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Introduction Lower respiratory tract infections (LRTI) due to parainfluenza (PIF) or respiratory syncytial virus (RSV) represent a major challenge for immunocompromised patients especially those after allogeneic stem cell transplantation (allo-SCT). Evidence-based guidelines for the management of respiratory virus infections in allo-SCT recipients are limited due to the paucity of effective antivirals and the lack of prospective randomized trials. In this study, we report our experience with the use of oral ribavirin with or without the addition of IVIgG in allo-SCT recipients with LTRI due to RSV and PIF. Patients and methods This is a retrospective study performed in 3 BMT centers in Athens, Greece (2 adult and 1 pediatric center). Review of medical records was performed with the aim to identify patients with RTIs who received treatment with oral ribavirin. LRTI was defined according to the European Conference on Infections in Leukaemia (ECIL-4) (Ref 1). Presence of RSV or PIF in specimens from nasopharyngeal wash and/or BAL was documented with the use of a PCR assay. Ribavirin was administered at a dose of 20-30mg/kg divided into 4 daily doses, with appropriate dose adjustment according to creatinine clearance. Treatment with ribavirin was continued until significant symptomatic and radiological improvement. Co-administration of IVIgG was at the discretion of the treating physician and was mostly based on the availability of IVIgG and on the severity of underlying illness. IVIgG was administered at a daily dose of 400mg/kg for a total dose of 2 g/kg. Informed consent was given by patients or their parents. Results Forty-nine episodes of LRTI due to RSV or PIF were reported during a nine-year period in 7 children and 37 adult patients after allo-SCT. Three children and 2 adult patients had a second LRTI due to RSV, 6, 8, 8, 10 and 12 months after the first episode. Patient's characteristics are shown in Table 1. All patients presented with fever and cough. In almost all of the patients auscultation revealed a prolonged expiratory phase, diffuse wheezing, while the presence of crackles was more prominent finding in patients presenting with pneumonia. Arterial blood gas analysis showed moderate to severe hypoxemia. In 32 out of 49 episodes oxygen supplementation was required while 7 patients required mechanical ventilation. All 49 episodes treated with oral ribavirin while in 37 cases ribavirin was administered in combination with IVIgG. High resolution CT-scan was performed in 41 episodes and revealed bilateral abnormalities in all patients examined. Small centrilobular nodules, bronchial wall thickening (32 out of 41 episodes) and ground-glass opacities (21 out of 41 episodes) were common findings, while a pattern of diffuse air-space consolidation was observed in 11 cases. In 43 LRTI episodes a rapid response to treatment in a median of 3 days (range, 2 - 5) was observed. Complete resolution of all symptoms and signs of disease occurred after a median of 9 days (7 - 20) of treatment. One patient with pneumonia due to PIF3 who needed mechanical ventilation had a complete resolution of all symptoms of disease. Six deaths attributable to LRTI were observed among a total of 49 episodes (all 6 patients were in need for mechanical ventilation). Disease relapse confirmed microbiologically occurred in only 1 patient with LRTI due to RSV 10 days after the end of treatment with ribavirin. One children developed bronchiolitis obliterans syndrome (BOS) 9 months after the resolution of RSV infection. Cryptogenic organizing pneumonia (COP) was observed in two patients 1 and 2 months after the complete resolution of LRTI due to PIF. Both patients had an excellent response to treatment with steroids. Conclusions Oral Ribavirin has an excellent efficacy and safety profile. Complete and fast resolution of infection occurred in 88% of cases. From the present study it is not clear if the addition of IVIgG offers any therapeutic benefit. References Fourth European Conference on Infections in Leukaemia (ECIL-4): Guidelines for Diagnosis and Treatment of Human Respiratory Syncytial Virus, Parainfluenza Virus, Metapneumovirus, Rhinovirus, and Coronavirus. Clin Infect Dis 2013;56(2):258-66 Disclosures Tsonis: Gilead: Other: Travel Grant; Astellas: Other: Travel Grants; Innovis: Other: Travel Grant; Pfizer: Other: Travel Grant; Takeda: Other: Travel Grant; Aenorasis: Other: Travel Grant. OffLabel Disclosure: Tabl Ribavirin. Current indication: Treatment of active hepatitis C in combination with interferon-a.

Mir-17-5p and mir-20a, members of the mir-17-92 family, down-regulate E2F1, which is over-expressed in myelodysplastic syndromes (MDS). Moreover, let-7a down-regulates KRAS, which is aberrantly expressed in MDS. We evaluated the expression of the aforementioned microRNAs in CD34+ cells of 43 MDS patients using real-time PCR and their target proteins (E2F1, MYC, BCL2, CCND1, and KRAS) by Western blot. Mir-17-5p and mir-20a were under expressed in high risk MDS patients, compared to low risk MDS patients. Similarly, let-7a was under expressed in patients with intermediate or high-risk karyotype. Interestingly, there was an inverse correlation between microRNA and the expression levels of their targets. Importantly, mir-17-5p and mir-20a constitute favorable prognostic factors in MDS, since their expression was associated with increased overall survival of MDS patients.

Central nervous system (CNS) involvement in patients with primary mediastinal large B-cell (PMLBCL) lymphoma is a rare event, occurring in approximately 6% of patients, on the basis of the review of the literature prior to induction of Rituximab. The aim of this retrospective study was to describe the incidence of CNS relapse among 100 consecutive patients with PMLBCL who were treated with R-CHOP ± RT in comparison to patients treated with CHOP ± RT (n = 45) in 11 hospitals in Greece. Two patients experienced a CNS relapse, representing an overall frequency of 2.0% in R-CHOP treated patients and a 2-year actuarial incidence of 2.3%. Both patients had isolated CNS relapses. The incidence of CNS relapse after CHOP without Rituximab was 2/45 (4.4%) for a 2-year actuarial incidence of 7.5% (p = 0.29). Again, both patients had isolated CNS relapses. Parenchymal-only localizations accounted for 3/4 cases. Risk factors for CNS involvement could include leukocytosis, poor performance status and higher age-adjusted International Prognostic Index, although their impact was weakened by competing risk survival analysis. Both patients relapsing after R-CHOP required CNS radiotherapy to achieve a complete remission and be forwarded to high-dose therapy and autologous stem cell transplantation: They are both alive and disease-free 18 and 23 months after CNS relapse. Both cases relapsing after CHOP without Rituximab were salvaged by CNS radiotherapy (one also received intrathecal chemotherapy) entering long-term remissions. In conclusion, CNS relapses are rare in PMLBCL tending to be isolated in the CNS, probably reflecting the persistence of latent CNS disease than dissemination of resistant disease. The impact of Rituximab in reducing CNS relapses remains unknown. Established risk factors for CNS involvement in aggressive lymphomas may not be helpful in assessing the risk of CNS recurrence in this disease. Routine CNS prophylaxis is not probably required in PMLBCL. Copyright © 2012 John Wiley & Sons, Ltd.

The disturbance of apoptosis molecular signaling pathways is involved in carcinogenesis. BCL2 family of proteins is the hallmark of apoptosis regulation. In the last decade, new members ofBCL2gene family were discovered and cloned and were found to be differentially expressed in many types of cancer. BCL2 protein family, through its role in regulation of apoptotic pathways, is possibly related to cancer pathophysiology and resistance to conventional chemotherapy. It is well known that leukemias are haematopoietic malignancies characterized by biological diversity, varied cytogenetics, different immunophenotype profiles, and diverse outcome. Current research focuses on the prognostic impact and specific role of these proteins in the pathogenesis of leukemias. The understanding of the molecular pathways that participate in the biology of leukemias may lead to the design of new therapies which may improve patients' survival. In the present paper, we describe current knowledge on the role of BCL2 apoptosis regulator proteins in acute and chronic leukemias.

Deregulation of cell cycle and apoptosis pathways are known contributors to the pathogenesis of myelodysplastic syndromes (MDS). However, the underlying mechanisms are not fully clarified. The aim of our study was to examine mRNA expression levels of cell cycle and apoptosis regulatory genes, as well as the percentage of apoptotic and S phase cells and to correlate the findings with clinical characteristics and prognosis. Sixty patients with MDS, classified according to FAB (17 RA, five RARS, 19 RAEB, nine RAEBT, ten CMML) and WHO (ten RA, three RARS, seven RCMD, two RCMD-RS, 11 RAEBI, eight RAEBII, ten CMML, and nine AML) were included in the study. We found increased expression of anti-apoptotic bclxL and mcl1 genes and decreased expression of p21 gene in MDS patients. Moreover, we found increased expression of anti-apoptotic mcl1 gene in patients with higher than Intermediate-1 IPSS group. Multivariate analysis confirmed that combined expression of apoptotic caspases 8, 3, 6, 5, 2, 7, and Granzyme B was decreased in MDS patients. Regarding cell cycle regulatory genes expression, we demonstrated increased expression of cyclin D1 in patients with CMML Increased combined expression of cyclins B, C, D1, and D2 was found in patients with cytogenetic abnormalities. The two pathways seem to be interconnected as shown by the positive correlation between CDKs 1, 2, 4, p21 and the level of apoptosis and positive correlation between apoptotic caspase 3 expression and the percentage of S phase cells. In conclusion, our study showed altered expression of genes involved in apoptosis and cell cycle in MDS and increased expression of cyclin D1 in patients with CMML.

Central nervous system (CNS) involvement in acute myeloid leukemia (AML) is a rare complication of the disease and is associated with poor prognosis. Sometimes the clinical presentation can be unspecific and the diagnosis can be very challenging. Here we report a case of CNS infiltration in a patient suffering from AML who presented with normal complete blood count and altered mental status.

Rosuvastatin, a statin indicated for patients with primary hypercholesterolemia, mixed dyslipidemia and familial hypercholesterolemia, is well tolerated by most patients. Its most common adverse effects are gastrointestinal derangement, muscle aches and hepatitis. One rare complication of statin treatment is severe thrombocytopenia. The case of a 65-year-old patient who developed severe thrombocytopenia while on rosuvastatin is presented, in addition to a review of the literature.

Parvovirus B19 is recognized as a rare cause of pure red cell aplasia (PRCA) in allogeneic stem cell (SCT) and solid organ transplant patients. We report a patient with Hodgkin's disease who developed PRCA due to parvovirus B19 after autologous SCT and who had an excellent response after treatment with gamma-globulin.

The p15 gene is a putative tumor suppressor gene that encodes a member of the cyclin dependent kinase inhibitors. Inactivation of p15 by promoter hypermethylation has been postulated as a possible way by which tumor suppressor genes are inactivated in cancer. In this study, we examined the methylation status of the p15 gene promoter in 34 patients with B-Chronic Lymphocytic Leukemia (B-CLL), by the Methylation-Specific Polymerase Chain Reaction. Selective methylation of the p15 gene promoter was found in 4/34 cases (11.8%). According to Rai staging, the four patients with methylated p15 were staged on diagnosis as: 1 on Stage 0, 1 in Stage I, 1 in Stage III, and 1 in Stage IV. Our results suggest that methylation of the p15 gene promoter can be detected in a small subset of B-CLL patients, at all stages of the disease.

The aim of the present study was to examine caspases, granzyme B and bcl-2 family mRNA expression and the degree of apoptosis in the bone marrow (BM) of 46 Myelodysplastic Syndromes (MDS) and to correlate our findings with clinical parameters. The degree of apoptosis was determined by Annexin V, whereas expression of genes was determined using a multiprobe RNase Protection System. A positive correlation was found between caspases 8, 5, 3, 2, 1 and the level of apoptosis. bfl1 and mcl1 levels were significantly higher in patients with BM blasts >5%. Cases with ratio of bid expression >1 compared to normal pool were associated with IPSS values ≤1.

Introduction Sepsis is the result of an uncontrolled inflammatory response to various stimuli such as bacterial infections. Sepsis can be complicated by hemodynamic instability, multi-organ dysfunction and hematological abnormalities. Patients with severe sepsis usually develop thrombocytopenia due to various reasons, and the presence of thrombocytopenia is considered as a negative prognostic marker. The aim of the present study was to determine the cytokine profile in serum of patients with severe sepsis and thrombocytopenia. Patients and Methods Serum cytokine profile was analyzed in a cohort of 112 consecutive patients with severe sepsis and/or septic shock treated in the intensive care unit (ICU) of our institute from October/2009 to September/2012. Patients were divided in two groups (A and B). Group A consisted of 43 patients with severe sepsis and thrombocytopenia (platelet count below 70X103/ìl), while group B consisted of 69 patients without thrombocytopenia. Patients with thrombocytopenia due to disseminated intravascular coagulation (DIC), suspected drug etiology, or due to any obvious etiology such as hematologic malignancy were excluded from our analysis. A cohort of 10 healthy volunteers served as control group. Serum levels of IFNã, IL-8, ICAM, VCAM, and SUPAR (soluble urokinase plasminogen activation receptor) were estimated by using Luminex xMAP technology. Statistical analyses were performed using NCSS software. The following variables were entered in a multiple logistic regression model: 1) age and sex, 2) active malignancy vs. not, 3) septic shock vs. severe sepsis, 4) APACHEII score, 5) SOFA score, 6) serum IFNã, ICAM, VCAM, IL-8, and SUPAR levels, and 7) platelet number. All parameters were estimated on day of admission. Results Hospital Mortality: Overall 65 out of 112 patients died during their hospital stay. The overall hospital mortality was 58%. In multivariate analysis non-survivors had higher APACHE score (p=0.01) and had lower platelet counts (p Plasma cytokine levels in patients with and without thrombocytopenia: Patients in group A had a different cytokine profile as compared with patients in group B. Serum levels of VCAM and IFNã were not different between group A and B. However, patients with sepsis and thrombocytopenia had statistically significantly higher serum levels of ICAM, IL-8 and SUPAR (p Predictive value of serum cytokine for thrombocytopenia: Serum levels of ICAM, IL-8, and SUPAR were good predictors of the presence of thrombocytopenia in patients with sepsis. The ROC curves of ICAM, IL-8, and SUPAR serum levels in predicting thrombocytopenia in patients with sepsis are shown in Figure 1. The AUCs were 0.785 (95% CI, 0.696 – 0.857, p Conclusion Thrombocytopenia is not an uncommon finding in patients with severe sepsis. Although DIC or drug reactions are well known causes of low PLT counts, the pathogenesis of thrombocytopenia in patients with sepsis remains poorly understood. High levels of ICAM, IL-8 and SUPAR are usually associated with severe endothelial dysfunction. In our study, we showed that patients with thrombocytopenia have a specific serum cytokine profile expression consistent with the presence of endothelial damage. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction: Cutaneous T-cell lymphomas (CTCL) represent a heterogeneous group of extranodal non-Hodgkin lymphomas, derived from skin-homing mature T-cells. Mycosis fungoides (MF) and Sézary syndrome (SS) are the commonest types and together comprise 54% of all CTCL. MF evolves from patches to infiltrated plaques and eventually tumors. SS is a lymphoma-leukemia syndrome characterized by erythroderma and the presence of a malignant T-cell clone in the peripheral blood and the skin. At present, no curative treatment for CTCL is available. Therefore current CTCL research efforts are focused on elucidating the molecular mechanisms of the disease’s pathogenesis and on identifying new pharmacological targets. Several drugs have shown potentially significant activity either alone or in combination with conventional agents. Their effectiveness and their mechanisms of action comprise a current research challenge for the improvement of CTCL therapy. The aim of this study was to investigate the possible alterations in the gene expression profile (focusing on DNA Damage Signaling and DNA Repair pathways) and cell death in CTCL cell lines after treatment with two chemotherapeutic agents, Bortezomib and Methotrexate. Methods: Three CTCL cell lines were used. MyLa, (MF), SeAx and Hut-78 (both SS). Cells were cultured in RPMI 1640 and were treated with either Bortezomib (10nmol/L) or Methotrexate (10μM) for 24h. Apoptosis was determined by flow cytometry using the Annexin V/PI method. Gene expression profiling following PCR arrays analysis was performed after total RNA extraction and purification from untreated and drug-treated cells. All RNA samples’ amplification, labeling and hybridization to RT2 Profiler PCR Arrays (DNA Damage Signaling and DNA Repair PCR array) (QIAGEN) were performed according to the manufacturer’s instructions. All data were analyzed using the appropriate RT2 Profiler PCR Array data analysis tool. Results: Hut-78, Seax and Myla cells responded with statistically significant enhanced apoptosis when treated for 24h with bortezomib, compared to untreated cells, while Methotrexate led to a rather moderate increase of apoptosis in Hut-78 and Seax cells and did not affect the apoptosis of Myla cells. Microarrays analysis after bortezomib treatment revealed a great effect in the expression profile of genes involved in almost all DNA repair pathways tested, in all three cell lines, with Hut-78 being affected the most. Specifically, in all cell lines, there was a significant down-regulation of a large number of genes involved in the Double Strand Breaks DNA Repair mechanism, (i.e. BRCA1, BRCA2, RAD50, RAD51, RAD51C, XRCC2, XRCC3, XRCC4, XRCC5 and XRCC6) as well as of genes involved in the Mismatch Repair pathway (i.e. MLH1, MLH3, MSH2, MSH5, MSH6) and the Nucleotide Excision Repair mechanism (i.e. DDB2, LIG1 and RAD23A), compared to untreated cells. On the contrary, bortezomib had a small effect on Base Excision repair mechanism, mostly downregulating the expression of XRCC1 gene in Hut-78 and Myla cells. Methotrexate treatment also led to a significant down-regulation of genes involved in the DSB (RAD50, XRCC4, XRCC6), MMR (MSH4) and NER (CDK7, RAD23A) repair mechanisms in Hut-78 cells but had a rather much more moderate effect on the expression profile of Seax and Myla cells, where fewer genes were affected. Conclusions: Our data clearly demonstrate a differential effect of bortezomib and methotrexate in terms of apoptosis induction on CTCL cells with bortezomib inducing apoptosis of both MF and SS derived cell lines and methotrexate being rather inactive on SS derived cells. We showed that both drugs, but mostly bortezomib significantly down-regulate a large number of genes involved in the DSB, MMR and NER mechanisms, suggesting a possible mechanism, among probably others, for the enhanced sensitivity to apoptosis of SS and MF cell lines after treatment. Bortezomib’s significant effect could be easily understood, since it is a well known proteasome inhibitor and has been previously related to inhibition of NF-kB and accumulation of pro-apoptotic proteins, while it has also been reported that cancer cells are more sensitive to proteasome inhibition than normal cells. Although these results need to be further confirmed, they appear very encouraging for understanding the mechanisms of action of these drugs in CTCL with the view to ameliorate their use in clinical practice. Disclosures No relevant conflicts of interest to declare.

Abstract 4965 Introduction. MicroRNAs are small non-coding RNAs that act at the post-transcriptional level, regulating protein expression by repressing translation or destabilizing mRNA target. Since their discovery, microRNAs have been associated with almost every normal cell function, including proliferation, differentiation and apoptosis. Several studies suggest that they have an important role in normal hematopoiesis and hematological malignancies. Let-7a negatively regulates the expression of Ras proteins, which are overexpressed in patients with Myelodysplastic syndromes (MDS). In addition mir-17-5p contributes to the down-regulation of E2F1, which is overexpressed in MDS. Purpose. The purpose of the current study was to evaluate the expression of let-7a and mir-17-5p in CD34 cells from the bone marrow of patients with MDS. Material and methods. We evaluated 29 patients with MDS (25 men, 4 women) with median age 73 years. FAB classification was as follows: 9 RA, 15 RAEB, 2 RAEB-t and 3 AML. According to IPSS our study included 9 patients with low, 8 with Int-1, 7 with Int-2 and 5 with high risk disease. We isolated CD34+ cells from bone marrow of patients using magnetic beads. Extraction of microRNA and total RNA was performed and cDNA of let-7a and mir-17-5p was amplified using real time PCR, to study the relative expression of these microRNAS according to the expression of RNU48 (a snoRNA used as control). The control group included donors of CD34+ cells for stem cell transplantation. Results. The median miR17-5p expression level in patients was lower compared to controls [23vs 47.4]. The median let-7a expression levels were higher in MDS patients than in healthy donors [32.85 vs 8]. However these differences were not significant as shown by the Mann-Whitney U test. Moreover, ROC analysis demonstrated that neither miR17-5p nor let-7a expression had significant discriminatory value to efficiently distinguish MDS patients from non-MDS. In addition Spearman analysis did not reveal any correlations between these two microRNA expression levels and other continuous variables examined in the current study (age, hemoglobin concentration, numbers of neutrophils, platelets, and BM blasts). Finally, we found no associations between either miR17-5p or let-7a expression and clinicopathological parameters of MDS patients using chi-square (χ2) or Fisher's exact test where appropriate. Conclusion. The micro RNA let-7 was overexressed although not significantly in CD34 cells from MDS patients demonstrating that this is not a mechanism contributing to the increased expression RAS proteins in MDS. The micro RNA-mir17-5 is underexpressed in CD34 cells and needs further investigation for the establishment of a role in the overepxression of E2F in MDS. The lack of significant differences in the expression levels of both micro RNAs could be related to the admixture of normal and dysplastic CD34 cells in the bone marrow of MDS or to the small sample size. Disclosures: No relevant conflicts of interest to declare.

Introduction: MDS constitute a heterogeneous group of clonal haematopoietic disorders characterized by ineffective haematopoiesis and an increased risk of transformation to acute myeloid leukaemia. Increased apoptosis has been associated with ineffective progenitor and maturing haematopoietic cell survival and associated cytopenias early in the disease whereas the progression of MDS to AML occurs concomitant with decreased apoptosis and an increased degree of neoplastic cell survival. The aim of the present study was to identify molecular alterations of cell cycle and apoptosis regulatory genes in the bone marrow cells of adult patients with de novo MDS and to correlate these with clinical characteristics. Patients and methods: A total of 60 cases of MDS, 40 males and 20 females, classified according to FAB classification as follows: 17 RA, 5 RARS, 19 RAEB, 9 RAEBT and 10 CMML, were included in our study. Twelve non-Hodgkin’s lymphoma patients without bone marrow involvement were used as normal controls. BM aspirates were obtained at diagnosis from the patients as well as the control group. We used RNAse Protection Assay to detect alterations of expression at the mRNA level of cell cycle regulatory genes, particularly CDK1, CDK2, CDK3, CDK4, p27, p21, PISSLRE, p16, cyclins A, B, C, D1, D2, D3, A1 as well as apoptosis regulatory genes, particularly caspases 1,2,3,5,6,7,8,9, Granzyme B, bclxL, bclxS, bfl1, bik, bak, bax, bcl2, mcl1. Moreover our study included estimation of apoptosis using the Annexin V affinity assay, as well as analysis of cell cycle by determining the percentage of cells in S phase using flow cytometry. Results: The median value of apoptosis for all MDS cases was 2,79% (range 0–34,9). A positive correlation was found between caspases 3, 5, 9, CDKs 1, 2 and 4, as well as p21 expression and the level of apoptosis. The median value of cells in S phase of the cell cycle was 10,6% (range 0,19–27,4). A positive correlation was found between caspase 3 expression levels and S phase. Patients of the IPSS ≥1 score group were associated with higher values of mcl1 gene expression. Cases with bone marrow blasts ≥5% showed higher bclw, mcl1 and bfl1 gene expression values. Regarding FAB classification, CMML correlated with higher cyclin D1 gene expression. bclxL and mcl1 gene median values were found higher in patients with MDS, compared to normal controls. On the contrary CDK3 and p21 gene median values were lower in patients compared to healthy individuals. Multivariate analysis revealed that combined expression of caspases 8, 3, 6, 5, 2, 7 and Granzyme B was lower in MDS patients compared to normals, as well as that combined expression of cyclins B, C, D1 and D2 was higher in patients with abnormal karyotype compatible with a higher proliferation rate and a higher probability of revealing karyotypic abnormalities. Conclusions: Our study demonstrated that the expression of the anti-apoptotic mcl1 was higher in MDS patients with IPSS>1 and the anti-apoptotic genes bclw, mcl1 and bfl1 had higher expression in cases with 35% blasts in the bone marrow. In the myeloproliferative CMML category a significantly higher cyclin D1 expression was found. Taking into consideration the multifactorial pathogenetic features of MDS, we consider the developing understanding of apoptosis and cell cycle function essential, with the view to proceed to molecularly targeted treatment and improved clinical outcome.

Introduction. Within the group of core binding factor (CBF) AML, the presence of the t(8;21)(q22;q22) confers a favorable prognosis based on high complete remission rates and high survival probabilities. However within this subgroup the presence of KIT mutations and in some studies specifically mutations at codon 816 in exon 17 have been associated with inferior event free survival, relapse free survival, cumulative incidence of relapse and overall survival. Dasatinib a dual SRC/ABL kinase inhibitor is an active agent already approved for the treatment of imatinib resistant or intolerant chronic myelogenous leukemia which has shown in vitro activity against KIT exon 17 mutations including the D816 imatinib resistant mutation. The aim of the present study was the investigation of the activity of dasatinib on cell proliferation and apoptosis of leukemic cell lines with or without KIT mutations. Materials and methods. The leukemic cell lines ME-1, NB4 and KASUMI were cultured in RPMI. Following RNA extraction RT-PCR was performed for the amplification of the extracellular (exon 8,9), transmembrane/juxtamembrane (exon 10,11) and tyrosine kinase 2 domains (exon 17,18) of c-Kit.Following sequencing only the KASUMI cell line derived from a t(8;21)(q22;q22) AML was found to bear the N822K KIT mutation at exon 17, also described in patients’samples. The KASUMI, the K562 cell line bearing the t(9;22) used as a positive control and the NB4 cell line without KIT mutations used as a negative control, were subsequently cultured under the presence of dasatinib at the concentrations of 1nM, 10nM, 100nM, 500 nM. Cell proliferation, was determined at 24, 48, 72 h using the Cell Proliferation Elisa, BrDU protocol and apoptosis was determined by the method of annexin using flow cytometry at the same time points. Results The BrDU value of K562 cells at 48h without the drug was 1.046 significantly higher compared to those of cells cultured under the presence of Dasatinib at 1nM, 10nM, 100nM, 500 nM (0.6485, 0,5647, 0,4770, 0.4755 respectively) (p Conclusion. Dasatinib is an effective suppressor of proliferation and inducer of apoptosis of the KASUMI cell line with the t(8;21)(q22;q22) and the N822K KIT mutation. These encouraging results need to be confirmed on patients’ cells with the view to integrate the drug in conventional chemotherapy regimens in future clinical trials.

8101 Background: To determine whether the PI3K/Akt signaling pathway is involved in the pathogenesis of mantle cell lymphoma (MCL), we investigated the phosphorylation status of Akt in primary MCL cases and cell lines. We also sought to determine whether loss of the phosphatase PTEN is the mechanism of Akt activation in MCL. Methods: We evaluated the protein levels of Akt, phosphorylated Akt and PTEN in 15 primary MCL cases and 2 cell lines by immunoblotting Results: Akt was phosphorylated in 3 of 15 MCL cases and in 2 of 2 mantle cell lymphoma cell lines. PTEN protein was expressed in 15 of 15 primary mantle cell lymphoma cases and in 2 of 2 MCL cell lines. Conclusions: We conclude that constitutive activation of the PI3K/Akt pathway contributes to the pathogenesis of MCL. Loss of PTEN protein expression is not the responsible mechanism for Akt activation. Alternative mechanisms of Akt activation are being evaluated to identify markers predictive for response to PI3K/Akt inhibitors in MCL. No significant financial relationships to disclose.

Background: MDS cover a range of clonal stem cell disorders characterized by ineffective hematopoiesis associated by excessive intramedullary apoptosis of hematopoietic cells. The bcl2 family of proteins constitutes an essential component of the apoptotic machinery at the level of mitochondria and is involved in the acitvation of caspases a family of cytosolic proteases, the effector molecules of apoptosis Aim: The aim of the present study was to examine caspases, granzyme B and bcl2 family mRNA expression and the degree of apoptosis in the bone marrow (BM) of adult de novo MDS and to correlate our findings with clinical parameters and prognosis. Methods. We studied 46 cases of MDS including 14 RA, 21 RAEB, 6 CMML and 5 RARS according to FAB criteria. The degree of apoptosis was determined by flow cytometry using the Annexin method and propidium iodine labelling (PI) on BM mononuclear cells. The expression of caspases 1, 2, 3,5,6,7,8 and 9 and Granzyme-B as well as of bclw, bclxL, bclxS, BFL1, BID, Bik, Bak, Bax, bcl2, Mcl1was determined using a multiprobe RNase Protection Assay System (Riboquant, BD Biosciences). A pool of RNA from normal bone marrow mononuclear cells was used as a normal control. The expression of each gene was compared to that of two housekeeping genes (GAPDH and L32) using the Image Master analysis Software. The level of each gene expression was compared to that obtained from the normal pool RNA. The expression of the genes and the degree of apoptosis were analyzed, taking into consideration haematological parameters, the FAB classification and the IPSS value. Results: The median value of apoptosis for all MDS cases was 4,07 (0.04–16.9) Apoptosis in the low risk group was higher but not significantly different from the high risk group (8.9 in the RA-RARS vs 4.3 in the RAEB, and CMML). A positive correlation was found between caspases 8, 5, 3, 2, 1 expression and the level ofapoptosis. Moreover a positive correlation was found between all caspases level of expression with the exception of caspase 7 that did not correlate with caspases 8 and 5. The same phenomenon was observed for the bcl2 family members with the exception of BFL1 that did not correlate with bclw, bclxS and bcl2. The level of expression as well as the percentage of positive cases for all caspases and granzyme B was not significantly different between different FAB subgroups and different IPSS risk categories. BFL1 and Mcl1 levels were significantly higher in patients with BM blasts >5%. Cases with ratio of BID gene expression >1 compared to normal pool were associated with IPSS values Conclusion. The expression level of caspases 1,2,3,5,6,8,9 and granzyme B did not correlate with the FAB, calssification and the IPSS risk groups in patients with MDS. The antiapoptotic genes BFL1 and Mcl1 were significanlty higher in cases with BM blasts >5% while cases overexpressing the proapoptotic gene BID were mostly represented by the low risk IPSS subgroups. Larger number of cases need to be examined to draw definite conclusion about the role of these apoptosis regulatory genes in the pathogenesis and prognosis of MDS.

Introduction: Mature T cell lymphomas are a rare and heterogeneous group of malignancies characterized by a poor prognosis. They usually present with advanced-stage disease and generally have a significantly worse outcome compared with aggressive B-cell lymphomas. The aim of the present study was the retrospective analysis of clinical and laboratory characteristics of patients with mature T cell lymphomas with the view to identify factors of prognostic significance. Patients and Methods: 39 patients with mature T cell lymphoma, classified according to WHO classification, diagnosed in our institution within a 17 year period were analyzed. Cases with anaplastic and cutaneous lymphomas were excluded. There were 25 males and 14 females with a median age of 61 (26–80). 3 patients had stage I,. 7 stage II, 10 stage III and 19 stage IV disease. B symptoms were present in 28/39 cases and bulky disease in 6/39 cases. All cases were risk stratified according to the International Prognostic Index (IPI). 9 patients had IPI 0/1, 9 had IPI 2, 10 had IPI 3 and 8 IPI 4/5. In 3 cases the IPI could not be defined. 21 patients were treated with CHOP like therapy, 11 with COP and 7 with other treatment. The size and the immunophenotype of the neoplastic cell population in the diagnostic biopsy (Τ4, Τ8 or mixed), the patients’ clinical characteristics and their laboratory findings were analyzed to identify factors of prognostic significance. Results. 17/39 patients (47.6%) achieved CR, 7/39 PR, while 6 had stable and 7 progressive disease. 2 patients died during induction treatment. 21/39 patients developed recurrent disease and the median disease free survival (DFS) was 78 months. Factors associated with significantly worse DFS were age above 60 (p=0.0005) and the presence of hepatomegaly at diagnosis (3 vs 78 months, p=0.0219), while the presence of extranodal disease was of borderline significance (p=0.0629). The patients’ median survival was 24 months. Factors associated with significantly shorter survival were female gender (14 vs 61 months, p=0.0396), the presence of supradiaphragmatic disease (p=0.0337) and eosinophilia at diagnosis (p=0.0467). Τhe IPI scoring system could distinguish between groups with a significant difference both in DFS and overall survival. DFS for IPI 4/5 was significantly shorter vs IPI 0-3 (p=0.0009). Patients with IPI 0/1 had a median survival 271 months, with IPI 2/3 24 months and with IPI 4/5 8 months (p=0.002). Histological characteristics like cell size or immunophenotype of the neoplastic cell were not important for prognosis. Conclusions. Mature T cell lymphomas constitute an heterogenous group of aggressive lymphomas that can be prognostically evaluated using clinical parameters. The IPI scoring system can identify subgroups with significant differences both in terms of DFS and overall survival. The analysis of larger number of cases or of histological characteristics may be helpful in the identification of other clinical or laboratory parameters that will increase the discriminative capacity of the IPI scoring system in this group of aggressive lymphomas.

Abstract 3792 Introduction: MicroRNAs are single, small non-coding RNA molecules of approximately 21–26 nucleotides, which regulate the expression of numerous genes. miRNAs may act either at the post-transcriptional or the post-translational level to repress gene expression; still, upregulation of gene expression has been noticed in some cases as a direct effect of miRNA function. The importance of miRNAs in carcinogenesis is emphasized by the association of cancers with alterations in miRNA expression. Many miRNAs, including let-7a and those of the miR-17-92 cluster (miR-17, miR-20a, etc.), have been shown or are predicted to affect the activities of targeted mRNAs encoding proteins that have oncogenic or anti-oncogenic functions. let-7a downregulates KRAS, while miR-17 and miR-20a downregulate E2F1. Both these proteins are overexpressed in myelodysplastic syndromes (MDS) and have been shown to be involved in the pathobiology of the disease. Purpose: In the current study, we examined the prognostic value of let-7a, miR-17 and miR-20a levels in MDS and their potential as novel molecular biomarkers. Furthermore, we investigated the protein expression levels of validated targets of these three miRNAs in bone marrow CD34+ cells of MDS patients. Material and Methods: We evaluated 43 patients with MDS (34 men, 9 women) with a median age of 73 years (range 45–87). According to WHO classification, 12 patients (27.9%) were diagnosed with RA, 6 (13.9%) RCMD, 8 (18.6%) with RAEB-I, 7 (16.3%) with RAEB-II, 8 (18.6%) with AML, and 2 (4.7%) with CMML. According to IPSS, 13 patients (32.5%) had low risk, 14 (35.0%) intermediate I risk, 6 (15.0%) intermediate II, and 7 (17.5%) high risk disease. WPSS classification was: 8 (23.5%) very low risk, 5 (14.7%) low risk, 8 (23.5%) intermediate, 9 (26.5%) high risk, and 4 (11.8%) very high risk. We isolated CD34+ cells from bone marrow mononuclear cells from MDS patients, as well as from peripheral blood of donors of CD34+ cells for stem cell transplantation, using magnetic beads. Extraction of small RNA-containing total RNA from CD34+ cells was performed and cDNA of let-7a, miR-17 and miR-20a was synthesized using specific primers. miRNA expression levels were determined using quantitative real-time PCR, the TaqMan® chemistry and the relative quantification (2−ΔΔCT) method. The snoRNA RNU48 was used as reference gene. Furthermore, total protein was extracted from CD34+ cells using a lysis buffer and subsequently quantified using the Bradford assay. Western blot analysis was carried out for MYC, E2F1, Cyclin D1 (CCND1), BCL2 and KRAS, while Actin was used as reference protein. Results: In MDS patients, let-7a expression levels were 0.053–506.1 copies/RNU48 copies, while miR-17 and miR-20a expression levels were 0.005–2694.5 and 0.003–3116.7 copies/103RNU48 copies, respectively. No significant differences were found between patients and controls regarding let-7a, miR-17 and miR-20a expression. let-7a underexpression was associated with high (>10%) bone marrow blasts percentage (P =0.036), presence of WHO classification subtypes with poor prognosis (RAEB-I, RAEB-II and AML) (P =0.020), and high IPSS (P =0.037). Furthermore, miR-17 underexpression was related to high (>10%) bone marrow blasts percentage (P =0.008), intermediate and/or high risk karyotype (P =0.018) and high IPSS (P =0.016). Moreover, miR-20a underexpression was associated with high IPSS (P =0.037) and WPSS (P =0.013). Interestingly, protein expression levels of all targets analyzed in the current study were shown to be lower in samples overexpressing let-7a, miR-17 and/or miR-20a, in comparison with the corresponding protein levels noticed in specimens showing lower expression of these three miRNAs. Conclusion: To the best of our knowledge, this is the first study showing that expression levels of let-7a, miR-17 and miR-20a are associated with established prognostic factors in MDS, including IPSS and WPSS. Furthermore, these three miRNAs seem to be implicated in the pathogenesis of the disease, most probably by finely tuning the expression of target proteins that are involved in highly important molecular pathways, therefore affecting key cellular functions, such as cell cycle control, apoptosis, cell proliferation, and regulation of gene expression. Undoubtedly, further studies are needed to confirm the present findings and clarify their association with the pathogenesis of different MDS subgroups. Disclosures: No relevant conflicts of interest to declare.