gamma levels. Although addition of IL-13-neutralizing antibodies did not alter granulocyte-colony-stimulating factor secretion by allostimulating AML blasts, altered blast proliferation was detected for certain patients. Thus, most T cell clones can release IL-13, and IL-13 can modulate cytokine responses during T cell recognition of allogeneic AML cells.

Background T cell mediated immune dysregulation is an established feature of Myelodysplastic syndrome (MDS). We have shown previously that the number of T regulatory cells (Tregs) are increased in high risk MDS and associate with disease progression. In low risk disease however, T helper-17 cells (Th17) are increased which correlates with augmented bone marrow apoptosis. Nevertheless, the role of other components of immune system in MDS pathogenesis is still emerging. Myeloid derived suppressor cells (MDSCs) are a group of immature myeloid cells with a potent immunosuppressive effect which are expanded in an inflammatory environment. MDSCs suppress T cells by depriving them of arginine and cysteine, as well as the secretion of Interleukin (IL)-10, Transforming Growth Factor (TGF)-β and induction of Tregs. While, the role of MDSCs in negatively regulating the anti-tumour immune responses and in determining the T cell polarity have been demonstrated in solid tumours, this information is lacking in MDS. The aim of this study was to investigate the number and function of MDSCs in MDS and potential contribution to immune dysregulation in MDS. Material and Methods MDSCs and Treg numbers were assessed in peripheral blood (PB) and bone marrow (BM). In total we have analysed PB from 24 MDS patients of whom 12 patients had concurrent BM samples. PB samples from 11 age-matched healthy donors (HD) were also included. The WHO subtypes were: RC, RARS or RCMD (n=16, 67%) and 8 patients (33%) had RAEB. Cell surface staining and flow-cytometry was performed following red cell-lysis using the following markers: Live/dead dye, (eBioscience), Lineage markers (CD3, CD16, CD19, CD20, CD56,), CD33, CD34, HLA-DR, CD11b, CD15, CD66b, CD14, CX3CR1, CXCR3 and CXCR4. MDSCs were stained for intracellular TGF-β and IL-10 following fixation and permeabilisation, and they also stained positive for Arginase-1. To evaluate MDSCs function, CFSE stained CD3+CD4+CD25- (T-effectors) from MDS patients were stimulated by anti-CD3/CD28 antibodies. Cells were cultured with: T-effectors alone, T-effectors + CD3+/CD4+/CD25high (Tregs), (ratio 2:1); T-effectors + Tregs + HLA-DR-/CD14+ (MDSC) (ratio 2:1:1). Results MDSCs in the PB were higher in patients with RAEB compared to both healthy donors (HD) and non-RAEB MDS (2.6% v 1.03% p=0.0004) & (2.6% v 2% p=0.02). The absolute numbers of MDSCs were also higher in RAEB compared to non-RAEB subtypes (0.17 x 109/L v 0.08 x 109/L, p=0.02) and in Intermediate (INTR)/High (HR)/Very high (VHR) risk groups in comparison with Very low (VLR)/ Low (LR) risk group (0.31 x 109/L v 0.08 x 109/L, p=0.01). MDSCs frequency in MDS patients was higher in BM than in PB (45% v 2.6%, p=0.002). The PB MDSCs were mainly Monocytic (M)-MDSCs (0.6 % in PB v 0.3% in BM, p=0.02). M-MDSCs were also expressing higher CX3CR1, (MFI 350 v 8849, p=0.008) and higher CXCR4 (MFI 1846 v 2498, p=0.0156) compared to G-MDSCs. Granulocytic (G)-MDSC percentages were increased in INT/HR/VHR compared to VLR/LR (0.7% v 0.2%, p=0.0089) and HD (0.2% v 0.7%, p=0.0385). G-MDSCs were also more frequent in the peripheral blood of RAEB compared to non-RAEB subtypes (0.5% v 0.1% p=0.0299). G-MDSCs from MDS patients produce higher amount of IL-10/TGF-β compared to Monocytic (M)- MDSCs, evaluated by intracellular staining (IL-10 R-MFI, 7.9 v 3.8, p=0.003 and TGF-β R-MFI 15 v 4.8, p=0.024). To evaluate the suppressive effect of MDSCs, autologous Tregs and T-effectors from MDS patients (one INTR and one LR) were co-cultured in the presence and absence of MDSCs. Presence of MDSCs, not only increased the T-effector suppression (figure 1) but also induced Tregs proliferation. In patients with RAEB we also found a positive correlation between absolute counts of MDSC and CD27+FOXP3+CD4+Tregs (Spearman R=0.9, p=0.0374). Discussion Our data show that MDSCs are increased in MDS patients, particularly in high-risk disease, compared to healthy donors. The expanded MDSCs express chemokine receptors and secrete IL-10 and TGF-β, and potentiate the suppressive effect of Tregs. In RAEB patients, numbers of MDSCs are positively correlated with Treg numbers. Data suggests that increased MDSCs play an important role in immunopathogenesis of MDS by suppressing the immune-surveillance against the dysplastic clone. Inhibition of MDSCs (i.e. by arginase inhibitor) could reverse the immunosuppressive environment and re-establish immune-surveillance in MDS. Disclosures: No relevant conflicts of interest to declare.

Introduction Dysregulation and mutations in signaling genes of cancer cells characterize more than half of the acute myeloid leukemia (AML) patients, and contribute to chemoresistance through regulation of cellular processes including apoptosis and DNA repair. We investigated if determination of single cell basal phosphorylation of signaling proteins reflected mutation of FLT3 and NPM1, cytogenetics, response to first course of chemotherapy or overall survival. Methods We employed flow cytometric single cell analysis of phosphoproteins central to signal transduction pathways in myeloid cancer cells and analyzed peripheral blood leukocytes from 93 acute myeloid leukemia (AML) patients. Blood samples from consecutively diagnosed AML patients with high peripheral blood blast counts (>7x109/L, >70% blasts) were collected after informed consent was given and biobanked by cryo-preservation. AML samples was thawed and equilibrated for one hour in a defined serum-free medium (StemSpan SFEM medium, Stem Cell Technologies) which include insulin and transferrin. All samples were viability controlled and validated for growth factor response. Basal phosphorylation was determined using 17 phosphorylation specific antibodies: All staining panels contain the same 4 surface antibodies and a live dead discriminator; CD33(P67.6) PerCP-Cy5.5, CD38(HB7) PE-Cy7, CD34(581) PE, CD45(MEM-28) PE-Dynamics590 and phospho c-PARP(Asp214) Alexa Flour 700. Two phosphospecific antibodies were added to each panel with the respective direct conjugated dye Alexa Flour 488 and Alexa Flour 647; p38(pT180/pY182) and ERK2(pT202/pY204), SRC(pY418) and Akt(pT308), PDK1(pS241) and Akt(pS473), STAT1(pY701) and ribosomal protein S6(pS235/36), STAT3(pY705) and STAT5(pY694), CREB(pS133) and STAT3(pS727), ribosomal protein S6(pS240) and NFkB(pS529), 4EBP(pT37/pT45) and STAT6(Y641), without p-antibody and JNK(pT183/pY185). The lowest median signal for each phosphoprotein in AML cells and lymphocytes, respectively, were used as reference value for calculation of basal phosphorylation. Hierarchical clustering with the use of complete linkage were created using TM4, and Principal Component Analysis was carried out using Unscrambler X (CAMO Software). Results Unsupervised clustering revealed two distinct signature clusters based on low or elevated phosphorylation level among AML cells. A similar cluster signature was absent in endogenous non-leukemic lymphocytes from the same patients. No correlations between basal phosphorylation and prognostic mutations (FLT3 or NPM1), cytogenetics or response to first course of chemotherapy were found. In AML patients treated with intensive chemotherapy (n=45) the cluster with low phosphorylation level (n=19) correlated with significant (p=0.007) shorter overall survival. Principal component analysis verified the cluster analysis and guided a reduction to only three phosphoproteins (STAT3, 4EBP1, ribosomal protein S6) with statistically significant (p=0.014) stratification of survival. Conclusion Leukemic cells demonstrated a phosphorylation profile that reflected survival of the intensively treated patients, but surprisingly not correlated with mutational status of FLT3, NPM1, cytogenetics or first course remission status. This suggests that phosphoprotein determination in leukemic cells provide prognostic information so far not available with current diagnostics. The robust and relatively simple method of single cell signal profiling should be tested in clinical trials to examine its feasibility in therapy response prediction. More extensive mutational and epigenetic analyses are needed in search for the molecular origin of the low/high signal profiles. (A) Unsupervised hierarchical cluster analysis based on three phospho protein analysis stratified in two distinct clusters of patients receiving standard intensive induction chemotherapy (n=45; daunorubicin + cytarabine or idarubicin + cytarabine (3+7) similar to HOVON AML protocols 103 and 102, respectively). (B) Kaplan-Meier plot was performed calculating P value with the use mantel-Cox log-rank test. Disclosures: No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) is a heterogeneous disease with poor prognosis. The overall survival rate in AML patients has changed little in the last 20 years with overall survival rates for young patients (< 60 years) remaining at 35%. However, for elderly patients (> 60 years), survival is less than 10%. Despite the fact that AML represents one of the most molecularly characterized malignancies, the standard therapeutic option is unaltered in the last 15 years i.e. we still solely rely upon a combination of anthracycline and Cytarabine (Ara-C) as the cornerstone of induction therapy. Recently many potentially drugable targets have been discovered and while responses have been observed, these are generally amongst sub-sets of patients with good to intermediate prognostic factors and most often in combination with conventional therapy. Elacytarabine (ELA; CP-4055) is a fatty acid derivative (elaidic acid ester) of Ara- C, currently in undergoing phase II clinical trial as second-course remission-induction therapy in combination with anthracycline. In this study we determined the maximum tolerated dose for the combination of ELA with Daunorubicin (DAN) in immunodeficient NOD-scid-IL2Rγcnull (NSG) mice. Subsequently, we compared the preclinical efficacy of the combination of ELA (30 mg/kg; q.d.x5) or Ara-C (500 mg/kg; q.d.x2) with DAN (2.5 mg/kg; q.d.x3) in a primary patient xenograft model of refractory AML following relapse after Ara-C+DAN therapy (n = 24). Efficacy was evaluated employing a novel time-domain imaging strategy utilizing targeted monoclonal antibodies conjugated to near infrared dye that specifically recognize and bind to epitopes on AML cells enabling non-invasive monitoring of disease progression following therapy. Similarly, bone marrow aspirates were assayed for AML content by flow cytometry (CD45+/34+/33+) and IHC analysis. The combination of ELA and DAN was well tolerated in NSG mice xenografted with primary patient AML cells. While longitudinal near infrared optical imaging of AML disease dissemination illustrated lower disease burden in Ara-C + DAN treated mice than controls (p ELA is more effective preclinically than Ara-C in combination with DAN in primary patient xenografts of refractory AML. ELA was found to be well tolerated and significantly increased survival in combination with Daunorubicin. Despite withdrawal of Elacytarabine as a single agent (following Phase III clinical trial earlier this year in relapsed and refractory AML versus investigators choice of treatment) our preclinical results demonstrate combined therapy of ELA+DAN may promise enhanced survival advantages over current standard therapy for AML patients. Disclosures: Popa: KinN Therapeutics AS: Employment. Gjertsen:KinN Therapeutics AS: Membership on an entity’s Board of Directors or advisory committees. McCormack:KinN Therapeutics AS: Membership on an entity’s Board of Directors or advisory committees.

Background: The overall survival rate in patients with acute myeloid leukaemia has changed little in the last 20 years with overall survival rates for young patients (60 years, survival statistics are less than 10% being cured (2). Although AML has become one of the most molecularly characterised malignancies, the core therapeutic option remains unchanged in the last 15 years i.e. We continue to be solely reliant upon a combination of anthracycline and Cytarabine (Ara-C) as the cornerstone of induction therapy. Recently many potentially drugable targets have been revealed (3-7) and while responses have been observed, these are generally amongst sub-sets of patients with good to intermediate prognostic factors and most often in combination with conventional therapy. Aims and methods: Elacytarabine (ELA; CP-4055) is a fatty acid derivative of Ara-C, currently in undergoing phase II and phase III clinical trial as second-course remission-induction therapy and in late stage AML, respectively. In this study we determined the MTD for the combination of ELA in combination with Daunorubicin (DAN) in NSG mice. Subsequently, we compared the preclinical efficacy of the combination of ELA (30 mg/kg; q.d.x5) or Ara-C (500 mg/kg; q.d.x2) with DAN (2.5 mg/kg; q.d.x3) in primary xenografts in NSG mice (n = 24) from relapsed (Ara-C+DAN) AML patients. Employing a novel time-domain imaging strategy (8) we non-invasively monitored disease progression following therapy. Similarly marrow aspirates were assayed for AML content by flow cytometry and IHC analysis. Results: The combination of ELA and DAN was well tolerated in NSG mice xenografted with primary patient AML cells. While longitudinal near infrared optical imaging of AML disease burden illustrated lower disease burden in Ara-C + DAN treated mice than controls (p Conclusions: The combination of ELA+DAN is well tolerated and significantly increase survival in comparison to Ara-C+DAN in primary patient xenografts of refractory AML. 1. Rowe JM, et al. Blood 2010; 116: 3147-56. 2. Burnett AK. Rev Clin Exp Hematol 2002; 6: 26-45; discussion 86-7. 3. Nencioni A et al. Leukemia 2007; 21: 30-6. 4. Bruserud O et al. Expert Opin Ther Targets 2006; 10: 51-68. 5. Burnett AK et al. Hematology Am Soc Hematol Educ Program 2007: 429-34. 6. Doepfner KT et al. Crit Rev Oncol Hematol 2007; 63: 215-30. 7. Tang R et al. BMC Cancer 2009; 9: 199. 8. McCormack E et al. Blood 2012; In Press. Citation Format: Tereza Osdal, Lars P. Jordheim, Mihaela Popa, Lene M. Vikebø, Andre Sulen, Siv Lise Bedringaas, Marit Liland Sandvold, Birgitte Booij, Øystein Bruserud, Bjørn Tore Gjertsen, Charles Dumontet, Emmet McCormack. Combination of elacytarabine and daunorubicin is significantly more effective than combination of cytarabine and daunorubicin in primary patient xenografts of AML. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3274. doi:10.1158/1538-7445.AM2013-3274

We show that cyclic AMP (cAMP) elevating agents protect blasts from patients with acute promyelocytic leukemia (APL) against death induced by first-line anti-leukemic anthracyclines like daunorubicin (DNR). The cAMP effect was reproduced in NB4 APL cells, and shown to depend on activation of the generally cytoplasmic cAMP-kinase type I (PKA-I) rather than the perinuclear PKA-II. The protection of both NB4 cells and APL blasts was associated with (inactivating) phosphorylation of PKA site Ser118 of pro-apoptotic Bad and (activating) phosphorylation of PKA site Ser133 of the AML oncogene CREB. Either event would be expected to protect broadly against cell death, and we found cAMP elevation to protect also against 2-deoxyglucose, rotenone, proteasome inhibitor and a BH3-only mimetic. The in vitro findings were mirrored by the findings in NSG mice with orthotopic NB4 cell leukemia. The mice showed more rapid disease progression when given cAMP-increasing agents (prostaglandin E2 analog and theophylline), both with and without DNR chemotherapy. The all-trans retinoic acid (ATRA)-induced terminal APL cell differentiation is a cornerstone in current APL treatment and is enhanced by cAMP. We show also that ATRA-resistant APL cells, believed to be responsible for treatment failure with current ATRA-based treatment protocols, were protected by cAMP against death. This suggests that the beneficial pro-differentiating and non-beneficial pro-survival APL cell effects of cAMP should be weighed against each other. The results suggest also general awareness toward drugs that can affect bone marrow cAMP levels in leukemia patients. publishedVersion

Chemokines and their receptors are involved in the recruitment of leukocytes to sites of inflammation. Recently, chemokine expression signatures have been reported to convey a prognostic value in myelodysplastic syndrome (MDS) patients. In the present study, we investigated the chemokine receptor repertoire on fresh peripheral blood lymphocytes from 31 (22 low-risk and 9 high-risk) patients affected by MDS. Chemokine receptor expression was studied in defined T-cell subsets using eight-color flow cytometry. MDS patients exhibited quantitative differences in peripheral lymphocyte subpopulations. In addition, T cells obtained from MDS patients expressed a chemokine receptor pattern suggesting a dominance of mature and activated T cells. This is illustrated by increased levels of CCR3, CCR5, CX3CR1 and/or by a decreased abundance of CCR7 in defined T-cell subsets. The T-cell subset distribution appears to differ between the peripheral blood and the bone marrow of MDS patients, suggesting a preferential recruitment of specific T-cell subsets to the latter compartment. Alteration in chemokine receptor expression can develop over time even in patients that are considered clinically stable. Elevated expression levels of CXCR4 by CD8+ cells were associated with prolonged patient survival and reduced numbers of bone marrow blasts. We conclude that immunological abnormalities in MDS also involve chemokine receptors on different subsets of T cells, and that these changes may have a prognostic value.

Interleukin 6 (IL6) secretion by blast cells derived from peripheral blood of patients with acute myelogenous leukemia (AML) was characterized. IL6 secretion showed a wide variation both when AML blasts were cultured in medium alone and in the presence of exogenous cytokines. The level of IL6 secretion was significantly correlated to secretion of other cytokines (IL1 alpha, IL1 beta, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor alpha). IL1 beta, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor and stem cell factor increased IL6 secretion, whereas IL4 caused decreased IL6 secretion together with increased release of IL1 receptor antagonist.

Potential anti-leukemia effects mediated by T cells or by natural killer (NK) cells were investigated in chronic myelogenous leukemia (CML) patients treated with interferon-alpha. Therapy-associated modulation of T cell and NK reactivity was monitored for one year from initiation in autologous mixed lymphocyte-tumor cell reactions and cytotoxicity directed against autologous CML cells, respectively. During the course of IFN-therapy, NK activity against autologous CML cells increased steadily, whereas T cell reactivity fluctuated randomly. Despite the high level of T cell reactivity to autologous tumor cells in short-term (6 days) culture, 1) they failed to respond to synthetic peptides corresponding to the bcr/abl fusion sequence of the patient, and 2) only one proliferative T cell clone (TCC) was isolated which specifically recognized HLA-DR-matched CML cells. This TCC appeared not to recognize synthetic peptides corresponding to the bcr/abl fusion sequence of the patient; the antigen to which it responds remains unknown. To assess potential immunogenicity of bcr/abl peptides, it was attempted to sensitize T cells from normal donors in vitro. Of 109 cell lines obtained from seven different donors, eleven showed peptide-dependent proliferation. Therefore, although these results show that it is possible to isolate apparently CML-specific T cells from patients, as well as to prime T cells against tumor-specific peptide in vitro, the frequency of such T cell-mediated reactivity appears low and its relevance to anti-leukemic effects questionable. On the other hand, the strong time-dependent enhancement of natural killing of autologous CML blasts during IFN-alpha treatment, a phenomenon not observed for T cell reactivity, suggests that natural immunity may be more important in controlling disease.

The in vitro effect of the dextroisomer r-verapamil on blast cells derived from patients with acute myelogenous leukemia (AML) was studied. R-verapamil caused a dose-dependent inhibition of AML blast proliferation in the presence of stem-cell factor, leukemia inhibitory factor, interleukin 4, interleukin 6, and interleukin 10 when these cytokines were tested both alone and in different combinations. R-verapamil also inhibited the growth of clonogenic AML blast cells. The antiproliferative effect was not specific for AML blast cells, because r-verapamil also inhibited cytokine-dependent proliferation of blast cells derived from patients with acute lymphoblastic leukemia. The inhibitory effects of r-verapamil and anti-IL1 serum were additive, suggesting that the antiproliferative effect of r-verapamil does not depend solely on inhibition of IL1-mediated effects. Although r-verapamil inhibited spontaneous AML blast proliferation, for a majority of patients it caused only minimal, if any, inhibition of spontaneous cytokine secretion (IL1 alpha, IL1 beta, TNF alpha, IL6) by AML blast cells. Thus, although inhibition of IL1 effects may contribute in certain patients to the antiproliferative effect of r-verapamil, mechanisms other than IL1 inhibition seem to be more important in mediating the effects of r-verapamil.

Serum concentrations of tumour necrosis factor-alpha (TNF-alpha) increase during septicaemia in previously healthy individuals. To investigate whether a similar increase in TNF-alpha can be seen in severely immunocompromised patients with acute leukaemia and chemotherapy-induced leukopenia, serum TNF-alpha was analysed in leukopenic patients with bacterial infections. Pretherapy serum levels of TNF-alpha were decreased in leukaemia patients compared with healthy controls, and serum TNF-alpha levels showed a further decrease when patients developed chemotherapy-induced leukopenia. When leukopenic patients developed bacterial infections, serum concentrations of TNF-alpha increased. Serum levels of TNF-alpha decreased when clinical signs of infection resolved during antibiotic therapy, but an increase occurred later in parallel with haematopoietic reconstitution.

Abstract 3599 Heat shock proteins (HSPs) maintain cellular homeostasis and function as molecular chaperones assisting protein folding and translocation. Their intracellular levels are up-regulated in response to a wide variety of insults allowing the cell to survive otherwise lethal conditions. Heat shock proteins are also released to the extracellular space and lead to a more efficient cross-presentation to specific T cells and may also initiate release of proinflammatory cytokines, stimulate NK cells and facilitate DC maturation. Extracellular Heat shock protein (HSP) 70 and HSP90 are characteristics of immunogenic apoptosis, and primary human acute myeloid leukemia (AML) cells release these mediators during spontaneous in vitro apoptosis. Primary AML cells also show constitutive release of several cytokines; these heterogeneous mediators interact through local cytokine networks that regulate growth and differentiation of normal as well as malignant hematopoietic cells. In the present study we investigated serum levels of HSP70/HSP90 and the serum cytokine profiles of patients with untreated AML and patients receiving disease-stabilizing treatment based on all-trans retinoic acid (ATRA) plus valproic acid. Serum samples from 82 untreated AML patients, including 42 patients receiving AML-stabilizing therapy, and 20 healthy controls were analysed. HSP and cytokine levels were determined by ELISA and multiplex analyses. Patients with untreated AML showed significantly increased HSP90 levels compared to healthy controls (Mann Whitney U-test, p AML patients showed an altered serum cytokine profile compared to healthy controls with significantly altered levels of several chemokines, interleukins, growth factors and immunomodulatory cytokines (Tab.1). These cytokine levels were usually increased compared to the healthy controls. Hierarchical cluster analysis of the untreated patients showed a close association between HSP70, HSP90 and Hepatocyte growth factor (HGF) levels, these mediators also showed a significant correlation (Spearman, p We conclude that both HSP levels and serum cytokine profiles are altered in untreated AML, and the modulation of these serum levels by AML-stabilizing chemotherapy may reflect a possible mechanism for the clinical effect of this treatment strategy. The table shows serum levels for 82 AML patients and 20 healthy controls where significant differences were found (Mann-Whitney U-test, p Disclosures: No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) frequently comprises mutations in genes that cause perturbation in intracellular signaling pathways, thereby altering normal responses to growth factors and cytokines. Such oncogenic cellular signal transduction may be therapeutic if targeted directly or through epigenetic regulation. We treated 24 selected elderly AML patients with all-trans retinoic acid for 2 days before adding theophylline and the histone deacetylase inhibitor valproic acid (ClinicalTrials.gov NCT00175812; EudraCT no. 2004-001663-22), and sampled 11 patients for peripheral blood at day 0, 2 and 7 for single-cell analysis of basal level and signal-transduction responses to relevant myeloid growth factors (granulocyte-colony-stimulating factor, granulocyte/macrophage-colony-stimulating factor, interleukin-3, Flt3L, stem cell factor, erythropoietin, CXCL-12) on 10 signaling molecules (CREB, STAT1/3/5, p38, Erk1/2, Akt, c-Cbl, ZAP70/Syk and rpS6). Pretreatment analysis by unsupervised clustering and principal component analysis divided the patients into three distinguishable signaling clusters (non-potentiated, potentiated basal and potentiated signaling). Signal-transduction pathways were modulated during therapy and patients moved between the clusters. Patients with multiple leukemic clones demonstrated distinct stimulation responses and therapy-induced modulation. Individual signaling profiles together with clinical and hematological information may be used to early identify AML patients in whom epigenetic and signal-transduction targeted therapy is beneficial.

Secretion of different cytokines may be an important T-cell effector mechanism for bone marrow engraftment, graft versus host disease and graft versus leukaemia effects after allogeneic bone marrow transplantation (BMT). Cytokine secretion and autocrine proliferative capacity of T-cell clones derived from leukaemia patients 3–6 weeks after allogeneic bone marrow transplantation were investigated. Only a minority of post-transplant T-cell clones (23/120; 19%) was capable of undergoing autocrine proliferation. By contrast, 21/65 (32%) normal control clones from the marrow donors derived under the same conditions were autocrine proliferative. All clones were interleukin-2 (IL-2) responsive. A majority (12/17; 71%) of autocrine proliferating post-transplant clones secreted detectable IL-2. Compared with control clones, CD4+ T-cell clones derived early after BMT produced decreased levels of interleukin-4 (IL-4) and interleukin-6 (IL-6), whereas secretion of interleukin-3 (IL-3) and granulocyte/macrophage colony-stimulating factor (GM-CSF) showed no significant difference. The small number (n = 8) of post-transplant CD8+ clones showed decreased production of IL-3, IL-4 and IL-6 compared with control clones, but normal secretion of GM-CSF. Neither CD4+ nor CD8+ T-cell clones secreted interleukin-7 (IL-7).

Cytokine-dependent AML cell proliferation was investigated in 16 patients. Dipyridamole and R-verapamil caused a dose-dependent inhibition of AML cell proliferation, and for both drugs the degree of inhibition was similar when testing various haematopoietic growth factors or growth factor combinations (IL3, G-CSF, GM-CSF, G-CSF + GM-CSF, TNF-alpha + GM-CSF). TNF-alpha alone increased AML cell proliferation for five patients, whereas four patients showed unaltered or decreased proliferation. Independent of this TNF-alpha effect, R-verapamil inhibited proliferation for all AML patients in the presence of TNF-alpha, whereas dipyridamole caused only a weak inhibition.

Abstract 2147 Introduction: Normal hematopoiesis takes place in the bone marrow niches where the hematopoietic stem cells are surrounded by stromal cells and extracellular matrix, and were both soluble mediators as well as cell-cell interactions seem to regulate proliferation and initial maturation. Among the various molecules participating in hematopoietic regulation are gap junctions (GJs) that are formed by connexins (Cxs) and represent intercellular communication channels (reviewed in Foss B et al., Stem Cell Dev. 18(6): 807–812, 2009). Acute myelogenous leukemia (AML) is characterized by bone marrow accumulation of immature leukemic cells, and both disease development as well as chemosensitivity of the leukemic cells seem to be affected neighbouring stromal cells in the bone marrow microenvironment. Studies of leukemic cell lines and animal models suggest that especially Cx43 and possibly Cx32 are involved in regulation of AML cell proliferation and differentiation, but the functional potential of Cxs in AML seems to be wider (reviewed in Foss B, et al., Biochim Biophys Acta. 1798(1):1-8, 2010). Still, the expression profile of Cxs by primary human AML cells are not well characterized. Methods: We characterized the mRNA and protein expression of various Cxs in primary human AML cells. The mRNA expression was investigated in microarray assay (n = 47) and the protein expression in the cell surface membrane by flow cytometry (n = 38). Results: The mRNA levels of Cx32 (mean 0.29, stdv ± 0.16), Cx43 (0.09 ± 0.21) and Cx45 (0.56 ± 0.25) showed very low levels whereas Cx37 showed higher expression (1.66 ± 0.93). The membrane expression was classified as positive (i.e. > 20% of AML cells stained positive) only for a minority of patients especially when investigating Cx32 (5 out of 38 patients examined) but also for Cx37 (13/38) and Cx43 (16/38), whereas the leukemic cells were classified as Cx45 positive for a majority of the patients (21/38). The mean fluorescence intensities (MFI) of the membrane expression for Cx32, Cx37, Cx43 and Cx45 were all significantly correlated (P Conclusions: These results show for the first time that primary AML cells express various Cxs on their cell membranes, but patients are heterogeneous and the expression is seen especially in AML cells with signs of monocytic differentiation. Disclosures: No relevant conflicts of interest to declare.

Abstract 4018 Several T cell abnormalities have been described in myelodysplastic syndromes (MDS), and such abnormalities may become important for identification of patients who will benefit from T cell targeting immunosuppressive treatment. Chemokine receptor repertoires are important in the regulation of both T cell migration and function and may therefore be important in the development of MDS. Materials and Methods: The chemokine receptor expression by circulating T cells were investigated by multicolor flow cytometry for patients with newly diagnosed low risk MDS (N=16) and for healthy controls (N=18). CD3+CD8+ and CD3+CD8- cells were examined for expression of CCR2-7, CXCR3-4 and CX3CR1 (only 7 unselected patients examined for CX3CR1 expression). CCR6 and CXCR4 expression was also investigated for specific T cell subsets defined by CD62L and CD45RA expression (naïve, central memory, effector memory and terminal effector). CX3CR1 expression was stratified for CD8+ and CD8- subpopulations according to high, low and negative expression of CCR5. Results: Chemokine receptor profiles showed several differences between low risk MDS patients and controls. Total CD8+ T cells from MDS patients showed increased expression of CCR3 (p=0.005) and decreased expression of CCR7 and CCR4 (p=0.023 and p=0.036 respectively), whereas the CD8- T cells from MDS patients showed increased expression of CX3CR1 (p=0.043). In contrast, CCR6 expression was increased only by CD8+ central memory T cells (p=0.044). Finally, CD8+CCR5- and CD8-CCR5high cells from MDS patients showed increased expression of CX3CR1 compared with the controls (p=0.011 and p=0.049). Discussion: CCR7 is mainly expressed by central memory and naïve T cells whereas CX3CR1 is especially expressed by cytotoxic effector lymphocytes independent of the lymphocyte subclass (i.e. CD4, CD8, delta/gamma and NK cells). The observed changes are in line with a shift from naïve/central memory to effector/effector memory dominance, especially in the CD8+ population. A similar shift has been described previously using CD45RA and CD62L (Zou et al, Leukemia 2009). Our median values are in line with such a shift. CCR6 expression is associated with IL17 production both for CD8+ and CD4+cells, and increased levels of circulating CD4+ IL17 producing cells in low risk MDS have been described. Conclusions: The chemokine receptor profiles of circulating T cells differ between low risk MDS patients and healthy controls, especially for the CD8+ T cell subset. These differences may be important for T cell trafficking and disease development, and they may reflect a shift from naïve to effector/effector memory cell dominance in low risk MDS. Disclosures: No relevant conflicts of interest to declare.

Myeloid leukemias are a heterogeneous group of diseases originating from bone marrow myeloid progenitor cells. Patients with myeloid leukemias can achieve long-term survival through targeted therapy, cure after intensive chemotherapy or short-term survival because of highly chemoresistant disease. Therefore, despite the development of advanced molecular diagnostics, there is an unmet need for efficient therapy that reflects the advanced diagnostics. Although the molecular design of therapeutic agents is aimed at interacting with specific proteins identified through molecular diagnostics, the majority of therapeutic agents act on multiple protein targets. Ongoing studies on the leukemic cell proteome will probably identify a large number of new biomarkers, and the prediction of response to therapy through these markers is an interesting avenue for future personalized medicine. Mass spectrometric protein detection is a fundamental technique in clinical proteomics, and selected tools are presented, including stable isotope labeling with amino acids in cell culture (SILAC), isobaric tags for relative and absolute quantification (iTRAQ) and multiple reaction monitoring (MRM), as well as single cell determination. We suggest that protein analysis will play not only a supplementary, but also a prominent role in future molecular diagnostics, and we outline how accurate knowledge of the molecular therapeutic targets can be used to monitor therapy response.

The effect of disodium cromoglycate on in vitro proliferative responses of peripheral blood mononuclear cells from healthy individuals, allergic patients with moderate serum IgE and patients with atopic dermatitis and high levels of serum IgE was investigated. Peripheral blood mononuclear cells were stimulated with mitogens (phytohaemagglutinin, Concanavalin A), recombinant interleukin-2, calcium ionophore + phorbol 12-myristate 13-acetate, purified protein derivative of tuberculin and allergens. It was possible to induce in vitro specific, allergen-triggered responses only in allergic individuals with moderate serum IgE and not in individuals with atopic dermatitis and high serum IgE. Generally, whenever the stimulatory signal(s) caused a significant proliferative response, disodium cromoglycate inhibited the proliferation. This inhibition was seen for all activation agents and for both healthy and allergic individuals. By contrast, for certain non- or low-responders (both healthy and allergic individuals) disodium cromoglycate seemed to amplify the proliferation to various activation signals. Only non- or low-responder cells derived from atopic dermatitis patients showed a biphasic kinetic response pattern when stimulated with the drug in combination with recombinant interleukin-2, recombinant interleukin-2 + ionophore or specific allergens.

Abstract 4151 Disease stabilisation, instead of cure, is proposed as the therapeutic strategy of choice in elderly or chemoresistant acute myeloid leukemia (AML). This approach may also be of particular benefit to patients for whom allogeneic bone marrow (re)transplantation is not an option. Previously, we have clinically investigated the addition of valproic acid (VPA) to various combination chemotherapies with initial results in AML indicating prolonged survival, with follow-up on a predominantly out-patient basis. Consequently, we aimed to identify further molecular targets of VPA, which may enhance its therapeutic efficacy through screening for VPA-modulated phosphoproteins in relevant preclinical models of AML, and validation of these targets in RNAi screen of Caenorhabditis elegans (C. elegans). Brown Norwegian Myeloid Leukemia (BNML) has previously been described as a particularly relevant preclinical rat model of AML. Indeed, leukemic rats treated with 170 mg/kg VPA twice-daily achieved therapeutic serum levels of VPA and demonstrated significant increases in survival in comparison to controls (p = 0.004). To screen for molecular targets of VPA effect in this responsive model, we investigated the differences in control and VPA treated BNML phosphoproteomes by difference gel electrophoresis (DIGE) separation and subsequent differential gel software analysis. This was achieved through harvest of phosphoproteins from leukemic blasts, isolated from the spleens of treated and control BNML rats by immobilized metal ion affinity chromatography (IMAC) and subsequent protein identification via Orbitrap mass-spectrometry. Significant differential expression of 9 phosphoproteins was found in VPA treated BNML rats compared to controls, including Tubulin α-1B chain (TBA1B) and Actin β (ACTB), indicating these genes as possible targets of VPA therapy. To validate the functionality of 7 of these genes, RNAi was performed in wild type Bristol N2 strain of C. elegans at larval stage L1, 24 hours prior to exposure to 15 mM VPA for 72 hours. Knockdown of 4 of 7 genes resulted in larval developmental arrest, defined as synthetic lethality. In order to ascertain if synthetic lethality induced by these 4 genes was resultant of apoptosis, we employed the CED-1::GFP transgenic reporter assay to quantify germline cell death following RNAi depletion and subsequent exposure to VPA (15 mM, 24 hours). Increased numbers of apoptotic corpses in the germline was determined for all genes examined. To further examine the role of p53 in the observed apoptotic induction we used the transgenic strain cep-1::CED-1::GFP, which expresses the C. elegans ortholog of p53, CEP-1. Successive RNAi knockdown of our 4 candidate genes, again effected increased basal number of apoptotic corpses independently of CEP-1. These results suggest that similar combinational treatment of AML may be beneficial, irrespective of p53 status. To further investigate this thesis in a human AML cell line, MOLM-13 cells were co-treated with VPA and small molecule inhibitors of prospective targets TBA1B and ACTB, namely paclitaxel, and cytochalasin B. Inhibition of actin polymerization or stabilisation of tubulin polymerization resulted in increased apoptosis when supplemented with VPA, as determined by DNA specific staining with Hoechst 33342. These results suggest that use of these combinations may be beneficial in the treatment of AML. In conclusion, this study indicates that phosphoproteomic screening of BNML and subsequent target verification in C. elegans worms has the potential to identify future drugable targets for effective combinatorial therapy with valproic acid in acute myeloid leukemia. Disclosures: No relevant conflicts of interest to declare.

Nutlin-3 is a small-molecule antagonist of MDM2 that induces non-genotoxic stabilization and activation of the tumor suppressor protein p53, resulting in therapeutic effects in tumor models comprising wild type TP53. Valproic acid (VPA) is an anti-convulsive drug with histone deacetylase (HDAC) inhibitor activity that induces differentiation and apoptosis in acute myeloid leukemia (AML) cells. Both therapeutic compounds indirectly affect the regulation of p53; nutlin-3 by inhibiting MDM2, the main negative regulator of p53, and VPA by inhibiting HDACs that participate in p53 deacetylation and destabilization. While mutations in TP53 occur in less than 10% of AML, over-expression of MDM2 is frequently observed. Furthermore, aberrant recruitment of histone deacetylases (HDACs) is seen in AML, leading to block of myeloid differentiation. We therefore hypothesized that concomitant inhibition of MDM2 and HDACs would synergistically induce p53-mediated apoptosis and inhibit tumor growth. We examined the anti-leukemic effects of nutlin-3 in combination with low doses valproic acid in AML cell lines, primary AML cells, and in an in vivo xenograft model using optical imaging. To evaluate the efficacy of the combination of nutlin-3 and VPA in AML cells expressing wild type TP53, the AML cell line MOLM-13 was treated with various concentrations of the two drugs both alone and in combinations (nutlin-3; 0.5–10 μM, VPA; 50–1000 μM, nutlin-3:VPA; 1:100) over different time periods (24–72h, nutlin-3 only for the 24 last hours). Synergistic or additive effects were detected in three distinct viability assays; 3H-thymidine incorporation was used to examine effect on proliferation, WST-1 was used to determine number of metabolic active cells in culture, and DNA specific staining with Hoechst 33342 was used to determine apoptosis after drug-treatment. In addition, MOLM-13 cells treated with the combination showed super additive induction of p53 and target genes. The optimal combination and time period found in MOLM-13 cells were tested in 40 different primary AML samples using the three different viability assays. Toxicity of the combination treatment was tested in normal peripheral blood lymphocytes, while preliminary toxicity of nutlin-3 and VPA alone and in combination on healthy NOD/SCID IL2γnull mice permitted determination of treatment regime. We developed an optical imagable model for in vivo evaluation of the combinational therapy by injecting NOD/SCID IL2γnull mice with MOLM-13 cells transfected with a tetracycline activated luciferase expressing construct (termed L192). Bioluminescent imaging was performed using a TD-SAMI (Time-domain small animal molecular imager). The efficacy of the combinational therapy was tested in 20 NOD/SCID IL2γnull mice injected with MOLM-13 L192 cells and divided into four groups; control, VPA (50 mg/kg b.i.d), nutlin-3 (200 mg/kg b.i.d) and nutlin-3 + VPA (200 mg/kg + 50 mg/kg) b.i.d. Treatment was scheduled for three weeks. The combination of nutlin-3 and VPA significantly inhibited disease development after one week, as monitored by in vivo imaging. Limitations due to nutlin related toxicity prevented further evaluation of continuous combinational therapy after 14 days. However, all treatment groups showed a significant increase in survival compared to the control group, with the combination group demonstrating decreased leukaemic burden as visualized by optical imaging and longer mean average survival time. VPA effect on survival was also tested in a BNML rat leukemia model, in which VPA-treatment (170 mg/kg) resulted in significant longer mean survival compared to the control. Together, the results suggest combined targeting of MDM2 and HDACs as a promising therapeutic approach in AML. Future studies will apply the established bioluminescent MOLM-13 AML xenograft model for further evaluation of the combinational therapy, using a different dosing regimen and scheduling. In addition, we will evaluate combinations of differing classes of HDAC inhibitors and MDM2 antagonists.

Background Two-dimensional gel electrophoresis (2DE) is a powerful technique to examine post-translational modifications of complexly modulated proteins. Currently, spot detection is a necessary step to assess relations between spots and biological variables. This often proves time consuming and difficult when working with non-perfect gels. We developed an analysis technique to measure correlation between 2DE images and biological variables on a pixel by pixel basis. After image alignment and normalization, the biological parameters and pixel values are replaced by their specific rank. These rank adjusted images and parameters are then put into a standard linear Pearson correlation and further tested for significance and variance. Results We validated this technique on a set of simulated 2DE images, which revealed also correct working under the presence of normalization factors. This was followed by an analysis of p53 2DE immunoblots from cancer cells, known to have unique signaling networks. Since p53 is altered through these signaling networks, we expected to find correlations between the cancer type (acute lymphoblastic leukemia and acute myeloid leukemia) and the p53 profiles. A second correlation analysis revealed a more complex relation between the differentiation stage in acute myeloid leukemia and p53 protein isoforms. Conclusion The presented analysis method measures relations between 2DE images and external variables without requiring spot detection, thereby enabling the exploration of biosignatures of complex signaling networks in biological systems.

Background α-interferon (IFN) is an effective treatment in polycythemia vera (PV) and essential thrombocythemia (ET). IFN can induce cytogenetic remissions in PV pts. However, 21–25 % of pts discontinue therapy due to side effects. Pegylated IFN has been suggested to have less side effects. Study design and patients: The Nordic MPD Study Group performed an open label phase II feasibility study of PegIntron® treatment in 42 MPD pts, 20 females and 22 males, median age 54 yr (range 29–77). Inclusion criteria were a platelet count of > 400x109/L in pts with symptoms or previous thrombosis (n=26) or >1000x109/L in asymptomatic pts without previous thrombosis (n=16). 22 pts had PV, 20 ET. 15 pts had previously received cytoreductive therapy; anagrelide (7), hydroxyurea (6), busulfan (1), P32 (1). Previous IFN therapy was not allowed. Median time from diagnosis to study start was 0.74 ys (0.01–30.2). Initial dose was 0.5 μg/kg once weekly. The treatment goal was a platelet count < 400x109/L in symptomatic patients and < 600 in asymptomatic patients (CR). If CR was not achieved within 12 weeks PegIntron was increased to 1 μg/kg/week. The dose was subsequently decreased to the lowest dose maintaining CR. Pts QoL was investigated using QLQ-C30 and HAD forms. Neutrophil PRV-1 mRNA expression was analyzed by quantitative RT-PCR prior to and after therapy. Results: Response to treatment was evaluated at 1,2,3,6,9 and 12 months. At 6 months, 29/42 pts (69 %) had achieved CR, after a median time of 83 days. The median dose at time of CR was 0.5 μg/kg/week. CR rate was not related to diagnosis or gender. 4/42 pts (10%) discontinued therapy early due to side effects, another 9 pts were taken off study at 6 months due to side effects or insufficient response. Only 4/14 responding PV pts required phlebotomy therapy, compared to 20/22 before PegIntron. 20/29 patients with CR at 6 months maintained CR at 12 months, whereas 9 pts had gone off study due to side effects. The mean platelet count was 875x109/L prior to therapy, and 512, 448, 362x109/L at 3,6 and 12 months in pts on therapy. No thrombotic or bleeding complications were observed. Side effects were common, the majority WHO grades I-II; fatigue (76 %), injection site reaction (64 %), flu-like symptoms (64 %), headache (56 %), muscle pain (51 %), depression (35 %), insomnia (24 %), hair loss (20 %). Mild elevations were noted of serum ALAT, creatinine and TSH in 19, 3 and 3 pts respectively. QoL data will be presented. Changes in PRV-1 expression were determined in 13 PRV-1 positive pts (9 PV, 4 ET) after 6 months and in 2 PV pts after 24 months. PRV-1 expression was normalized in 3/4 ET pts at 6 months and in 1/2 PV pts after 24 months treatment. Conclusion: We found PegIntron to be effective in reducing the platelet count in 29/42 pts (69 %) and 20 pts (48 %) remained in CR at 1 year. 22 pts (52 %) had discontinued therapy at 12 months, mainly due to side effects, a higher rate than in previous trials. Although analyzed in a small number of pts, reversal of PRV-1 positivity nonetheless suggests that PRV-1 quantification may be useful as a molecular marker of therapeutic efficacy.

During the last decade high-dose Ara-C (HIDAC; single doses of 3 g/m(2)) and autologous stem cell transplantation have been increasingly used as postremission therapy in adult acute myelogenous leukemia (AML). Controlled clinical trials have demonstrated a long-term disease-free survival of 40%-50% for patients treated with at least two courses of HIDAC. Other studies have demonstrated that postremission autologous bone marrow transplantation results in a disease-free survival equal to or better than conventional chemotherapy. However, autotransplantation with mobilized peripheral blood stem cells (PBSC) would now be preferred instead of autologous bone marrow, due to the shorter hematopoietic reconstitution period. The results reviewed in the present article suggest that HIDAC and autologous PBSC transplantation can be combined in the postremission treatment of adult AML, and this combination therapy may also reduce minimal residual disease and the risk of posttransplant relapse. From the available studies it cannot be concluded whether graft purging further reduces the relapse risk. However, the possible advantage of combination therapy with repeated courses of HIDAC and autologous PBSC transplantation needs to be demonstrated in prospective clinical trials before it can be recommended as a part of the routine treatment in AML.

The proliferative T lymphocyte responses to two different mumps antigen preparations, S (nucleocapsid) and V (viral envelope), were characterized. Eight patients with Type 1 (insulin-dependent) diabetes mellitus were all found to be responders to S and V mumps antigen. Among the 64 healthy individuals, 52 were classified as responders to the S antigen and 50 responders to the V antigen. No difference was found between T lymphocytes from Type 1 diabetic patients and those from healthy individuals as regards the effect of indomethacin on the mumps-specific response. The majority of the mumps-specific T lymphocytes seemed to be restricted by epitopes on the HLA-DR molecules. The frequency of mumps antigen-reactive T lymphocytes (ARTL) was found to be low when the response was restricted by DR3-associated elements, and high when it was restricted by DR4-associated elements, compared to the frequency of ARTL when other DR-associated elements restricted the mumps-specific response. For the majority of the individuals tested, the DR-associated hypo- and hyper-responsiveness was found with both the S and the V mumps antigens.

Twelve insulin-dependent diabetes mellitus (IDDM) patients and healthy controls, who all carried the serologically defined DR3 and DR4 antigens, were compared with respect to other HLA polymorphisms. No significant differences between patients and controls were found by typing for HLA-Dw determinants by homozygous cell typing, nor by studies of their genomic DR beta polymorphism using different restriction enzymes. In contrast, certain DR beta polymorphism using different restriction enzymes. In contrast, certain DR4-associated genomic DQ beta fragments had a significantly different distribution among the IDDM patients than among the controls. Furthermore, when the distribution of all DQ beta-specific fragments which demonstrated polymorphism in our material was taken into account, nine of the 12 DR3, 4 IDDM patients demonstrated a similar DQ beta polymorphism compared with only two out of the 12 DR3, 4 controls (P = 0.006; corrected P = 0.037). Cells from patients and controls who demonstrated this IDDM-associated DQ beta polymorphism stimulated each other significantly less in reciprocal MLC tests, compared with the responses seen when their cells were confronted with cells from the DR3, 4 individuals with other genomic DQ beta polymorphisms.

A patient with multiple myeloma and amyloidosis was admitted to hospital following successful cardiopulmonary resuscitation at home. No disturbances in heart rhythm were seen during the first 48 hours of continuous telemetric ECG recording. The patient died from ventricular asystole due to complete atrioventricular block, while he was on a 24-hour Holter monitoring the fifth night in hospital. Patients with known cardiac amyloidosis and syncope should undergo long-term ECG recordings, preferably by telemetry. Repeated registrations may be necessary to discover disturbances in heart rhythm.

An increased frequency of mumps-specific T lymphocytes restricted by DR4-associated elements has previously been described in DR4 heterozygotes. Here we report that the preferential restriction elements may be present on DR4 molecules expressing either Dw4 or Dw14. No particular genomic DQ-beta-polymorphism was associated with the preferential restriction elements.

By priming in vitro with allogeneic HLA-DR compatible and also HLA-A,B mostly compatible lymphoid cells, PLT cells resulted in recognizing a group of non D/DR allelic antigens provisionally named K, L, M and N. To improve discrimination these bulk primed typing reagents were cloned and expanded. By typing of previously SB typed lymphoblastoid B cell lines (LCL) the provisional specificities could be identified as SB1, 4, 3 and 2, respectively. Typing of 186 unrelated Norwegians gave the following gene frequences: SB1: 0.05, SB2: 0.16, SB3: 0.13, SB4: 0.42 and SB blank: 0.24. No triplets were found, the calculated gene frequencies fit with Hardy-Weinberg equilibrium, and typing of a B-DR recombinant family confirmed that the SB locus is situated centromeric to B. Associations between SB and A, B, DR antigens in the same material were generally weak, the most significant associations found were between SB1-DR3 and SB4-DR2.

While B lymphocytes can bind antigen directly, T lymphocytes recognize antigen only when presented together with major histocompatibility complex (MHC)-en-coded class I or II molecules in the membrane of antigen-presenting cells (APC). This is called MHC restriction and was first shown in animal studies (see [5, 25] for references). The parts of the MHC molecule which are co-recognized together with antigen are often called restriction elements.

Verapamil, a calcium-inhibitory drug, was found to inhibit T-lymphocyte proliferation in mixed lymphocyte culture and the proliferative response to phytohaemagglutinin and mumps antigen. It also inhibited production of interleukin 2 (IL-2). To exert inhibition, verapamil had to be added early in the culture period. Verapamil also had a relatively small inhibitory effect on IL-2-dependent growth. The effects were clearly seen only at concentrations exceeding the therapeutic serum level of verapamil.