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The gene encoding for transcription factor ETV6 presents recurrent lesions in hematologic neoplasms, most notably the ETV6-RUNX1 rearrangement in childhood B-ALL. The role of ETV6 for normal hematopoiesis is unknown, but loss of its function probably participates in oncogenic procedures. In myeloid neoplasms, ETV6-locus (12p13) deletions are rare but recurrent; ETV6 translocations are even rarer, but those reported seem to have phenotype-defining consequences. We herein describe the genetic and hematologic profile of myeloid neoplasms with ETV6 deletions (10 cases), or translocations (4 cases) diagnosed in the last ten years in our institution. We find complex karyotype to be the most prevalent one among patients with 12p13 deletion (8/10 patients), with most frequent coexisting anomalies being monosomy 7 or deletion 7q32 (5/10), monosomy 5 or del5q14-15 (5/10) and deletion/inversion of chromosome 20 (5/10), and most frequent point-mutation being TP53 mutation (6/10 patients). Mechanisms of synergy of these lesions are unknown. We describe the entire genetic profile and hematologic phenotype of cases with extremely rare ETV6 translocations, confirming the biphenotypic T/myeloid nature of acute leukemia associated to ETV6-NCOA2 rearrangement, the association of t (1;12) (p36;p13) and of the CHIC2-ETV6 fusion with MDS/AML, and the association of the ETV6-ACSL6 rearrangement with myeloproliferative neoplasm with eosinophilia. Mutation of the intact ETV6 allele was present in two cases and seems to be subclonal to the chromosomal lesions. Decoding the mechanisms of disease related to ETV6 haploinsufficiency or rearrangements is important for the understanding of pathogenesis of myeloid neoplasms and fundamental research must be guided by observational cues.

Instructive editions from the late 19th and early 20th century include various annotations regarding musical and interpretative aspects, such as articulation, bowings, fingerings, dynamics, note values, or vibrato As a popular medium at the time, instructive editions were often in the centre of contemporary discussions and attracted the attention of musicians and music teachers, bequeathing us a wide corpus of valuable sources. Joseph Joachim was arguably the most prominent violinist and a sought-after pedagogue in the German-speaking world at the time. Hitherto unknown letters as well as revisited statements by Joachim lead to new insights regarding his attitude towards instructive editions: he viewed them - despite his (few) publications in this genre - very critically, as he was convinced that detailed instructions would limit the freedom of the performer. He instead preferred editions without annotations, but interpreted the music freely andd variably in what he considered the spirit of the composer. Joachim's attitude thus poses general questions as to the role and freedom of performance and interpretation in the second half of the nineteenth century.

Background: To evaluate the effect of various, everyday intensive care unit (ICU) practices on glucose levels in critically ill pediatric patients with the use of a continuous glucose monitoring system. Methods: Seventeen sensors were placed in 16 pediatric patients (8 male). All therapeutic and diagnostic interventions were recorded and 15 minutes later, a flash glucose measurement was obtained by swiping the sensor with a reader. Glucose difference was calculated as the glucose value 15 minutes after the intervention minus the mean daily glucose value for each individual patient. Additionally, the consciousness status of the patient (awake or sedated) was recorded. Results: Two hundred and five painful skin interventions were recorded. The mean difference of glucose values was higher by 1.84 ± 14.76 mg/dL (95% CI: −0.19 to 3.87 mg/dL, P = .076). However, when patients were categorized regarding their consciousness level, mean glucose difference was significantly higher in awake state than in sedated patients (4.76 ± 28.07 vs −2.21 ± 15.77 mg/dL, P Conclusions: Diagnostic and therapeutic procedures in the ICU, especially when performed in an awake state, exacerbate the stress and lead to a significant rise in glucose levels.

The adaptive response of bacterial cells to changing environmental conditions depends on the behavior of single cells within the population. Exposure of Listeria monocytogenes to sublethal acidic conditions in foods or in the gastrointestinal track of the host may induce injuries relevant to difficult physiological states within the dormancy continuum. In this study, exposure to acidic conditions (acetic-AA and hydrochloric acid-HCl adjusted to pH 3.0, 2.7, 2.5 at 20 °C for 5 h) was used to evaluate injury of L. monocytogenes, Scott A strain. To differentiate the resistant sub-population from the total, Tryptic Soy Agar with 0.6 % Yeast Extract (TSAYE) supplemented or not with 5 % NaCl were comparatively used. Sublethally injured cells were detected by comparing plate counts with fluorescence microscopy, using combinations of CFDA (viability) and Propidium-Iodide (death). Effect of acid stress on the relative transcription of clpP, mazE, mazF, relA, gadC, gadD, gadB, sigB, inlA and prfA upon transition of total population into different physiological stages was evaluated through RT-qPCR. AA treated cells showed measurable logarithmic reduction at pH 2.7 and 2.5, while there was a significant percentage of CFDA

Hereditary erythrocytes disorders include a large group of conditions with heterogeneous molecular bases and phenotypes. We analyzed here a case series of 155 consecutive patients with clinical suspicion of hereditary erythrocyte defects referred to the Medical Genetics Unit from 2018 to 2020. All of the cases followed a diagnostic workflow based on a targeted next-generation sequencing panel of 86 genes causative of hereditary red blood cell defects. We obtained an overall diagnostic yield of 84% of the tested patients. Monogenic inheritance was seen for 69% (107/155), and multi-locus inheritance for 15% (23/155). PIEZO1 and SPTA1 were the most mutated loci. Accordingly, 16/23 patients with multi-locus inheritance showed dual molecular diagnosis of dehydrated hereditary stomatocytosis/xerocytosis and hereditary spherocytosis. These dual inheritance cases were fully characterized and were clinically indistinguishable from patients with hereditary spherocytosis. Additionally, their ektacytometry curves highlighted alterations of dual inheritance patients compared to both dehydrated hereditary stomatocytosis and hereditary spherocytosis. Our findings expand the genotypic spectrum of red blood cell disorders and indicate that multi-locus inheritance should be considered for analysis and counseling of these patients. Of note, the genetic testing was crucial for diagnosis of patients with a complex mode of inheritance.

Smoking is associated with impaired fasting glucose levels and increased insulin resistance.Post-cessation weight gain (PSWG) highly concerns diabetic patients since it leads to poor control of diabetic parameters, discourages them to quit and can be a cause of relapse. Identification of this population9s characteristics could help improve smoking cessation rates. We recruited smokers with DM (type 1 and 2), who agreed to take varenicline and attend an intense smoking cessation program once a week given by multidisciplinary team. 41 (14 women, 27 men) diabetic smokers accepted to follow the program. The participants were very addicted (FTND 9/11, 30cigs/day), motivated (8,1/10) but lacking confidence (5,1/10). Characteristics shown on Fig 1a. After 3 months 25 participants quit smoking (61%), 7 reduced more than half the cigarettes smoked daily, while 9 participants quit the program. Interestingly, patients who quit the program showed higher levels of psychopathology compared to those who continued. Changes of body weight and rest metabolism rate (RMR) were not significant after smoking cessation (RMR measurements for DM 2 only, N=21) (Fig 1b). HDL was significantly increased. Findings were partly attributed to an increase of physical activity level reported after strong recommendations (Fig 1c). Diabetics succeeded in quitting (61%), ameliorated lipidemic profile, without a statistically significant PSWG by improving physical activity.

Signal transducer and activator of transcription (STAT) proteins have been intensively studied in hematologic malignancies, and the efficacy of agents against STATs in lymphomas is already under research. We investigated the expression of total STAT5 and STAT5b in peripheral blood samples of patients with chronic lymphocytic leukemia (CLL) in correlation with the presence of Epstein–Barr Virus (EBV) and its major oncoprotein (latent membrane protein 1, LMP1). The EBV load was measured in the peripheral blood by real‐time PCR for the BXLF1 gene and the levels of LMP1 by PCR and ELISA. Western blotting was performed for total STAT5 and STAT5b in protein extracts. STAT5b was only expressed in patients (not in healthy subjects) and STAT5 but particularly STAT5b expression was correlated with the presence of the virus (77.3% vs. 51.2%, P = 0.006 for STAT5b) and to the expression of LMP1 (58.3% vs. 21.6%, P = 0.011 for STAT5b). Moreover, the expression of STAT5b and the presence of EBV and LMP1 were strongly negatively correlated with the overall survival of the patients (log‐rank test P = 0.011, 0.015, 0.006, respectively). Double positive (for EBV and STAT5b) patients had the lowest overall survival (log‐rank test P = 0.013). This is the first report of a survival disadvantage of EBV+ patients with CLL, and the first time that STAT5b expression is correlated with survival. The correlation of STAT5 expression with the presence of the virus, along with our survival correlations defines a subgroup of patients with CLL that may benefit from anti‐STAT agents.

This study evaluates the efficiency of iron-based oxy-hydroxides to remove antimony from groundwater to meet the requirements of drinking water regulations. Results obtained by batch adsorption experiments indicated that the qualified iron oxy-hydroxide (FeOOH), synthesized at pH 4 for maintaining a high positive charge density (2.5 mmol OH−/g) achieved a residual concentration of Sb(III) below the EU drinking water regulation limit of 5 μg/L by providing an adsorption capacity of 3.1 mg/g. This is more than twice greater compared either to similar commercial FeOOHs (GFH, Bayoxide) or to tetravalent manganese feroxyhyte (Fe-MnOOH) adsorbents. In contrast, all tested adsorbents failed to achieve a residual concentration below 5 μg/L for Sb(V). The higher efficiency of the qualified FeOOH was confirmed by rapid small-scale column tests, since an adsorption capacity of 3 mg Sb(III)/g was determined at a breakthrough concentration of 5 μg/L. However, it completely failed to achieve Sb(V) concentrations below 5 μg/L even at the beginning of the column experiments. The results of leaching tests classified the spent qualified FeOOH to inert wastes. Considering the rapid kinetics of this process (i.e., 85% of total removal was performed within 10 min), the developed qualified adsorbent may be promoted as a prospective material for point-of-use Sb(III) removal from water in vulnerable communities, since the adsorbent’s cost was estimated to be close to 30 ± 3.4 €/103 m3 for every 10 μg Sb(III)/L removed.

Background Epstein-Barr virus (EBV) is a ubiquitous pathogen that chronically infects B lymphocytes and is implicated in the pathogenesis of lymphoproliferative diseases. Latent membrane protein 1 (LMP1), the major oncoprotein of the virus, has been shown to inhibit apoptosis and trigger survivin expression in malignant cell lines. LMP1 expression has been detected in patients with chronic lymphocytic leukemia, but its properties have not been studied in patients with low-grade B-cell lymphomas. Recent data show that LMP1 can simultaneously induce and inhibit apoptosis in B cells. We detected LMP1 messenger RNA (mRNA) in patients with leukemic low-grade B-cell lymphoma and correlated the expression of the antiapoptotic molecule survivin to that of LMP1 in this group of patients. Patients and Methods Peripheral whole blood from 64 patients with low-grade B-cell lymphoma was tested by quantitative reverse transcriptase–polymerase chain reaction (PCR) for the presence of the BXLF-1 gene of EBV, and positive samples were tested by conventional PCR for LMP1 expression. Accordingly, survivin mRNA levels were measured by quantitative reverse transcriptase PCR in all samples and compared between LMP1-positive (LMP1 + ) and LMP1 − patients. Results The BXLF-1 gene was detected in 27 of 64 patients (42%). LMP1 was expressed in 22 of 27 (81%) EBV + patients. Survivin expression was found to be 6.36 times higher in LMP1 − patients than in LMP1 + patients ( P = .008). Conclusion Our results imply that in patients with non–EBV-related leukemic low-grade B-cell lymphoma, LMP1 expression is possibly correlated to apoptosis, as indicated by the lower survivin mRNA levels in LMP1 + patients.

The general purpose of this study is to thoroughly examine decentralization in education according to the literature and previous research, and to discuss the applicability of educational decentralization practices in Turkey. The literature was reviewed for the study and findings reported. It has been observed that decentralization in education practices were realized in many countries after the 1980’s. It is obvious that the educational system in Turkey has difficulty in meeting the needs, and encounters many problems due to its present centralist state. Educational decentralization can provide effective solutions for stakeholder engagement, educational financing and for problems in decision making and operation within the education system. However, the present state of local governments, the legal framework, geographical, cultural and social features indicate that Turkey’s conditions are not ready for decentralization in education. A decentralization model realized in the long run according to Turkey’s conditions, and as a result of a social consensus, can help resolve the problems of the Turkish education system.

The efficacy and safety of rituximab against B-cell lymphomas is well established. However, there has been an increased incidence of infectious complications after rituximab treatment, mostly hepatitis B reactivation and progressive multifocal leukoencephalopathy. This is the case of a 67-year-old patient with primary central nervous system lymphoma, who developed cytomegalovirus meningoencephalitis after receiving high-dose chemotherapy and rituximab. As there was no evidence of lymphoma relapse or additional immunosuppression, besides his previous treatment, an association between rituximab and cytomegalovirus meningoencephalitis cannot be ruled out.

BCL6 is essential for normal antibody responses and is highly expressed in germinal centre B-cells. Constitutive expression due to chromosomal translocations or mutations of cis-acting regulatory elements contributes to diffuse large B-cell lymphoma. BCL6 expression is therefore tightly regulated in a lineage- and developmental-stage-specific manner, and disruption of normal controls can contribute to lymphomagenesis. In order to discover potential cis-acting control regions we carried out DNase I-hypersensitive site mapping. Gel-shift assays and chromatin immunoprecipitation of the core region of a hypersensitive site 4.4 kb upstream of BCL6 transcription initiation (HSS-4.4) showed an E-box element-binding ZEB1 (zinc finger E-boxbinding homeobox 1) and the co-repressor CtBP (C-terminal binding protein). As compared with peripheral blood B-cells, ZEB1, a two-handed zinc finger transcriptional repressor, is expressed at relatively low levels in germinal centre cells, whereas BCL6 has the opposite pattern of expression. Transfection of ZEB1 cDNA caused a reduction in BCL6 expression and a mutated ZEB1, incapable of binding CtBP, lacked this effect. siRNA (small interfering RNA)-mediated knockdown of ZEB1 or CtBP produced an increase in BCL6 mRNA. We propose that HSS-4.4 is a distal promoter element binding a repressive complex consisting of ZEB1 and CtBP. CtBP is ubiquitously expressed and the results of the present study suggest that regulation of ZEB1 is required for control of BCL6 expression.

Bcl-6 is essential for germinal centre development and normal antibody responses, and has major roles in controlling B-cell proliferation and differentiation. Bcl-6 expression is tightly controlled, but neither the nature of all the regulatory signals nor their interactions are known. Bcl-6 expression is induced in Bcr-Abl expressing lymphoid cell lines by the tyrosine kinase inhibitor, imatinib. We show that p38 MAPK mediates induction of Bcl-6 following inhibition of Bcr-Abl by imatinib. Next we analyze p38 function in a germinal centre B-cell line, Ramos. p38 is phosphorylated under basal conditions, and studies with p38 inhibitors show that it induces Bcl-6 expression. Membrane bound CD40 ligand activates p38 but also other MAPK pathways that strongly repress Bcl-6 and the overall effect is reduction in Bcl-6 expression. Surprisingly soluble CD40 ligand induces Bcl-6 by activating p38 without activating the repressive pathways. Hence different types of CD40 signalling are associated with varying effects on Bcl-6 expression. Transcription reporter assays demonstrate p38 responsive sequences at about 4.5 kb from the transcription start site. Immunocytochemistry of tonsil sections show phosphorylated p38 in a minor population of germinal centre B-cells. We demonstrate for the first time that p38 induces Bcl-6 transcription, but increased protein expression occurs only when the strong pathways repressing Bcl-6 are not activated.

The aim of this work is to develop biopharmaceutics classification systems for new molecular entities (BCS-NMEs) and marketed drugs (BCS-MD). The kinetics of gastrointestinal (GI) wall permeation and dissolution were re-considered theoretically. The relationships between the solubility/dose ratio and the fractions of dose dissolved and absorbed, were also examined. Mean time calculations for drug dissolution (MDT) and permeation (MPT) of the GI wall were analyzed in respect to the mean intestinal transit time (MITT) to identify a cutoff point for drug dissolution and GI wall permeation. Dissolution experiments for marketed drugs were carried out. NMEs were classified into four classes of BCS-NMEs, based on solubility/dose ratio and apparent permeability estimates. A physiologically based cutoff time point for dissolution and permeation was used to differentiate rapidly from slowly dissolving-permeating marketed drugs, which were classified into four classes of BCS-MD using their dissolution index (DI = MITT/MDT) and permeation index (PI = MITT/MPT) values as follows: I (DI ≥ 3, PI ≥ 3), II (DI < 3, PI ≥ 3), III (DI ≥ 3, PI < 3) and IV (DI < 3, PI < 3). In conclusion, two classification systems were developed, one for NMEs based on solubility/dose ratio and permeability estimates and one for marketed drugs based on MDT and MPT estimates.

A reaction-limited model for drug dissolution is developed assuming that the reaction at the solid-liquid interface is controlling the rate of dissolution. The dissolution process is considered as a bidirectional chemical reaction of the undissolved drug species with the free solvent molecules, yielding the dissolved species of drug complex with solvent. This reaction was considered in either sink conditions, where it corresponds to the unidirectional case and the entire amount of the drug is dissolved, or reaching chemical equilibrium, which corresponds to saturation of the solution. The model equation was fitted successfully to dissolution data sets of naproxen and nitrofurantoin formulations measured in the paddle and basket apparatuses, respectively, under various experimental conditions. For comparative purposes these data were also analyzed using three functions based on the diffusion layer model. All functions failed to reveal the governing role of saturation solubility in the dissolution process associated with the diffusion layer model when the conditions for the valid estimation of saturation solubility, established theoretically in this study, were met by the experimental set up employed. Overall, the model developed provides an interesting alternative to the classic approaches of drug dissolution modeling, quantifying the case of reaction-limited dissolution of drugs.

Bcl-6 is a transcription factor that is normally expressed in germinal centre B cells. It is essential for the formation of germinal centres and the production of high-affinity antibodies. Transcriptional downregulation of Bcl-6 occurs on terminal differentiation to plasma cells. Bcl-6 is highly expressed in B-cell non-Hodgkin's lymphoma and, in a subset of cases of diffuse large cell lymphoma, the mechanism of Bcl-6 overexpression involves interruption of normal transcriptional controls. Transcriptional control of Bcl-6 is, therefore, important for normal antibody responses and lymphomagenesis, but little is known of the cis-acting control elements. This report focuses on a region of mouse/human sequence homology in the first intron of Bcl-6, which is a candidate site for such a control element. We demonstrate that poly-(ADP-ribose) polymerase-1 (Parp-1) binds in vitro and in vivo to specific sequences in this region. We further show that PARP inhibitors, and Parp-1 knockdown by siRNA induce Bcl-6 mRNA expression in Bcl-6 expressing cell lines. We speculate that Parp-1 activation plays a role in switching off Bcl-6 transcription and subsequent B-cell exit from the germinal centre.

The protein tyrosine phosphatase SHP-1 dephosphorylates BCR-ABL1, thereby serving as a potential control mechanism of BCR-ABL1 kinase activity. Pathways regulating SHP-1 expression, which could be exploited in the therapeutics of TKI-resistant chronic myeloid leukemia (CML), remain unknown. Moreover, the questions of whether there is any kind of SHP-1 deregulation in CML, contributing to disease initiation or evolution, as well as the question of prognostic significance of SHP-1, have not been definitively answered. This study shows moderately lower SHP-1 mRNA expression in chronic phase CML patients in comparison to healthy individuals and no change in SHP-1 mRNA levels after successful TKI treatment. Mutational analysis of the aminoterminal and phosphatase domains of SHP-1 in patients did not reveal genetic lesions. This study also found no correlation of SHP-1 expression at diagnosis with response to treatment, although a trend for lower SHP-1 expression was noted in the very small non-responders' group of the 3-month therapeutic milestone.

The hypomethylating agent 5-azacytidine has been the standard-of-care for patients with higher-risk myelodysplastic syndrome (MDS) during the past few years. Its efficacy has been proven in large clinical trials, and its safety has been shown to be superior to that of conventional treatments.We conducted a retrospective study on the efficacy and safety of 5-azacytidine in 44 consecutive patients with MDS and acute myeloid leukemia treated with 5-azacytidine during a 63-month period. We recorded the clinical and laboratory characteristics of the patients and we analyzed the response to treatment, overall survival and adverse events during treatment.The median overall survival was 13 months, while serious adverse events consisted mostly of neutropenic infections.We reached two possibly valuable conclusions: Younger patients (73 years), as well as patients receiving treatment at longer than 28-day intervals had a significantly higher overall survival.

Previous findings from our group based on Monte Carlo simulations indicated that Fickian drug release from Euclidian or fractal matrices can be described with the Weibull function. In this study, the entire drug release kinetics of various published data and experimental data from commercial or prepared controlled release formulations of diltiazem and diclofenac are analyzed using the Weibull function. The exponent of time b of the Weibull function is linearly related to the exponent n of the power law derived from the analysis of the first 60% of the release curves. The value of the exponent b is an indicator of the mechanism of transport of a drug through the polymer matrix. Estimates for b< or =0.75 indicate Fickian diffusion in either fractal or Euclidian spaces while a combined mechanism (Fickian diffusion and Case II transport) is associated with b values in the range 0.75

Poly (ADP-ribose) polymerase 1 (PARP-1) has a central role in the repair of DNA breaks and is a promising treatment target in malignancy. We measured PARP1 mRNA levels by a SYBR-green-based PCR in the bone marrow of 74 patients with myelodysplastic syndrome (MDS) and correlated them to their demographic, hematologic and prognostic characteristics. The median PARP1 mRNA levels were correlated to the type of MDS (2008/2016 WHO classification, P=0.005) and to the IPSS score (P=0.002). A correlation was also found with the IPSS-R score (P=0.011) and the cytogenetic risk (P=0.008). In all cases, higher PARP1 levels were correlated with a higher risk category. Moreover, we found a significant survival disadvantage for patients with high PARP1 levels (median survival of 37.4 months versus ‘not reached’ for low PARP1 levels, P=0.0001, and a 5-year survival rate of 29.8 versus 88.9%, respectively). PARP1 mRNA levels were found to be the stronger predictor of survival in multivariate analysis. These correlations have never been reported in the past and may render PARP1 a prognostic factor to be incorporated in the current prognostic systems for MDS, also laying the basis for clinical trials evaluating PARP1 inhibitors in higher-risk MDS.

Background and aims Premature infants are at high risk for neurodevelopmental impairment (NDI) even in the absence of known brain complications of prematurity. Evaluation of the effectiveness of therapeutic interventions in association to neurodevelopmental outcome is required to improve or prevent the neurodevelopmental consequences of prematurity. The Bayley-III is currently the most commonly applied measurement tool for assessing early development both in clinical practice and research settings. Objective To evaluate the relationship between known risk factors and early performance on the Bayley Scales of Infant Development-Third Edition at 12 months adjusted age in premature infants. Methods. Prospective study in a cohort of premature infants with gestational age ≤32 weeks, who underwent comprehensive developmental assessment using the five domains of Bayley Scales, cognitive, language, motor, social emotional and adaptive behavior at 12 months corrected age. Developmental scores were evaluated in relation to environmental influences, therapeutic interventions or practices and complications of prematurity. Results Composite and Subscale scores for the cognitive, language and motor scales were below the 50th percentile, with no significant differences among them. Scores for the social-emotional and adaptive behavior, which are derived from the parent-report questionnaires, were near the average and significantly higher than the scores derived by the examiners. Multiple regression analyses showed that blood transfusions, apart from severely abnormal head ultrasound, gender, being small for gestational age and duration of invasive mechanical ventilation and oxygen administration were consistently related to neurodevelopmental outcome. Conclusions Bayley-III assessments are important for getting early information about development following premature birth. Parents may overestimate children's performance. Neurodevelopmental outcome is related to several environmental, biological or medical conditions associated with prematurity. Adoption of therapeutic strategies targeting known neonatal risk factors could positively affect neurodevelopmental outcome.

Chronic lymphocytic leukemia (CLL) is considered a malignancy resulting from defects in apoptosis. For this reason, targeting apoptotic pathways in CLL may be valuable for its management. Poly [ADP-ribose] polymerase 1 (PARP1) is the main member of a family of nuclear enzymes that act as DNA damage sensors. Through binding on DNA damaged structures, PARP1 recruits repair enzymes and serves as a survival factor, but if the damage is severe enough, its action may lead the cell to apoptosis through caspase activation, or necrosis. We measured thePARP1mRNA and protein pretreatment levels in 26 patients with CLL and the corresponding posttreatment levels in 15 patients after 3 cycles of immunochemotherapy, as well as in 15 healthy blood donors. No difference was found between the pre- and posttreatment levels of PARP1, but we found a statistically significant relative increase of the 89 kDa fragment of PARP1 that is cleaved by caspases in the posttreatment samples, indicating PARP1-related apoptosis in CLL patients after treatment. Our findings constitute an important step in the field, especially in the era of PARP1 inhibitors, and may serve as a base for future clinical trials with these agents in CLL.

Mastocytosis is a myeloproliferative neoplasm characterized by clonal expansion of abnormal mast cells, ranging from the cutaneous forms of the disease to mast cell leukemia. In a significant proportion of patients, systemic mastocytosis (SM) coexists with another hematologic malignancy, termed systemic mastocytosis with an associated hematologic nonmast cell lineage disorder (SM-AHNMD). Despite the pronounced predominance of concomitant myeloid neoplasms, the much more unusual coexistence of lymphoproliferative diseases has also been reported. Imatinib mesylate (IM) has a role in the treatment of SM in the absence of the KITD816V mutation. In the setting of SM-AHNMD, eradicating the nonmast cell malignant clone greatly affects prognosis. We report a case of an adult patient with SM associated with B-lineage acute lymphoblastic leukemia (B-ALL). Three cases of concurrent adult ALL and mastocytosis have been reported in the literature, one concerning SM and two concerning cutaneous mastocytosis (CM), as well as six cases of concomitant CM and ALL in children.

The role of latent Epstein-Barr virus (EBV) infection in the pathogenesis of low-grade B cell non-Hodgkin lymphoma (B-NHL) has not been studied. We therefore investigated the incidence of latent EBV infection in a group of patients with leukemic low-grade B-NHL, as well as the incidence of viral latent membrane protein 1 (LMP1) oncoprotein expression in the same patient group. Furthermore, in an attempt to elucidate the role of this viral oncoprotein in non-EBV-related lymphomas, we correlated the expression of LMP1 with the level of oxidative stress, a parameter related to apoptosis. In the present study we detected lower levels of oxidative stress in the sera of LMP1-positive patients. This possibly implies an anti-apoptotic role of this viral oncoprotein in low-grade B cell lymphomas. However, LMP1 expression status did not affect expression of the major anti-apoptotic gene BCL-2.

Tyrosine kinase inhibitors (TKIs), namely imatinib mesylate (IM) and recently approved second-generation TKIs dasatinib and nilotinib, are currently considered the treatment of choice for newly-diagnosed chronic phase chronic myelogenous leukemia (CP-CML). Although treatment with TKIs has not yet been proven curative, it certainly accomplishes a sustained control of the disease in the vast majority of patients. More than a decade after the successful launching of IM in first-line treatment of CP-CML and the subsequent introduction of second-generation TKIs in this setting, the question of the possibility of TKI cessation in a specific subset of patients has emerged. Side-effects of TKIs, along with some patients' wish to abandon the drugs and the rising financial burden upon healthcare systems, have led to the dilemma whether IM can be safely withdrawn after achieving deep molecular remissions and which patients are suitable for this discontinuation. We examined the data of our patients with CML in search of potential canditates for cessation of TKI therapy and identified their characteristics. We also performed a thorough review of the relevant literature. Eight out of fifty patients were discriminated on grounds of sustained complete molecular response (CMR) exceeding 12 months, most of them with a low or intermediate Sokal score at diagnosis. The median interval from IM initiation to CMR was almost 2 years and the median duration of detected CMR reached 6.5 years. Based on the promising results of prospective clinical trials reporting successful cessation of treatment with TKIs on selected subgroups of patients, we decided to proceed to interruption of therapy in the specific subset of our patients and closely monitor their response.

We report here the interesting case of a 76-year-old man with severe proteinuria who was diagnosed with systemic mastocytosis accompanied by a clonal non-mast-cell lineage haematological disorder (a non-secretory plasma cell dyscrasia). This is a unique report of systemic mastocytosis with a non-secretory plasma cell dyscrasia and nephrotic syndrome. The pathophysiological relevance between these entities along with the probability of occult amyloidosis is discussed.

Background Substance users seeking help at the Counseling Station of the Psychiatric Hospital of Thessaloniki are admitted to the Detoxification Unit after a waiting time of about three weeks. Of the 532 users that have attended the Counseling Station in 2004, 270 were admitted to the Detox unit, 52 were referred to the dual diagnosis unit, 23 to the outpatient programme, and the rest interrupted their treatment against professional advice. The Unit admits patients over 18 years of age. Aim of this study was an attempt to determine any changes in the socio-demographic characteristics of our patients in the course of time.

The aim of the study is to develop modified, branched versions of the Noyes-Whitney and the Weibull equations, including explicitly the solubility/dose parameter, for the analysis of dissolution data, which reach the plateau either at infinite or finite time.The modified Weibull function is applied to the analysis of experimental and literature dissolution data. To demonstrate the usefulness of the mathematical models, two model drugs are used: one highly soluble, metoprolol, and one relatively insoluble, ibuprofen.The models were fitted successfully to the data performing better compared with their classic versions. The advantages of the use of the models presented are several. They fit better to a large range of datasets, especially for fast dissolution curves that reach complete dissolution at a finite time. Also, the modified Weibull presented can be derived from differential equations, and it has a physical meaning as opposed to the purely empirical character of the original Weibull equation. The exponent of the Weibull equation can be attributed to the heterogeneity of the process and can be explained by fractal kinetics concepts. Also, the solubility/dose ratio is present explicitly as a parameter and allows to obtain estimates of the solubility even when the dissolution data do not reach the solubility level.The use of the developed branched equations gives better fittings and specific physical meaning to the dissolution parameters. Also, the findings underline the fact that even in the simplest, first-order case, the speed of the dissolution process depends on the dose, a fact of great importance in biopharmaceutic classification for regulatory purposes.

Introduction The hypomethylating agent 5-azacytidine (AZA) has been the standard of care for higher risk Myelodysplastic Syndromes (MDS) for the last few years. Its efficacy has been proven in large clinical trials, and its safety has been shown to be superior to that of conventional treatments. We have conducted a retrospective study about the efficacy and safety of 5-azacytidine, as reported and analyzed in our center. Patients and Methods Forty four consecutive patients with MDS or Acute Myeloid Leukemia (AML) with 20-30% bone marrow blasts that were treated with AZA during the last 63 months were included in the study. The clinical and laboratory characteristics of the patients were recorded, and the efficacy and safety data were analyzed. Results The epidemiologic and hematologic characteristics of the patients are shown in Table 1. The median overall survival was 13 months (1-101) and there was no primary treatment failure (Table 2). Serious adverse events consisted mostly of neutropenic infections (blood stream and pneumonia) (Table 3). Discussion Treatment with AZA offered a favorable (complete and partial) response in 34.1% of the patients, and an overall survival of 13 months, with generally predictable toxicities, although hospitalization was frequently inevitable during the first treatment cycles, when supportive treatment was a significant part of the management. A valuable observation is that there was a considerable decrease in the patients’ transfusion needs following treatment (p Table 1. Epidemiologic and hematologic characteristics. Male: Female ratio 30:14 (2.1 : 1) Age, Median (Range) 73 (54-81) WHO classification of MDS/AML, N (%) RAEB-I RAEB-II RCMD-RS RCMD RARS CMML AML 9 (20.5) 18 (40.9) 2 (4.5) 3 (6.7) 1 (2.3) 4 (9.1) 7 (15.9) IPSS classification, N (%) Low Intermediate-1 Intermediate-2 High Not Applicable (AML) 0 (0) 3 (6.8) 29 (65.9) 5 (11.4) 7 (15.9) Complete Blood Count Parameters, Median (Range) Hemoglobin (g/dL) Absolute Neutrophil Count (x109/L) Platelet count (x109/L) 8.55 (4.5 - 12.5) 1.08 (0.0 – 16.3) 80.0 (2 – 820) Transfusion dependence, N (%) 39 (88.6) Transfusions per month, Median (Range) 3 (0 – 7) Table 2. Efficacy data AZA cycles, Median (Range) 5 (1-22) Actual AZA dose (mg/m2/cycle), Median (Range) 75 (59-75) Actual cycle duration (days), Median (Range) 28 (28-40) Dose reductions due to sustained neutropenia, N (%) 6 (13.6) Temporary AZA interruption, N (%) 26 (59.1) Reason Sustained cytopenia 10/26 (38.5) Neutropenic Infection 15/26 (57.7) Hemorrhagic Complication 1/26 (3.8) Permanent AZA discontinuation, N (%) 23/44 (52.3) Reason AML transformation 17/23 (73.9) Recurrent or severe infection 4/23 (17.4) Pyoderma gangrenosum 1/23 (4.3) Allogeneic Bone Marrow Transplantation 1/23 (4.3) AZA cycles till response (according to the IWG criteria), Median (Range) 4 (1 – 7) Response (IWG criteria), N (%) Complete response Partial response Stable disease Failure 7 (15.9) 8 (18.2) 29 (65.9) 0 (0) Overall survival (months), Median (Range) 13 (1 – 101) Post treatment transfusion dependence, N (%) 34 (77.3) Transfusions per month (post-treatment), Median (Range) 1 (0 – 5) Death rate, N (%) 29/44 (65.9) Cause of death, N (%) Infection Hemorrhage Cardiac dysrhythmia 24/29 (82.8) 3/29 (10.3) 2/29 (6.9) Table 3. Safety data Clinical adverse events, N (%) 29/44 (65.9) Neutropenic Infections 26/29 (89.7) Bloodstream Infection 9/26 (34.6) Lower respiratory infection 10/26 (38.5) Neutropenic Fever 8/26 (30.1) Septic shock 2/26 (7.7) Hemorrhagic events 2/29 (6.7) Cerebral hemorrhage (Grade 5) 1/2 (50.0) Epistaxis (Grade 3) 1/2 (50.0) Other (pyoderma gangrenosum) 1/29 (3.4) Laboratory incidents1, N (%) 44/44 (100) All grades Grades 3/4 Neutropenia 36/44 (81.8) 34/44 (77.3) Anemia 44/44 (100) 24/44 (54.5) Thrombocytopenia 31/44 (70.5) 21/44 (47.7) Supportive treatment (during AZA administration), N (%) GCSF administration 16/44 (36.4) Erythropoietin administration 7/44 (15.9) Red blood cell transfusions 39/44 (88.6) Red blood cell transfusions (units/cycle), Median (range) 3 (0-7) Pooled random donor platelet transfusions 17 (38.6) 1According to the CTCAE Version 4.0 Disclosures No relevant conflicts of interest to declare.

Background Tyrosine kinase inhibitors (TKIs) are the current treatment of choice for chronic myeloid leukemia (CML). However, approximately 15% of patients fail first and second line therapy, creating the need for the development of additional pharmacological targets in CML. Kinases acting downstream of BCR-ABL as well as anti-apoptotic proteins such as BCL-2 are being in vitro investigated as therapeutic targets. Previous research has yielded contradictory results on the role of c-myc in CML. In one study, conditional overexpression of myc was shown to confer resistance to imatinib-induced apoptosis via downregulation of the cell cycle regulator p27Kip1. In another study, myc induced p53-dependent apoptosis of leukemia-initiating cells (LICs) in CML. However, there has been so far no attempt to correlate myc transcript and protein levels with those of the bcr-abl transcript. Mcl-1, a bcl-2 family member, has been shown to promote survival of Ph+ lymphoblasts. Mcl-1 mRNA and protein levels are downregulated by imatinib in Ph + cell lines, but its expression in CML patient samples has not been investigated. Aims We aimed to test whether expression of myc and mcl-1 is influenced by the levels of the BCR-ABL transcript and therefore compared transcript levels of these two individual genes before and during treatment with TKIs in CML patients. Methods We used RNA isolated from whole blood samples of thirty-six (36) patients with CML, obtained at diagnosis and during treatment with a TKI, at least three months after initiation of treatment. All samples collected during treatment had a bcr-abl/abl ratio less than 2%, while samples collected at diagnosis had bcr-abl/abl ratios ranging from 33 to 130 %. RNA samples were subjected to reverse transcription and cDNA was used in quantitative Real-Time PCR reactions employing Taqman assays for myc, mcl-1 and hprt1 (housekeeping gene). Fold induction or repression of genes after treatment was calculated via the ΔCt method, after calculation of PCR efficiencies. Statistical analysis was performed with the use of the Wilcoxon rank-sum test. Results Median mRNA levels of myc were shown to be increased 2.2 times and median mRNA levels of mcl-1 were shown to be increased 2.6 times after treatment with imatinib, at a statistically significant level (p value Conclusions New treatment targets, either BCR-ABL independent or targets lying downstream of BCR-ABL, are needed in the field of CML therapy in order to circumvent the problem of TKI resistance. In our study, transcription of the genes myc and mcl-1 was shown to be moderately induced by imatinib treatment, which means that non-leukemic Ph-negative white blood cells have a higher amount of myc and mcl-1 transcripts compared to Ph+ leukemic white blood cells. Whole blood RNA was used in the study, as the BCR-ABL kinase has been shown to be expressed in the mature progeny of all haemopoietic lineages except some T cell clones. A study of the protein levels of the two genes would also be warranted in order to elucidate their potential pathogenetic or protective role in CML. In vitro functional studies with specific pharmacological inhibitors would subsequently show if the MYC and MCL-1 proteins could become targets to activate or inhibit in CML. Disclosures: No relevant conflicts of interest to declare.

Introduction Poly [ADP-ribose] polymerase 1 (PARP-1) has a central role in the repair of single-stranded DNA (ssDNA) breaks, thus protecting the cell from genomic instability. This enzyme is a promising target in anticancer treatment. The aim of the present study is to investigate the role of PARP1 in B chronic lymphocytic leukemia (B-CLL) patients pre- and post- treatment. Patients and Methods Blood samples were collected from B-CLL patients before treatment and following 3 cycles of immunochemotherapy (chosen by the treating physician according to common clinical practice). Quantification of PARP1-mRNA levels was performed by a SYBR-green based PCR performed on BIORADCFX96 (BIORAD). The results were expressed in correlation to the mRNA levels of the endogenous housekeeping gene of beta actin (ACTB). The detection of the gene product was performed by Western Blot using anti-PARP1 antibody (clone 47-258; Calbiochem). We detected the 70 KD protein segment that results from the in vivo fractionation of PARP1 by caspase-3. The protein bands were visualized using an enhanced chemiluminescence detection system (Amersham Biosciences) and quantitated by densitometry using the gel analysis software ImageJ. We correlated the PARP1 protein levels to those of a group of healthy volunteers. We also studied the differences between PARP1-mRNA and protein levels before and after treatment and correlated them to clinical response. The related samples Wilcoxon Signed Rank test (IBM SPSS statistics, version 19.0) was used for the statistical analysis of the results. Results Samples were obtained from 24 B-CLL patients before treatment and from 15/24 following 3 cycles of immunochemotherapy. The patients' characteristics are shown in Table 1. Pre-treatment PARP1 protein levels were similar to those of 15 healthy volunteers (p=0.426). No statistically significant difference was noted between the PARP1-mRNA and protein levels following treatment (p=0.507 and 0.650 respectively) (Table 1). Multivariate analysis did not reveal statistically significant differences in the mRNA and protein levels in correlation to the stage of disease, the peripheral blood lymphocyte count, the LDH levels, the response to treatment and the overall prognosis of the patients. Conclusions The expression of PARP1 in patients with B-CLL seems to be comparable to that of healthy persons. We could not distinguish patients displaying reduced levels of PARP1, as previously reported by others. Moreover, PARP1 expression is not affected by immunochemotherapy, irrespective of stage and response to treatment. Thus, this molecule seems not to participate in the mechanisms involved in DNA repair and apoptosis that are impaired in B-CLL. The study is still ongoing with the addition of more subjects. Disclosures: No relevant conflicts of interest to declare.

Background Chronic myeloid leukemia (CML) is a disease of the haemopoietic stem cell, hence its hallmark chromosomal rearrangement is detected in different haemopoietic lineages. Due to reported altered adhesion of haemopoietic cells to bone marrow stroma in CML, the cytogenetic profile and properties of bone marrow and adipose tissue mesenchymal cells have been the interest of our current study. Previous research has shown that the BCR-ABL fusion gene is not present in bone marrow mesenchymal stem cells; however, it has been detected in endothelial cells and in Flk1+ hemangioblastic progenitors. Aims To study the properties of mesenchymal stem cells in CML, we initially investigated the presence of the BCR-ABL fusion gene in bone marrow and adipose tissue mesenchymal stem cells (MSCs) of patients with CML at diagnosis. Although previous studies have yielded negative results, the numbers of patients studied have been limited. Moreover, the presence of BCR-ABL in hemangioblastic progenitors in some cases of CML patients highlights the possibility that the lesion can sometimes be detected in a progenitor cell much more primitive than the haemopoietic stem cell. Adipose tissue MSCs were studied concomitantly with those of bone marrow in order to establish possible cell motility between the two compartments during disease evolution. Methods We isolated mesenchymal stem cells from bone marrow and adipose tissue mononuclear cells of four CML patients via cell culture. MSCs were isolated from the mononuclear cell fraction via serial medium changes removing non-adherent cells, followed by at least three passages. Patient samples were collected before the onset of therapy; three of the patients were diagnosed in chronic phase and one in blast crisis. High purity of MSCs from haemopoietic cell contaminants (>94% in all cases) was confirmed by flow cytometry; mesenchymal stem cells were positive for CD90, CD73 and CD105 and negative for CD34 and CD45. FISH analysis was performed to detect the BCR-ABL gene (probe LSI BCR/ABL1, ES Dual Color Translocation, Vysis). For each sample, 500 nuclei were examined. Results The BCR-ABL fusion gene was detected in 15% of the bone marrow MSCs’ nuclei of one patient, who had chronic phase CML. The CD34+/CD45+ cell population percentage in the final MSC population isolated from this patient was only 1,5% on flow cytometry analysis, which means that this result cannot be accounted for by haemopoietic cell contamination of MSCs. The other three patients did not carry the translocation in their bone marrow MSCs upon FISH analysis. Adipose tissue MSCs of all four patients were negative for BCR-ABL. Conclusions We have shown the presence of the BCR-ABL fusion gene in bone marrow mesenchymal stem cells of one CML patient. This result implies an occurrence of the t (9;22) (q11;134) translocation at a more primitive level in some cases of the disease, so that the bone marrow stroma can be involved in the malignant clone. Because there is still limited knowledge about the role of MSCs, whether BCR-ABL–positive or negative, in the development of the malignant haemopoietic clone in CML, our study will be extended to study the cytogenetic and gene expression profile of mesenchymal stem cells in a larger number of CML patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 4833 Introduction. The Epstein-Barr virus has been implicated in the pathogenesis of certain human B-cell neoplasms, such as Burkitt's lymphoma, Hodgkin's disease and post-transplant lympho-proliferative disorders. Persistent latent EBV infection is, however, frequent and therefore its role is of interest in all types of B cell malignancies. In low-grade B cell lymphomas there are few reports for its potential role in higher grade transformation and its association with stereotypic BCRs in CLL. The mechanisms of EBV-associated B cell transformation are probably associated with its proteins expressed during latency; one of the most studied is the LMP1 oncoprotein, which is considered as an anti-apoptotic factor (activator of NF-êB). Recent studies, however, show evidence of coexisting apoptotic properties of LMP1. The level of oxidative stress reflects activation of caspase-mediated apoptotic pathways. Aims and methods. We measured the levels of oxidative stress in low-grade B cell lymphoma patient samples and correlated them with the expression of the LMP1 oncoprotein in order to study apoptotic functions of LMP1. Whole blood samples from 48 patients aged 51–87 (median age 74 years, 25 males, 23 females) without treatment in the previous six months were examined (chronic lymphocytic leukemia: 27, marginal zone lymphoma: 12, mantle cell lymphoma: 4, hairy cell leukemia: 2, follicular lymphoma: 2, lymphoplasmacytic lymphoma: 1). Latent EBV infection was detected with RT-PCR for the viral BXLF1 gene. LMP1 expression was quantitated with Real-Time PCR in EBV-positive patients. The levels of oxidative stress were quantitated in the sera of all patients with the use of a peroxide measuring kit (PerOx TOS/TOC kit by Immundiagnostik) and compared between the LMP1-positive (13) and LMP1-negative (35) group of patients with the use of 2-tailed Mann-Whitney test. Results. Of the fourty-eight (48) patients tested, nineteen (19) were EBV-positive. Thirteen (13) of the nineteen (19) EBV-positive ones expressed LMP1. Oxidative stress was found to be significantly higher in LMP1-negative vs LMP1-positive patients (372.3 vs 261.4 micromol/L, p=0.014). Discussion. The role of LMP1 expression is under investigation in the non EBV-related low grade B cell lymphomas. In the present study we examined a potential effect of LMP1 expression on oxidative stress and found that levels of oxidative stress were lower in LMP1-positive vs LMP1-negative patients with low-grade B cell lymphomas, reflecting an anti-apoptotic function of LMP1. In accordance with this result, LMP1 has been shown to upregulate BCL-2 using the NF-êB pathway. BCL-2 is a major inhibitor of the initiation of caspase-related apoptotic pathways and BCL-2 upregulation inhibits apoptosis resulting in lower levels of oxidative stress. However, in a study of sixty-four patients with low grade B cell lymphomas, we recently showed that LMP1 expression increases the levels of the apoptotic marker survivin, confirming that LMP1 may also possess an apoptotic function, as has been shown by another recent study on cell lines. Conclusion. The lower oxidative stress in the LMP1-expressing low grade B cell lymphoma samples shows evidence of an apoptotic function of the oncoprotein in this group of diseases. Disclosures: No relevant conflicts of interest to declare.