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Multivariate analysis of prognostic factors for overall survival in the patient cohort (n=74)

Molecular characterization of the G-CSF-inducible Stat3/5 double positive (DP) and double negative (DN) subpopulations.

Purpose: Azacitidine is the mainstay of high-risk myelodysplastic syndromes (MDS) therapy, but molecular predictors of response and the mechanisms of resistance to azacitidine remain largely unidentified. Deregulation of signaling via Stat3 and Stat5 in acute myeloid leukemia (AML) is associated with aggressive disease. Numerous genes involved in cell signaling are aberrantly methylated in MDS, yet the alterations and the effect of azacitidine treatment on Stat3/5 signaling in high-risk MDS have not been explored.Experimental Design: We assessed longitudinally constitutive and ligand-induced phospho-Stat3/5 signaling responses by multiparametric flow cytometry in 74 patients with MDS and low blast count AML undergoing azacitidine therapy. Pretreatment Stat3/5 signaling profiles in CD34+ cells were grouped by unsupervised clustering. The differentiation stage and the molecular properties of the CD34+ G-CSF–inducible Stat3/5 double-positive subpopulation were performed by flow cytometry and quantitative real-time PCR in isolated MDS progenitors.Results: The pretreatment Stat3/5 signaling profiles in CD34+ cells correlated strongly with response and cytogenetics and independently predicted event-free survival. We further identified a CD34+ G-CSF–inducible Stat3/5 double-positive subpopulation (DP subset) whose pretreatment levels were inversely associated with treatment response and cytogenetics. The kinetics of the DP subset followed the response to azacitidine and the disease course, whereas its molecular characteristics and cellular hierarchy were consistent with a leukemia propagating cell phenotype.Conclusions: Our findings provide a novel link among Stat3/5 signaling and MDS pathobiology and suggest that the Stat3/5 signaling biosignature may serve as both a response biomarker and treatment target. Clin Cancer Res; 22(8); 1958–68. ©2015 AACR.

Mutations analysis of the TET2 and TP53 coding regions using Next-Generation Sequencing (NGS)

BackgroundMyelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML) are characterised by abnormal epigenetic repression and differentiation of bone marrow haematopoietic stem cells (HSCs). Drugs that reverse epigenetic repression, such as 5-azacytidine (5-AZA), induce haematological improvement in half of treated patients. Although the mechanisms underlying therapy success are not yet clear, induction of endogenous retroelements (EREs) has been hypothesised.MethodsUsing RNA sequencing (RNA-seq), we compared the transcription of EREs in bone marrow HSCs from a new cohort of MDS and chronic myelomonocytic leukaemia (CMML) patients before and after 5-AZA treatment with HSCs from healthy donors and AML patients. We further examined ERE transcription using the most comprehensive annotation of ERE-overlapping transcripts expressed in HSCs, generated here by de novo transcript assembly and supported by full-length RNA-seq.ResultsConsistent with prior reports, we found that treatment with 5-AZA increased the representation of ERE-derived RNA-seq reads in the transcriptome. However, such increases were comparable between treatment responses and failures. The extended view of HSC transcriptional diversity offered by de novo transcript assembly argued against 5-AZA-responsive EREs as determinants of the outcome of therapy. Instead, it uncovered pre-treatment expression and alternative splicing of developmentally regulated gene transcripts as predictors of the response of MDS and CMML patients to 5-AZA treatment.ConclusionsOur study identifies the developmentally regulated transcriptional signatures of protein-coding and non-coding genes, rather than EREs, as correlates of a favourable response of MDS and CMML patients to 5-AZA treatment and offers novel candidates for further evaluation.

Acute myeloid leukemia (AML) is a heterogeneous group of clonal myeloid disorders characterized by intrinsic molecular variability. Pretreatment cytogenetic and mutational profiles only partially inform prognosis in AML, whereas relapse is driven by residual leukemic clones and mere morphological evaluation is insensitive for relapse prediction. Measurable residual disease (MRD), an independent post-diagnostic prognosticator, has recently been introduced by the European Leukemia Net as a new outcome definition. However, MRD techniques are not yet standardized, thus precluding its use as a surrogate endpoint for survival in clinical trials and MRD-guided strategies in real-life clinical practice. AML resistance and relapse involve a complex interplay between clonal and immune cells, which facilitates the evasion of the leukemic clone and which is not taken into account when merely quantifying the residual leukemia. Multiparameter flow cytometry (MFC) offers the possibility of capturing an overall picture of the above interactions at the single cell level and can simultaneously assess the competence of anticancer immune response and the levels of residual clonal cells. In this review, we focus on the current status of MFC-based MRD in diverse AML treatment settings and introduce a novel perspective of combined immune and leukemia cell profiling for MRD assessment in AML.

Azacytidine (AZA) remains the mainstay of therapy for patients with high-risk Myelodysplastic syndrome (HR-MDS). The antineoplastic effects of AZA are thought to be mediated by two core mechanisms,...

Background/Aim: Several studies have documented the effects of isothiocyanates (ITCs) on cancer prevention by inducing oxidative stress, activating apoptosis, affecting cell cycle regulation, etc. Previously, we have shown that ITCs, administered at low concentrations by the means of double-bolus are capable of potentiating cytotoxicity in human malignant melanoma (A375) cells by inducing apoptosis. The aim of the present study was to further investigate the effect of the treatment of A375 cells with ITCs on cell cycle progression and the levels of various cell cycle regulators. Materials and Methods: Cell cycle analysis was performed by means of flow cytometry whereas western immunoblotting was used to determine the expression levels of these protein regulators. Results: Our data showed an increase in the number of cells in the G2/M phase accompanied by a decrease in the G0/G1 phase, while several cell-cycle regulators were shown to be differentially expressed upon exposure to ITCs. \ud Conclusion: ITCs induced cell cycle arrest in A375 cells.

5-azacytidine (5-AZA) is considered the standard of care for patients with high-risk myelodysplastic syndromes (MDS) and patients with acute myeloid leukemia (AML) not candidate for intensive chemotherapy. However, even after an initial favorable response, almost all patients relapse, with the exact mechanisms underlying primary or secondary 5-AZA resistance remaining largely unknown. Several reports have previously demonstrated the significance of hypoxia in the regulation of both physiological and malignant hematopoiesis. In MDS, high hypoxia inducible factor 1α (Hif-1α) expression has been correlated with poor overall survival and disease progression, while its involvement in the disease's pathogenesis was recently reported. We herein investigated the possible association of the Hif-1α signaling pathway with response to 5-AZA therapy in MDS/AML patients. Our data demonstrated that 5-AZA-responders present with higher Hif-1α mRNA and protein expression compared to 5-AZA-non-responders/stable disease patients, before the initiation of therapy, while, interestingly, no significant differences in Hif-1α mRNA expression at the 6-month follow-up were observed. Moreover, we found that 5-AZA-responders exhibited elevated mRNA levels of the Hif-1α downstream targets lactate dehydrogenase a (LDHa) and BCL2 interacting protein 3 like (BNIP3L), a further indication of an overactivated Hif-1a signaling pathway in these patients. Kaplan-Meier survival analysis revealed a significant correlation between high Hif-1α mRNA expression and better survival rates, while logistic regression analysis showed that Hif-1α mRNA expression is an independent predictor of response to 5-AZA therapy. From the clinical point of view, apart from proposing Hif-1α mRNA expression as a significant predictive factor for response to 5-AZA, our data offer new perspectives on MDS combinational therapies, suggesting a potential synergistic activity of 5-AZA and Hif-1α inducers, such as propyl hydroxylases inhibitors (PHDi).

CD4+ T cells orchestrate immune responses and are actively engaged in shaping tumor immunity. Signal transducer and activator of transcription (STAT) signaling controls the epigenetic tuning of CD4+ T-cell differentiation and polarization, and perturbed STAT signaling networks in CD4+ T cells subvert antitumor immunity in malignancies. Azacitidine (AZA), the mainstay therapy for high-risk myelodysplastic syndromes (HR-MDS), affects CD4+ T-cell polarization and function, but whether this contributes to AZA efficacy is currently unknown. By using functional proteomic, transcriptomic, and mutational analyses in 73 HR-MDS patients undergoing AZA therapy, we demonstrate that responding patients exhibited a coordinated CD4+ T-cell immune response and downregulated the inflammatory cytokine signaling pathways in CD4+ T cells after AZA, in contrast to nonresponders who upregulated the same pathways. We further observed an AZA-mediated downregulation of intereukin-6 (IL-6)—induced STAT3 phosphorylation in CD4+FOXP3− conventional T cells (Tcons) that correlated independently with better response and survival, whereas it was also not associated with the mutation number and profile of the patients. The AZA-induced downregulation of IL-6/STAT3 axis in Tcons restored the STAT signaling architecture in CD4+ T-cell subsets, whereas STAT signaling networks remained disorganized in patients who upregulated IL-6/STAT3 activity in Tcons. Given the pivotal role of CD4+ T cells in adaptive immunity, our findings suggest that the downregulation of the IL-6/STAT3 pathway in Tcons potentially constitutes a previously unrecognized immune-mediated mechanism of action of AZA and sets the scene for developing rational strategies of AZA combinations with IL-6/STAT3 axis inhibitors.

The role of dietary probiotic strains on host anti-cancer immune responses against experimental colon carcinoma was investigated. We have previously shown that Lactobacillus casei administration led to tumor growth suppression in an experimental colon cancer model. Here, we investigated the underlying immune mechanisms involved in this tumor-growth inhibitory effect. BALB/c mice received daily live lactobacilli per os prior to the establishment of a syngeneic subcutaneous CT26 tumor. Tumor volume, cytokine production, T cell differentiation and migration, as well as tumor cell apoptosis were examined to outline potential immunomodulatory effects following L. casei oral intake. Probiotic administration in mice resulted in a significant increase in interferon gamma (IFN-γ), Granzyme B and chemokine production in the tumor tissue as well as enhanced CD8+ T cell infiltration, accompanied by a suppression of tumor growth. Cytotoxic activity against cancer cells was enhanced in probiotic-fed compared to control mice, as evidenced by the elevation of apoptotic markers, such as cleaved caspase 3 and poly (ADP-ribose) polymerase 1 (PARP1), in tumor tissue. Oral administration of Lactobacillus casei induced potent Th1 immune responses and cytotoxic T cell infiltration in the tumor tissue of tumor-bearing mice, resulting in tumor growth inhibition. Thus, the microorganism may hold promise as a novel dietary immunoadjuvant in raising protective anti-cancer immune responses.

Murine tumor models have played a fundamental role in the development of novel therapeutic interventions and are currently widely used in translational research. Specifically, strategies that aim at reducing inter-animal variability of tumor size in transplantable mouse tumor models are of particular importance. In our approach, we used magnetic nanoparticles to label and manipulate colon cancer cells for the improvement of the standard syngeneic subcutaneous mouse tumor model. Following subcutaneous injection on the scruff of the neck, magnetically-tagged implanted cancer cells were manipulated by applying an external magnetic field towards localized tumor formation. Our data provide evidence that this approach can facilitate the formation of localized tumors of similar shape, reducing thereby the tumor size's variability. For validating the proof-of-principle, a low-dose of 5-FU was administered in small animal groups as a representative anticancer therapy. Under these experimental conditions, the 5-FU-induced tumor growth inhibition was statistically significant only after the implementation of the proposed method. The presented approach is a promising strategy for studying accurately therapeutic interventions in subcutaneous experimental solid tumor models allowing for the detection of statistically significant differences between smaller experimental groups.

Several studies have documented the effects of isothiocyanates (ITCs) on cancer prevention by inducing oxidative stress, activating apoptosis, affecting cell-cycle regulation, etc. Previously, we have shown that ITCs, administered at low concentrations by the means of double-bolus are capable of potentiating cytotoxicity in human malignant melanoma (A375) cells by inducing apoptosis. The aim of the present study was to further investigate the effect of the treatment of A375 cells with ITCs on cell-cycle progression and the levels of various cell cycle regulators.Cell-cycle analysis was performed by means of flow cytometry whereas western immunoblotting was used to determine the expression levels of these protein regulators.Our data showed an increase in the number of cells in the GITCs induced cell-cycle arrest in A375 cells.

The beneficial effects of two potential probiotic lactobacillus strains, namely Lactobacillus pentosus B281 and L. plantarum B282 were examined and the mechanisms of action were investigated. Quantitative analysis and confocal microscopy showed that both strains exhibited a significant higher adherence to Caco-2 cells in comparison to the reference strain L. casei ATCC 393. Treatment with conditioned media (CM) of the two strains caused significant reduction of cell proliferation, as demonstrated by Sulforhodamine B (SRB) and clonogenic assays. Moreover, the CM of the two strains induced a G1-phase arrest and down-regulation of specific cyclin genes, as indicated by flow cytometry and real-time PCR analysis. To begin elucidating the nature of the bacterial components conveying these responses, the anti-proliferative effect of heat-treated CM was analysed. The anti-proliferative activity of heat-treated CM was similar to the non-heated CM in a time- and dose-dependent manner, indicating the presence of thermostable bioactive compounds.

Purpose: Azacitidine is the mainstay of high-risk myelodysplastic syndromes (MDS) therapy, but molecular predictors of response and the mechanisms of resistance to azacitidine remain largely unidentified. Deregulation of signaling via Stat3 and Stat5 in acute myeloid leukemia (AML) is associated with aggressive disease. Numerous genes involved in cell signaling are aberrantly methylated in MDS, yet the alterations and the effect of azacitidine treatment on Stat3/5 signaling in high-risk MDS have not been explored. Experimental Design: We assessed longitudinally constitutive and ligand-induced phospho-Stat3/5 signaling responses by multiparametric flow cytometry in 74 patients with MDS and low blast count AML undergoing azacitidine therapy. Pretreatment Stat3/5 signaling profiles in CD34+ cells were grouped by unsupervised clustering. The differentiation stage and the molecular properties of the CD34+ G-CSF–inducible Stat3/5 double-positive subpopulation were performed by flow cytometry and quantitative real-time PCR in isolated MDS progenitors. Results: The pretreatment Stat3/5 signaling profiles in CD34+ cells correlated strongly with response and cytogenetics and independently predicted event-free survival. We further identified a CD34+ G-CSF–inducible Stat3/5 double-positive subpopulation (DP subset) whose pretreatment levels were inversely associated with treatment response and cytogenetics. The kinetics of the DP subset followed the response to azacitidine and the disease course, whereas its molecular characteristics and cellular hierarchy were consistent with a leukemia propagating cell phenotype. Conclusions: Our findings provide a novel link among Stat3/5 signaling and MDS pathobiology and suggest that the Stat3/5 signaling biosignature may serve as both a response biomarker and treatment target. Clin Cancer Res; 22(8); 1958–68. ©2015 AACR.

Foxp3+ T regulatory cell (Tregs) are central in the pathobiology of recurrent spontaneous abortions (RSA). Signal transducer and activator of transcription (STAT) proteins instruct Treg differentiation and polarization, but the STAT signaling architecture of Tregs in RSA and its modifications by lymphocyte immunotherapy (LIT) are yet unknown. By using single-cell phospho-specific flow cytometry we show that the STAT signaling biosignature of Tregs in women with RSA was characterized by marked downregulation of the IFNα/pSTAT1&5, IL-6/pSTAT1&3 and IL-2/pSTAT5 signaling nodes compared to age-matched fertile females. LIT partially restored all of these signaling axes in Tregs only in women who achieved pregnancy after treatment. Both the pretreatment biosignature of Tregs and its modulations by LIT were associated with therapeutic success. We conclude that STAT signaling pathways in Tregs are actively involved in the pathophysiology of RSA and may serve as a predictive tool for selecting patients who may benefit from LIT.

It has been suggested that myeloid neoplasms with isolated isochromosome 17q [ MN i(17q)] comprise a distinct entity with poor prognosis . However, literature reports show a considerable clinical and molecular heterogeneity. We describe a 58-year-old male patient who was diagnosed as refractory anemia with multilineage dysplasia and ringed sideroblasts with isolated i(17q) . Though he initially responded well to erythropoietin, he gradually progressed to an aggressive form of MDS/MPN refractory to azacytidine and died 29 months after first diagnosis. Notably, in contrast to disease advancement, his karyotype reverted to normal, whereas his mutational profile remained unchanged. To our knowledge this is the first report of karyotype normalization during disease progression in patients with MN i(17q) , suggesting that the i(17q) anomaly is dispensable for the leukemic transformation and highlighting the underlying clinical and molecular complexity which both have to be resolved before the establishment of MN with isolated i(17q) as a distinct entity.

Lactobacillus pentosus B281 and Lactobacillus plantarum B282 are two Lactobacillus strains previously isolated from fermented table olives. Both strains were found to possess probiotic properties and displayed desirable technological characteristics for application as starters in novel functional food production. In the present study the anti-proliferative and immunostimulatory activities of the two strains were investigated. Firstly, we demonstrated that live L. pentosus B281 and L. plantarum B282 significantly inhibited the growth of human colon cancer cells (Caco-2) in a time- and dose-dependent manner. By employing the air pouch system in mice, we showed that administration of both strains led to a rapid and statistically significant infiltration of leukocytes in the air pouch exudates. The phenotypical characterisation of the recruited immune cells was performed by flow cytometry analysis. We demonstrated that the majority of the infiltrated leukocytes were neutrophils. Finally by using the Mouse Cytokine Array Panel A Detection Antibody cocktail, we showed that both strains induced the expression of granulocyte-colony stimulating factor, interleukin (IL)-1α, IL-1β, IL-6, chemokine (C-X-C motif) ligand (CXCL)-1, chemokine (C-C motif) ligand (CCL)-3, CCL-4, and CXCL-2 and diminished the expression levels of soluble intercellular adhesion molecule, macrophage colony-stimulating factor and metallopeptidase inhibitor 1. Our results showed that both strains display anti-proliferative and immunostimulatory properties equal or even better in some cases than those of established and commonly used probiotic strains. These findings further support the probiotic character of the two strains.

Th17 cells play a crucial role in neutrophilic inflammation and tissue injury in non-cystic fibrosis (non-CF) bronchiectasis. Clarithromycin demonstrates anti-inflammatory and immunomodulatory properties but the effect of long-term clarithromycin prophylaxis on the Th17 response in non-CF bronchiectasis is still unexplored. Th17 response was studied in 22 patients with stable non-CF bronchiectasis receiving daily 500-mg clarithromycin for 12 weeks. We analysed IL-17 concentrations in exhaled breath condensate (EBC) and peripheral blood Th17 cells, whereas functional parameters and clinical data were recorded in parallel. Both, post-treatment absolute counts of CD4+IL17+ cells in peripheral blood and IL-17 levels in EBC decreased significantly (post-treatment CD4+IL17+ mean 2.418 ± 0.414 cells/μl versus pre-treatment 3.202 ± 0.507 cells/μl, p = 0.036 and post-treatment IL-17 mean levels 7.16 ± 0.47 pg/ml versus pre-treatment 9.32 ± 0.47 pg/ml, p

Azacytidine (AZA), the mainstay of therapy in high risk Myelodysplastic syndromes (HR-MDS), affects CD4+ T-cell polarization and function, but the effect of these changes on tumor immunity is unclear. Signal transducer and activator of transcription (STAT) proteins are key regulators of differentiation and polarization of CD4+ T-cells in both health and cancer, but the STAT signaling architecture of CD4+ T-cell subsets in HR-MDS and its modulation by AZA are currently unknown. We applied single-cell phosphospecific flow cytometry in peripheral blood mononuclear cells from 67 HR-MDS patients at various time-points during AZA therapy. Unsupervised clustering of pretreatment STAT signaling profiles (SPs) of CD4+ T-cells revealed three signaling clusters (SCs), mainly differing in the potentiated responses of STAT3 to IL-6 stimulation (IL-6/STAT3 node). Compared to SC#1 and SC#3, patients in SC#2 displayed higher IL-6/STAT3 levels, higher levels of naïve (TN, p=0.05) and lower levels of PD1+ (p=0.04) and central memory ( TCM, p=0.04) CD4+ T-cells, and longer median overall survival (mOS, p=0.028, fig 1A). Moreover, comparisons of single signaling nodes revealed that the IL-6/STAT3 node correlated inversely with PD1+ (p=0.02) and IL-4+ (p=0.04) and positively with naïve CD4+ (p=0.04) and IFNγ+CD8+ T-cells (p=0.01). No other differences in clinicobiologic parameters, CD4+ and CD8+ T-cell subpopulations (FOXP3, IFNγ, IL-4, IL-17, Perforin and Helios) were noted among the 3 SCs and all other single nodes. To assess the effect of AZA on STAT signaling, we clustered the fold fold-change of pre- versus 6-month post-AZA SPs in CD4+ T-cells. Again the IL6/STAT3 node was the only differentiator among the clusters, and, by single node analysis, downregulation of IL6/STAT3 at 6th cycle (n=26) was associated with better response to AZA (p=0.02) and longer mOS (p=0.03), compared to upregulation of the same node (n=22); the latter also accompanied by an increase of IFNγ+CD8+ cells after AZA, (p=0.02, fig 1B). Further supporting a direct and beneficial modulation of the IL-6/STAT3 axis in CD4+ T-cells by AZA, the kinetics of IL-6/STAT3 during AZA therapy revealed a marked downregulation of the former node both at day15 (p=0.04) and cycle 6 after AZA (p=0.04) in responders (n=5), while no changes were observed in non-responders (n=7). We further compared the transcriptional profiles of isolated bone marrow CD4+ T-cells between responders (n=4) and non-responders to AZA (n=4) by RNA-seq, both prior and after AZA. No significant differences in pretreatment gene expression were identified. By contrast, 105 genes were differentially expressed at cycle 6 compared to pretreatment in responders (FDR To trace the molecular background associated with the differential regulation of the IL-6/STAT3 pathway we constructed mutational profiles by targeted DNA sequencing of 156 genes in blood mononuclear cells. No associations were found between pre or post-treatment IL-6/STAT3 node and mutational burden. By contrast, mutations in RNA splicing and STAG2 correlated with lower (p=0.02) and higher (p=0.017) pretreatment levels of IL6/STAT3, respectively. Notably, all 5 patients with NPM1/DNMT3A double mutation upregulated significantly IL6/STAT3 after AZA (p=0.03, fig 1D). Collectively, our results reveal for the first time that downregulation of the IL-6/STAT3 signaling axis in CD4+ T cells may represent an immune-mediated mechanism of action of AZA. However, the antileukemic activity of IL-6/STAT3LowCD4+ T-cells appears to be independent from modulation of common metrics of tumor immunity, as, paradoxically, the detrimental IL-6/STAT3 upregulation was linked with an expansion of antitumor T cell subsets and decrease of the immunosuppressive ones. The IL-6/STAT3 axis is notoriously pro-tumorigenic and pharmacologic inhibition of various individual modules of this pathway in cancer is under development. Our findings may serve as a guidepost for the ongoing investigation of IL-6/STAT3 axis inhibition as a therapeutic strategy to overcome azacytidine resistance in HR-MDS. Figure 1 Disclosures Vassilakopoulos: Celgene / GenesisPharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; WinMedica: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Pappa:Gilead: Honoraria, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Research Funding; Novartis: Honoraria, Research Funding, Speakers Bureau; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene / GenesisPharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Research Funding. Papaemmanuil:Celgene: Research Funding. Kotsianidis:Celgene: Research Funding.

Probiotic microorganisms such as lactic acid bacteria (LAB) exert a number of strain-specific health-promoting activities attributed to their immunomodulatory, anti-inflammatory and anti-carcinogenic properties. Despite recent attention, our understanding of the biological processes involved in the beneficial effects of LAB strains is still limited. To this end, the present study investigated the growth-inhibitory effects of Lactobacillus casei ATCC 393 against experimental colon cancer. Administration of live Lactobacillus casei (as well as bacterial components thereof) on murine (CT26) and human (HT29) colon carcinoma cell lines raised a significant concentration- and time-dependent anti-proliferative effect, determined by cell viability assays. Specifically, a dramatic decrease in viability of colon cancer cells co-incubated with 10(9) CFU/mL L. casei for 24 hours was detected (78% for HT29 and 52% for CT26 cells). In addition, live L. casei induced apoptotic cell death in both cell lines as revealed by annexin V and propidium iodide staining. The significance of the in vitro anti-proliferative effects was further confirmed in an experimental tumor model. Oral daily administration of 10(9) CFU live L. casei for 13 days significantly inhibited in vivo growth of colon carcinoma cells, resulting in approximately 80% reduction in tumor volume of treated mice. Tumor growth inhibition was accompanied by L. casei-driven up-regulation of the TNF-related apoptosis-inducing ligand TRAIL and down-regulation of Survivin. Taken together, these findings provide evidence for beneficial tumor-inhibitory, anti-proliferative and pro-apoptotic effects driven by this probiotic LAB strain.

The role of thrombophilia and LMWH use in pregnancy loss (PL) and pregnancy complications (PC) is debated. In this retrospective study from a single center we analyzed the clinical outcome of pregnancies in relation to thrombophilic factors and the use of LMWH, aspirin and folic acid in 143 women followed up for a total of 173 pregnancies referred to our center from 2003 to 2016. Methods: Women were referred to our unit for: more than 2 unexplained PL (n=96, 78 experienced only early PL, 11 had only late PL, 7 had both early and late), one pregnancy loss(n=45) or one pregnancy complication (placenta abruption, intrauterine growth restriction, eclampsia, n=2). Mutations in Factor V-Leiden (FVL, G1691A), Prothrombin (PTG, G20210A) and MTHFR (C677T, A 1206C) were checked by DNA hybridization Kit. Plasma levels of antithrombin-III, protein-C, free Protein-S, APCR, FVIII, FXII, PT aPTT, fibrinogen, homocysteine and La-test were measured by photometry (DACO). Anticardiolopin and anti-β2GPI antibodies (IGG and IGM) were measured by ELISA in serum (APLA). End points were live birth and pregnancy complications. The prevalence of thrombophilia in our cohort was similar with previous studies and 34/143 (23,4%) women were negative for all thrombophilic factors. We observed mutations in FVL(11,6%), PTG (9,6%), MTHFR (homozygous or double heterozygous, 33,3%) and deficiencies of AT-III (3,3%), Prot-C (1,6%), Prot-S (8,8%), APS (8,7%). Combined severe trombophilic factors were found in 31 women (21,5%) (FVL+PTG 4/31, Natural Anticoagulants one out 3 Def + MTHFR 3/31, APS + MTHFR 2/31, FVL+MTHFR 16/31, PTG + MTHFR 6/31). We then separated our cohort into women with 2 complications. The second group had significantly higher incidence of FVL mutation (12,5 vs 8,3%, p=0.05) and deficiencies of AT-III and Free Prot-S ( 6,5 vs 0 %, p=0.01) compared to the first one. By contrast, women in the first group had higher incidence of La-test (12,5 vs 4,5%, p=0,03), APLA ( 12 vs 6,6%, p=0.03) and Prot.C deficiency (4,5 vs 0%, p=0.01). In univariate analysis both hereditary and acquired thrombophilic factors did not correlated with pregnancy outcome (live birth or pregnancy complications). Only age as a continuous parameter correlated negatively with live birth and positively with pregnancy complications (p=0.01 and p=0.025, respectively), whereas high BMI as a continuous parameter also negatively affected live births (p= 0.049). Logistic regression analysis reveals that the age of 35 is the cut off for unfortunate pregnancy outcome. Pregnancies were proceeded with (n=143, 81,7%) or without (n=32, 18,3%,) LMWH. The decision to use LMWH were based in a positive thrombophilia screening test (n=84) or to prior history >2 pregnancy complications with negative trombophilia testing (n=59). Concomitant use of ASA was prescribed in 78 pregnancies (dose less than 100 mg/day) and concomitant follic acid in 143 pregnancies. The percentage of live births were identical in women treated with LMWH (87,4%) or not (87,5%, p=0.9). In multivariate analysis, the only factor that was strongly correlated to live birth was the duration of LMWH treatment (odds ratio, OR =3,567, 95% CI (1.845, 6,894), p= 0,01) and the titration of the dose with anti-Xa (OR=5,138, 95% CI (1,717, 15,376), p = 0,01, fig.1a). By ROC analysis the duration of LMWH which correlated to live birth was ≥ 5.5 months(fig. 1b). The addition of ASA was insignificant for live birth (p=0.7), while the duration (>6months) of follic acid also appeared to add a benefit in combination with LMWH (p=0.01). Moreover, pregnancies proceeded without LMWH exhibited higher rates of pregnancy complications (18,75 vs 11,2%, p=0.08) and prematurity (14,3 vs 8,8%, p=0.05). In summary, our findings argue against hereditary thrombophilia screening in the cases of previous pregnancy loss or pregnancy complications. On the contrary, testing for APS even after the first event might be of value as this population often has laboratory evidence of APS and may benefit from proper anticoagulation. The use of LMWH and folic acid but not of ASA was related to less pregnancy complications or prematurity, whereas proper titration of LMWH by using anti-Xa and long duration of therapy were the only important factors for successful pregnancy outcome. Disclosures No relevant conflicts of interest to declare.

Hypomethylating agents (HMA) such as azacytidine and decitabine are the mainstay of treatment for higher risk myelodysplastic syndromes (MDS) and are also used to treat older, unfit patients with acute myeloid leukemia (AML). Being cytidine analogues, both azacytidine and decitabine are incorporated into DNA of highly proliferating cells leading to genome-wide decrease of methylation levels (Stresemann & Lyko., 2008; Gnyszka et al., 2013), whereas azacytidine is additionally incorporated into RNA molecules. Although several putative modes of action have been suggested for HMA, the precise mechanism underlying treatment success or failure remains incompletely understood. One possible mechanism of HMA action is through 'viral mimicry' of transcriptionally repressed endogenous retroelements (EREs), which is thought to trigger innate immune pathways. EREs comprise nearly half of the human genome and their transcriptional activity is repressed by diverse mechanisms including DNA methylation. According to the 'viral mimicry' hypothesis, HMA induce unphysiological levels of ERE transcription in transformed cells, which in turn generated nucleic acid species, such as double-stranded RNAs from complementary ERE transcripts, activating innate immune sensors. Although support and a mechanistic basis for this hypothesis is provided from a number of in vitro studies, in vivo evidence from the clinical use of HMA is currently lacking. To explore the possible involvement of EREs in the HMA mode of action, we have compared the transcriptional profiles of CD34+ HSCs isolated from bone marrow samples of healthy donors (n=9) and patients diagnosed with AML (n=9), chronic myelomonocytic leukemia - II (CMML-II, n=9) or high-risk MDS (n=11). For MDS and CMML, samples were obtained before, 15 days (D15) after the initiation of azacytidine and/or after cycle 6. Our analysis revealed that ERE transcription, measured as a proportion of the total polyA-selected transcriptome, is globally repressed in untreated MDS and CMML, in line with the proposed epigenetic repression that characterizes these conditions. Treatment with azacytidine had a measureable effect in overall ERE transcription in HSCs from MDS and CMML patients, which by the 6th cycle was raised to levels equivalent to those seen in HSCs healthy controls. Comparable results were also obtained following analysis of a publicly available dataset from CD34+ HSCs isolated from MDS and CMML patients prior to and after the 6th cycle of azacytidine treatment (GSE76203). However, despite noticeable upregulation of overall ERE transcription relative to gene transcription by azacytidine, the therapeutic response was not correlated with or predicted by ERE activity. Indeed, ERE transcriptional activation was frequently observed in azacytidine-treated patients who failed to respond to treatment, whereas it was frequently low or absent in patients who attained complete remission (figures 1a & b). It remained theoretically possible that a therapeutic response to azacytidine depended on the transcriptional activation of a select few ERE loci with innate immune stimulatory properties, which might have been masked by the analysis of global ERE activity. However, few individual ERE loci differed in their activity between patients who responded or not to azacytidine treatment. Moreover, our analysis failed to detect induction of either interferon-inducible genes or interferon-inducible EREs, irrespective of treatment outcome(figures 2a & b). Together, our current results do not support a role for transcriptional activation of EREs or for innate sensing of their nucleic acid products in the therapeutic response of MDS and CMML patients to azacytidine. Investigation of alternative potential mechanisms of azacytidine is therefore warranted. Disclosures No relevant conflicts of interest to declare.

Philadelphia chromosome-negative Myeloproliferative neoplasms (Ph−MPN) are accompanied by a markedly increased risk for development of chronic lymphocytic leukemia (CLL) compared to the general population. However, the pattern of onset and the biological characteristics of CLL in patients with coexistent Ph−MPN are highly heterogeneous rendering questionable if the above association reflects a causal relationship between the two disorders or merely represents a random event. By analyzing 82 patients with Ph−MPN and 100 age-matched healthy individuals we demonstrate that MPN patients have an almost threefold higher prevalence of, typically low-count, CLL-like monoclonal B lymphocytosis (MBL) compared to normal adults. The clone size remained unaltered during the disease course and unaffected by the administration of hydroxycarbamide, whereas no patient with Ph−MPN/MBL progressed to CLL during a median follow up of 4 years. Monoclonal B cells in Ph−MPN/MBL patients and normal individuals and in four more patients with coexistence of overt CLL and MPN displayed heterogeneous biological characteristics, while the JAK2V617F mutation was absent in isolated lymphocytes from Ph−MPN patients with coexistence of CLL. Despite its clinical and biological variability, the increased incidence of MBL in Ph−MPN patients along with the one reported for CLL further enforces the notion of a shared pathophysiology among the two malignancies via a common genetic link and/or microenviromental interactions.

Expression of CD25 antigen on CD34+ cells is an independent predictor of outcome in late-stage MDS patients treated with azacitidine

Objective: Both normal and malignant stem cells maintain lower levels of reactive oxygen species (ROS), but the redox state in acute myeloid leukemia (AML) has not been thoroughly characterized and the role of ROS in leukemogenesis is still unclear. Herein, we report the identification of a rare but distinct ROSlow subpopulation in primary CD34+ AML samples. Methods: We analysed the ROS state of a number of AML samples by flow cytometry using the redox-sensitive fluorescence dye 2’7;-dichlorodihydrofluorescein diacetate. We FACS-sorted the ROSlow cells and investigated their immunophenotype, in vivo engraftment potential as well as their ability to withstand chemotherapeutic treatment. Results: Compared to the total CD34+ cells the ROSlow subset contained significantly more CMP-like and less GMP-like progenitors and could establish leukemia in NOD/SCID mice. Additionally, ROSlow cells bore a chemoresistant phenotype as they were more quiescent than total CD34+ AML cells, and showed increased in vitro chemoresistance and markedly higher GM-CSF-induced phosphorylation of STAT5. Conclusions: Thus, the ROSlow subpopulation arises as a novel candidate for cell-specific therapeutic targeting in AML. Further studies will help to ascertain the exact role of the ROSlow subset in the pathobiology and clinical management of AML.

CD4+ T cells hold a central role in tumor immunity by orchestrating and regulating antitumor responses. Azacytidine can apparently modulate tumor immunity in myelodysplastic syndrome (MDS) patients and affect CD4+ cell polarization, mainly in responding patients. Signal transducer and activator of transcription (STAT) proteins have essential roles in the epigenetic control of T helper (TH) cell differentiation and aberrant STAT signaling is involved in the pathobiology of numerous malignancies by compromising tumor immunity. MDS have a strong immunopathogenetic component, but the CD4+ cell STAT signaling network and the effect of hypomethylating therapy on the former have not yet been investigated. We applied phospho-specific flow cytometry to explore the alterations of STAT signaling in CD4+ cells during azacytidine treatment and addressed their association with clinical and biological parameters. Peripheral blood mononuclear cells of 26 late-stage MDS and low blast count AML (LBC-AML) patients and 7 age-matched healthy individuals were obtained before and 15 days after azacytidine initiation. According to WHO classification, two patients had RCMD (8%), 10 (38%) had RAEB-II, 8 (31%) CMML-II and 6 (23%) LBC-AML. Based on the IWG criteria patients were divided into responders (CR, PR and hematologic improvement, n=12) and non-responders (stable disease and failure, n=14). We applied a modified protocol of phospho-specific flow cytometry to measure either basal (untreated) or potentiated (after stimulation with IL-6, IFNá and IL-2 for 15') phospho-STAT1, 3 and 5 levels with simultaneous staining for CD3 and CD4. The following potentiated, i.e. target/stimuli, nodes were studied: pSTAT1/IL-6, pSTAT1/IFNá, pSTAT3/IL-6, pSTAT5/IFNá and pSTAT5/IL-2. In the same patients TH cell subsets were measured by intracellular staining with IFNã, IL-4, FOXP3 and IL-17A along with CD3 and CD4. Comparisons were performed by Mann Whitney, Kruskal Wallis or Wilcoxon Signed-Rank test as appropriate. Clustering of signaling profiles (SPs) was performed with hierarchical cluster analysis and correlations by using ÷2 or Fisher Exact tests. Unsupervised clustering of the CD4+ SPs in MDS patients and controls revealed 4 signaling clusters (SC1-4, figure 1). No differences were noticed among the SCs in relation to treatment response, cytogenetics, IPSS, WPSS and IPSS-R and transfusion requirements. By contrast, the disease subtypes were nonrandomly distributed among the clusters (p=0.036). Most RAEB-II patients segregated in cluster 2, which displayed high basal levels of pSTAT5 and intense response of pSTAT1 to IL-6 and IFNá and of pSTAT5 to IFNá, whereas all healthy subjects were enclosed in SC4 which was characterized by low basal levels of all pSTATs and powerful response of pSTAT1 and 5 to IFNá and of pSTAT3 to IL-6. Also, SCs showed similar levels of TH cells, with the exception of SC1 which was associated with markedly higher levels of FOXP3+/IL-17+ cells both on day 0 (0.29% of total CD4+ cells, range 0.04%-0.54%, p=0.04) and day15 (0.31%, range 0.12%-0.35%, p=0.04) after azacitidine administration compared to other SCs. Interestingly, the levels of FOXP3+/IL-17+ cells downregulated significantly on day 15 in responders (0.07%, range 0.04%-0.4% vs 0.04%, range 0.02%-0.16%, p=0.03), whereas non responders showed a non-significant decrease (0.13%, range 0.01%-0.54% vs 0.08%, range 0.04%-0.31, p=0.2). We further performed cluster analysis of patients SPs 15 days after azacytidine initiation. Patients were separated in two SCs, while normal controls still formed a different cluster (SC3, Figure 2), indicating that, at least on day 15, azacytidine was not able to restore the abnormal STAT signaling in CD4+ cells. In conclusion, we demonstrate that the STAT signaling biosignature of CD4+ cells in late stage MDS patients is abnormal and also differs among the various disease subtypes. Moreover, consistent with recent data (Bontkes HJ, 2012, Constantini B et al, 2012), azacytidine treatment appears to affect mainly the FOXP3/TH17 axis, particularly in responders. Our results further suggest a clinically relevant immunomodulatory activity of azacytidine. Figure 1. Heatmap of pretreatment signaling clusters (SC) in CD4+ cells of patients and normal subjects. Figure 1. Heatmap of pretreatment signaling clusters (SC) in CD4+ cells of patients and normal subjects. Figure 2. Heatmap of SCs in CD4+ cells of patients and normal subjects on day 15 after azacytidine initiation. Figure 2. Heatmap of SCs in CD4+ cells of patients and normal subjects on day 15 after azacytidine initiation. Disclosures Kotsianidis: Genesis Pharma Hellas: Honoraria.

Expression of CD25 on blasts of AML patients has been shown to hold independent prognostic value for both survival and response to induction therapy. Patients with MDS-related AML have generally higher CD25 expression from de novo AML patients, but though there is paucity of a serviceable biomarker of outcome in MDS patients treated with azacytidine, the prognostic value of CD25 has not yet been investigated. Bone marrow samples of 61 patients with intermediate-2/high risk IPSS, high/very high WPSS and low blast count AML were obtained before and 15 days (D15) after the initiation of treatment. All patients received azacytidine in a non clinical trial setting at an initial dose of 75mg/m2 SC for 7 days on 28-day cycles. CD25 expression was assessed by 4-color flow cytometry on total CD34+ blasts, committed progenitors (Lin-CD38+CD34+) and leukemic stem cells (LSC, Lin-CD38-CD34+). Positivity was defined as a CD25 expression of ≥ 20%. Statistical comparisons were done by ÷2, one-way ANOVA and paired or unpaired t-test as appropriate, and survival with Kaplan-Meier analysis and log-rank test. Overall survival (OS) was defined as the time from azacytidine initiation to death from any cause and event-free survival (EFS) as the time from diagnosis to disease progression, relapse or death. Multivariate survival analysis was based on Cox’s proportional hazards model using a backward stepwise selection procedure with entry and removal criteria of p=0.05 and p=0.10, respectively. As shown in table 1 the cohorts of CD25- and CD25+ patients were well balanced for most known predictive factors and characteristics, except sex. Compared to CD25+ patients the CD25- ones have significantly longer OS (16.2 vs 8.8 months, respectively, p=0.04) and EFS (12.8 vs 6.66 months, p=0.04) in univariate analysis. Multivariate analysis confirmed the independent predictive power of CD25 for OS and EFS (p=0.006 and p=0.009, respectively), whereas heavy transfusion requirements (p=0.003 and p=0.002) and age>75 (p=0.02 and p=0.02) were also independent predictors. The average expression of CD25 in CD34+ blasts of all patients was 21.6%±24%. Compared to committed progenitors, LSCs displayed higher expression (19.4%±23.7% vs 24.1%±28.2%, respectively, p=0.027). Interestingly, on D15 CD25 was downregulated in LSCs (p=0.03) but remained stable in committed progenitors (p=0.8, n=18), indicating a particular sensitivity of the CD25+ subset of LSCs in azacytidine.Table 1Patient characteristics. N/A: not applicable/not available.CD25- (n=36)CD25+ (n=25)p-valueAge72,5 (53,4-83.5)72,9 (52-81.7)0.2 >6531(86%)17(68%) 15%18(50%)9(36%) ≤15%18(50%)16(54%)Transfusions ≥ 4 per month0.48 Yes23(4%)17(4%) No13(4%)8(4%)Response0.4 CR & PR12(33%)4(16%) Hematologic improvement5(14%)4(16%) Stable disease8(22%)5(20%) Failure11(31%)12(48%)Figure 1(A) OS and EFS according to CD25 positivity status. (B) OS and EFS according to transfusion requirements.Figure 1. (A) OS and EFS according to CD25 positivity status. (B) OS and EFS according to transfusion requirements. Collectively, our findings reveal an independent prognostic role for CD25 in MDS patients treated with azacytidine. In addition, the differential expression and epigenetic modulation of CD25 in the LSC compartment support the investigation of therapeutic strategies using monoclonal antibody targeting combined with epigenetic agents. Disclosures: Kotsianidis: Genesis Hellas: Honoraria, Research Funding. Spanoudakis:Genesis Hellas: Honoraria. Tsatalas:Genesis Hellas: Honoraria.

T regulatory cells (Tregs) safeguard against autoimmunity, but may compromise antitumor immunity. Aberrant immune signaling via signal transducer and activator of transcription (STAT) proteins is involved in disease pathobiology in numerous malignancies. Azacytidine treatment affects Treg levels, particularly in responders, but its effect on STAT signaling is unknown. We explored the alterations of STAT signaling profile (SP) of Tregs during azacytidine treatment and their association with clinical and biological parameters. Peripheral blood mononuclear cells of 19 late-stage MDS patients were obtained before and 15 days after azacytidine initiation. According to WHO, one had RAEB-I (5%), 9(47%) patients had RAEB-II, 7(37%) CMML-II and 2(10%) AML/MDS. Based on the IWG criteria patients were divided into responders (CR, PR and hematologic improvement, n=8) and non-responders (stable disease and failure, n=11). We applied a modified protocol of phospho-specific flow cytometry to measure either basal (untreated) or potentiated (after stimulation with IL-6 and IL-2 for 15’) phospho-STAT1, STAT3 and STAT5 levels with simultaneous staining for FOXP3. The following potentiated, i.e. target/stimuli, nodes were studied: pSTAT1/IL-6, pSTAT3/IL-6 and pSTAT5/IL-2. Comparisons were performed by paired, unpaired t-test or one-way ANOVA as appropriate. Clustering of SPs was performed with hierarchical cluster analysis and was correlated with response, disease subtype, and cytogenetics by using ÷2 or Fisher Exact tests. The pretreatment levels of Tregs were significantly higher in non-responders compared to responders (8.4%±0.42% vs 5.5%±0.46%, p=0.004). By contrast, azacytidine treatment significantly upregulated Tregs at d15 only in responders (7.1±0.47%, p=0.03), whereas non-responders exhibited a mild, but non-significant increase (9.7%±1.22%, p=0.18). Unsupervised clustering of the Treg SPs revealed 4 clusters. No differences were noticed in the SP of Tregs in relation to treatment response, cytogenetics, IPSS, WPSS and IPSS-R. By contrast, the disease subtypes were nonrandomly distributed among the clusters. Most CMML-2 patients segregated in cluster 4, which displayed high basal levels of phospho-STAT1 and 5, but weak potentiated responses, whereas the cluster 2 was enriched in RAEB-II patients and was characterized by very high basal levels of pSTAT5 and relatively intense response of pSTAT3 in IL-6 (Fig 1). Intriguingly, the pretreatment levels of Helios+ Tregs were significantly higher in patients of cluster 2 (37.9%±7.7% of total Tregs) compared to patients in clusters 1, 3 and 4 (23.1±2.3%, 22.8%±2.4% and 22.2%±2.1%, respectively, p=0.035) indicating a higher level of Treg activity and, potentially, immune suppression in these patients. In addition, the clustering of the ratios of Treg SPs after/before azacytidine treatment revealed a distinct epigenetic modulation of Treg signaling via STATs in CMML-2 compared to all other patients (p=0.036), characterized by downregulation of pSTAT1 and pSTAT5 basal levels and upregulation of potentiated responses at day 15 after first azacytidine administration (Fig 2).Figure 1Pretreatment SPs of Tregs in 19 patients.Figure 1. Pretreatment SPs of Tregs in 19 patients.Figure 2Ratio of the SPs of Tregs 15 days after/before azacytidine initiation in 19 patients.Figure 2. Ratio of the SPs of Tregs 15 days after/before azacytidine initiation in 19 patients. Collectively, consistent with recent data (Constantini B et al, 2012), we observed both arithmetical and functional alterations of Tregs in MDS patients after azacytidine treatment. Moreover, both the pretreatment SP and the differential modulation of STAT signaling in the Tregs of CMML-2 and RAEB-II patients, argue for a potentially diverse epigenetic regulation of tumor immunity among the various MDS subtypes. Disclosures: Tsatalas: Genesis Hellas: Honoraria. Spanoudakis:Genesis Hellas: Honoraria. Kotsianidis:Genesis Hellas: Honoraria, Research Funding.