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The aim of this study was to investigate the effects of a non-standard, intermittent imatinib treatment in elderly patients with Philadelphia-positive chronic myeloid leukaemia and to answer the question on which dose should be used once a stable optimal response has been achieved. Seventy-six patients aged ⩾65 years in optimal and stable response with ⩾2 years of standard imatinib treatment were enrolled in a study testing a regimen of intermittent imatinib (INTERIM; 1-month on and 1-month off). With a minimum follow-up of 6 years, 16/76 patients (21%) have lost complete cytogenetic response (CCyR) and major molecular response (MMR), and 16 patients (21%) have lost MMR only. All these patients were given imatinib again, the same dose, on the standard schedule and achieved again CCyR and MMR or an even deeper molecular response. The probability of remaining on INTERIM at 6 years was 48% (95% confidence interval 35–59%). Nine patients died in remission. No progressions were recorded. Side effects of continuous treatment were reduced by 50%. In optimal and stable responders, a policy of intermittent imatinib treatment is feasible, is successful in about 50% of patients and is safe, as all the patients who relapsed could be brought back to optimal response.

The aim of this work was to assess whether "modeled microgravity" affects cell response to ionizing radiation, increasing the risk associated with radiation exposure. Lymphoblastoid TK6 cells were irradiated with various doses of gamma rays and incubated for 24 h in a modeled microgravity environment obtained by the Rotating Wall Vessel bioreactor. Cell survival, induction of apoptosis and cell cycle alteration were compared in cells irradiated and then incubated in 1g or modeled microgravity conditions. Modulation of genomic damage induced by ionizing radiation was evaluated on the basis of HPRT mutant frequency and the micronucleus assay. A significant reduction in apoptotic cells was observed in cells incubated in modeled microgravity after gamma irradiation compared with cells maintained in 1g. Moreover, in irradiated cells, fewer G2-phase cells were found in modeled microgravity than in 1g, whereas more G1-phase cells were observed in modeled microgravity than in 1g. Genomic damage induced by ionizing radiation, i.e. frequency of HPRT mutants and micronucleated cells, increased more in cultures incubated in modeled microgravity than in 1g. Our results indicate that modeled microgravity incubation after irradiation affects cell response to ionizing radiation, reducing the level of radiation-induced apoptosis. As a consequence, modeled microgravity increases the frequency of damaged cells that survive after irradiation.

The in vivo effect of recombinant human erythropoietin (rHuEpo) and granulocyte colony-stimulating factor (G-CSF) combined treatment on CD34(+) cells was evaluated by fluorescence in situ hybridization (FISH) in 13 myelodysplastic syndrome (MDS) patients with known cytogenetic abnormalities. After treatment, responsive patients presented a significantly lower proportion of FISH abnormal CD34(+) cells than before treatment (P = 0.003), and in comparison with unresponsive cases (P = 0.007). Response to treatment was associated with a reduced degree of apoptosis in CD34(+) cells (P = 0.021): however, no difference in telomere length was observed in responsive patients after growth factor administration. Although the number of patients analysed was relatively small, the present data suggest that, in MDS patients, response to rHuEpo and G-CSF may be related to the proliferation of karyotypically normal but potentially defective CD34(+) progenitor cells.

The powerful methyl(trifluoromethyl)dioxirane (1b) was employed to achieve the direct oxyfunctionalization of 2,4-didehydroadamantane (5), spiro[cyclopropane-1,2'-adamantane] (9), spiro[2.5]octane (17), and bicyclo[6.1.0]nonane (19). The results are compared with those attained in the analogous oxidation of two alkylcyclopropanes, i.e., n-butylcyclopropane (11) and (3-methyl-butyl)-cyclopropane (14). The product distributions observed for 11 and 14 show that cyclopropyl activation of alpha-C-H bonds largely prevails when no tertiary C-H are present in the open chain in the tether; however, in the oxyfunctionalixation of 14 cyclopropyl activation competes only mildly with hydroxylation at the tertiary C-H. The application of dioxirane 1b to polycyclic alkanes possessing a sufficiently rigid framework (such as 5 and 9) demonstrates the relevance of relative orientation of the cyclopropane moiety with respect to the proximal C-H undergoing oxidation. At one extreme, as observed in the oxidation of rigid spiro compound 9, even bridgehead tertiary C-H's become deactivated by the proximal cyclopropyl moiety laying in the unfavorable "eclipsed" (perpendicular) orientation; at the other end, a cyclopropane moiety constrained in a favorable "bisected" orientation (as for didehydroadamantane 5) can activate an "alpha" methylene CH2 to compete effectively with dioxirane O-insertion into tertiary C-H bonds. Comparison with literature reports describing similar oxidations by dimethyldioxirane (1a) demonstrate that methyl(trifluoromethyl)dioxirane (1b) presents similar selectivity and remarkably superior reactivity.

Polyurethane-resin doming is currently one of the fastest growing markets in the field of industrial graphics and product identification. Semi-rigid bio-based polyurethanes were prepared deriving from soybean oil as a valuable alternative to fossil materials for digital doming and applied to digital mosaic technology. Bio-resins produced can favorably compete with the analogous fossil polymers, giving high-quality surface coatings (ascertained by SEM analyses). In addition, polyurethane synthesis was accomplished by using a mercury- and tin-free catalyst (the commercially available zinc derivative K22) bringing significant benefits in terms of cost efficiency and eco-sustainability.

Genomic instability leads to a wide spectrum of genetic changes, including single nucleotide mutations, structural chromosome alterations, and numerical chromosome changes. The accepted view on how these events are generated predicts that separate cellular mechanisms and genetic events explain the occurrence of these types of genetic variation. Recently, new findings have shed light on the complexity of the mechanisms leading to structural and numerical chromosome aberrations, their intertwining pathways, and their dynamic evolution, in somatic as well as in germ cells. In this review, we present a critical analysis of these recent discoveries in this area, with the aim to contribute to a deeper knowledge of the molecular networks leading to adverse outcomes in humans following exposure to environmental factors. The review illustrates how several technological advances, including DNA sequencing methods, bioinformatics, and live-cell imaging approaches, have contributed to produce a renewed concept of the mechanisms causing genomic instability. Special attention is also given to the specific pathways causing genomic instability in mammalian germ cells. Remarkably, the same scenario emerged from some pioneering studies published in the 1980s to 1990s, when the evolution of polyploidy, the chromosomal effects of spindle poisons, the fate of micronuclei, were intuitively proposed to share mechanisms and pathways. Thus, an old working hypothesis has eventually found proper validation.

This study compares the profitability of cooperatives and investor-owned firms in the Italian wine sector. From a review of the financial ratios that have traditionally been applied in previous studies, we identify the key factors that affect firm profitability (proxied by sales growth) and analyse them for the five-year period from 2008 to 2012. Italian wine cooperatives offer a particularly suitable environment in which to apply our study because they have benefitted from EU regulation and several supporting measures since 2008, allowing them to invest in infrastructure and improve efficiency in order to produce quality wine, grow their brands, and penetrate export markets. In particular, this study expands the body of knowledge on this topic by focusing on the factors that affect the profitability of cooperatives and investor-owned firms and by considering time series data. We find that the EU support measures for cooperatives have led to an increase in their financial performance since 2008. Moreover, cooperatives typically have lower liquidity levels and significantly high debt as a proportion of net equity compared with investor-owned firms. Hence, consistent with the findings in the literature, the influence of financial performance on profitability is clearly related to business type.

In previous experiments performed on anaesthetised rats, we demonstrated that whisking neurons responsive to spontaneous movement of the macrovibrissae are located within the trigeminal mesencephalic nucleus (Me5) and that retrograde tracers injected into the mystacial pad of the rat muzzle extensively labelled a number of Me5 neurons. In order to evaluate the electrophysiological characteristics of the Me5-whisker pad neural connection, the present study analysed the Me5 neurons responses to artificial whisking induced by electrical stimulation of the peripheral stump of the facial nerve. Furthermore, an anterograde tracer was injected into the Me5 to identify and localise the peripheral terminals of these neurons in the mystacial structures. The electrophysiological data demonstrated that artificial whisking induced Me5 evoked potentials as well as single and multiunit Me5 neurons responses consistent with a direct connection. Furthermore, the neuroanatomical findings showed that the peripheral terminals of the Me5 stained neurons established direct connections with the upper part of the macrovibrissae, at the conical body level, with fibres spiralling around the circumference of the vibrissae shaft. As for the functional role of this sensory innervation, we speculated that the Me5 neurons are possibly involved in encoding and relaying proprioceptive information related to vibrissae movements to other CNS structures.

Nuclear invaginations, also referred to as fishmouth or cuplike nuclei, have long been identified in microgranular APL, myelomonocytic and monocytic AMLs. More recently, this typical morphological feature has been associated with NPM1 and FLT3 mutations, as well as with the lack of CD34 and HLA-DR expression. In this study, we retrospectively analyzed the morphologic, immunophenotypic, cytogenetic, and molecular features of 68 patients with AML. A cuplike nuclear invagination was detected in more than 10% of blast cells in 15 (22%) cases. Our data show that a cuplike morphology is associated with FLT3-ITD positivity, as well as with the loss of CD34 and HLA-DR expression. The results were not significantly modified when a higher cutoff of cuplike cells was used. Our results are not sufficient to suggest that cuplike AML could represent a distinct subtype, but further investigations could yield a better characterization of this feature in patients with AML.

The purpose of a financial statement is to assist the analyst to assess businesses in a neutral way, without being influenced by external factors that are not technically relevant to the evaluation process. Otherwise, assessing a company is not a neutral process because the analyst uses his or her cognitive baggage - made up of prejudice, stereotypes and expectations - in the assessment process. These factors enter into the decision–making process and influence assessment (Kahneman and Tversky, 2000). The research hypothesis of this paper is as follows: the decision–making process of financial analysts is influenced by external labels (in this case, the company name) in their evaluation process. The survey has been conducted on university students in an experiment through the submission of questionnaires to verify whether a simple name change affects the way a company is assessed. The responses show that the effect of name recognition has a significant influence on company evaluation.

Background/Aims: The protein kinase Akt2/PKBβ is a known regulator of macrophage and dendritic cell (DC) migration. The mechanisms linking Akt2 activity to migration remained, however, elusive. DC migration is governed by Ca2+ signaling. We thus explored whether Akt2 regulates DC Ca2+ signaling. Methods: DCs were derived from bone marrow of Akt2-deficient mice (akt2-/-) and their wild type littermates (akt2+/+). DC maturation was induced by lipopolysaccharides (LPS) and evaluated by flow cytometry. Cytosolic Ca2+ concentration was determined by Fura-2 fluorescence, channel activity by whole cell recording, transcript levels by RT-PCR, migration utilizing transwells. Results: Upon maturation, chemokine CCL21 stimulated migration of akt2+/+ but not akt2-/- DCs. CCL21-induced increase in cytosolic Ca2+ concentration, thapsigargin-induced release of Ca2+ from intracellular stores with subsequent store-operated Ca2+ entry (SOCE), ATP-induced inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ release as well as Ca2+ release-activated Ca2+ (CRAC) channel activity were all significantly lower in mature akt2-/- than in mature akt2+/+ DCs. Transcript levels of IP3 receptor IP3R2 and of IP3R2 regulating transcription factor ETS1 were significantly higher in akt2+/+ than in akt2-/- DCs prior to maturation and were upregulated by LPS stimulation (1h) in akt2+/+ and to a lower extent in akt2-/- DCs. Following maturation, protein abundance of IP3R2 and ETS1 were similarly higher in akt2+/+ than in akt2-/- DCs. The IP3R inhibitor Xestospongin C significantly decreased CCL21-induced migration of akt2+/+DCs and abrogated the differences between genotypes. Finally, knock-down of ETS1 with siRNA decreased IP3R2 mRNA abundance, thapsigargin- and ATP-induced Ca2+ release, SOCE and CRAC channel activation, as well as DC migration. Conclusion: Akt2 upregulates DC migration at least in part by ETS1-dependent stimulation of IP3R2 transcription.

Dendritic cells (DCs), key players of immunity, are regulated by glycogen synthase kinase GSK3. GSK3 activity is suppressed by PKB/Akt and SGK isoforms, which are in turn stimulated by the PI3K pathway. Exposure to bacterial lipopolysaccharides increases cytosolic Ca(2+)-concentration ([Ca(2+)]i), an effect augmented in DCs isolated from mutant mice expressing PKB/SGK-resistant GSK3α,β (gsk3(KI) ). Factors affecting [Ca(2+)]i include Ca(2+)-release from intracellular stores (CRIS), store-operated Ca(2+)-entry (SOCE) through STIM1/STIM2-regulated Orai1, K(+)-dependent Na(+)/Ca(2+)-exchangers (NCKX), K(+)-independent Na(+)/Ca(2+)-exchangers (NCX) and calbindin-D28k. The present study explored whether PKB/SGK-dependent GSK3α, β-activity impacts on CRIS, SOCE, NCKX, NCX or calbindin. DCs were isolated from gsk3(KI) mice and respective wild-type mice (gsk3(WT) ), [Ca(2+)]i estimated from Fura2 fluorescence, Orai1, STIM1, STIM2 as well as calbindin-D28k protein abundance determined by Western blotting and mRNA levels quantified by real time PCR. As a result, thapsigargin-induced CRIS and SOCE were significantly blunted by GSK3-inhibitors SB216763 (1-10 µM, 30 min) or GSK-XIII (10 µM, 30 min) but were significantly lower in gsk3(WT) than in gsk3(KI) DCs. Orai1, STIM1 and STIM2 protein abundance was significantly lower and calbindin-D28k abundance significantly higher in gsk3(KI) than in gsk3(WT) DCs. Activity of NCKX and NCX was significantly higher in gsk3(KI) than in gsk3(WT) DCs and was significantly increased by SB216763 (1 µM, 30 min) or GSK-XIII (10 µM, 30 min). Treatment of gsk3(WT) DCs with SB216763 (1 µM, 4-24 h) or GSK-XIII (10 µM, 4-24 h) did not significantly modify the protein abundance of Orai1, STIM1 and STIM2. The present observations point to a dual role of GSK3 in the regulation of Ca(2+) in DCs. Acute inhibition of GSK3 blunted the increase of [Ca(2+)]i following CRIS and SOCE and stimulated NCKX/NCX activity. However, expression of PKB/SGK-resistant GSK3α, β downregulated the increase of [Ca(2+)]i following CRIS and SOCE, an effect at least partially due to downregulation of Orai1, STIM1 and STIM2 expression as well as upregulation of Na(+)/Ca(2+)-exchanger activity and calbindin D28k expression.

A novel human cDNA sequence has been isolated from human testis cDNA library. This sequence, named PD1, reveals an open reading frame encoding a protein of 520 amino acids. A partial sequence similarity has been found with the RBM gene involved in the regulation of human spermatogenesis. Northern blot analysis for PD1 mRNA from several human tissues demonstrated two distinct transcripts of 2.7 (more abundant) and 4.0 kb and revealed that PD1 is expressed in testis and to a lesser extent also in spleen, thymus, and prostate. Immunohistochemical analysis of human testis showed that this protein is detected in the cytoplasm of Sertoli cells. Antibodies against a rhPD1 fragment were used for Western blot analysis, which confirmed the presence of a 60-kDa molecule in crude extract of human testicular cells obtained from fine-needle aspiration and showed different patterns in various testiculopathies, suggesting a role for such gene in human spermatogenesis.

The effect of different 1,3-butadiene (BD) inhalation doses, 130, 250 and 500 ppm, on somatic cells of mice was studied. Two different cell populations with diverse replicative and differentiative activities, namely splenocytes and peripheral blood reticulocytes, were examined and micronucleus (MN) frequencies were estimated. In splenocytes, different postinhalation time intervals were studied with regard to MN induction and characterisation. BD was found to be clastogenic by inducing increased micronucleus frequencies in both cell compartments and also to induce cytotoxicity at the highest level of exposure. In mouse splenocytes, BD has also shown a weak aneugenic effect at a short time interval after the exposure. Postinhalation time influences the induction of chromosome damage in stimulated splenocytes treated in vivo, since MN frequency decreases with time; in addition, BD has shown its aneugenic and cytotoxic potential only at 2 days after exposure.

The genotoxicity of diepoxibutane (DEB) and epoxybutene (EB), two of the main metabolites of 1,3-butadiene, was tested in the germ and somatic cells of the mouse by applying an MN assay in early spermatids, and in peripheral blood reticulocytes of a subgroup of the same animals. DEB (0.17 and 0.35 mmol/kg) and EB (0.35, 0.70 and 1.04 mmol/kg) were administered i.p. In the germ cell assay, significant increases of MN were observed after treatment of premeiotic S-phase cells with both butadiene metabolites, but DEB was shown to be more powerful than EB in the induction of chromosomal damage. A weak effect of the same compounds was also found after treatment of late spermatocytes, approaching the meiotic divisions. From the MN assay in peripheral blood reticulocytes, a statistically significant increase of the frequency of MN was detected at each dose tested for both chemicals. However, the results have again shown that DEB is much more efficient than EB in inducing chromosome damage.

Common fragile sites are hotspots for chromosome instability and co-localize to cancer genomic rearrangements. Whether these loci may be considered stable in human subjects under physiological conditions remains an open question. Here we show by molecular combing that a small but significant percentage of normal human cells carry an abnormal sequence pattern within the tumor suppressor gene FHIT (3p14.2) at FRA3B. Each sequence variation represents a unique pattern within a normal cell population, and therefore it would remain undetected or not interpreted by genome-wide analyses. Remarkably, the region is the same as in FHIT rearrangements described in tumors. By analyses on several normal cell lines (proliferating and resting primary lymphocytes, primary fibroblasts, lymphoblastoid cells including clonal cell cultures) we verified that: (a) each cell type displays altered sequence patterns at FHIT; (b) the presence of abnormal sequence patterns is specific for the FHIT locus; and (c) FHIT instability occurs de novo during cell proliferation, and heterogeneous sequence variants progressively accumulate in the cell populations. FHIT has been widely investigated in cancer cells, but to our knowledge this is the first direct evidence of spontaneous and recurrent occurrence of genomic instability at this gene in human subjects, at the same region involved in cancer rearrangements. Our results suggest that common fragile site activity is not restricted to in vitro cell culture and that genomic instability may pre-exist in normal cells in the absence of exogenous replication stress.

The function of dendritic cells (DCs), antigen-presenting cells regulating naïve T-cells, is regulated by cytosolic Ca²⁺ concentration ([Ca²⁺]i). [Ca²⁺]i is increased by store-operated Ca²⁺ entry and decreased by K⁺-independent (NCX) and K⁺-dependent (NCKX) Na⁺/Ca²⁺ exchangers. NCKX exchangers are stimulated by immunosuppressive 1,25-dihydroxyvitamin D3 [1,25(OH)₂D₃], the biologically active form of vitamin D. Formation of 1,25(OH)₂D₃ is inhibited by the antiaging protein Klotho. Thus 1,25(OH)₂D₃ plasma levels are excessive in Klotho-deficient mice (klothohm). The present study explored whether Klotho deficiency modifies [Ca²⁺]i regulation in DCs. DCs were isolated from the bone marrow of klothohm mice and wild-type mice (klotho+/+) and cultured for 7-9 days with granulocyte-macrophage colony-stimulating factor. According to major histocompatibility complex II (MHC II) and CD86 expression, differentiation and lipopolysaccharide (LPS)-induced maturation were similar in klothohm DCs and klotho+/+ DCs. However, NCKX1 membrane abundance and NCX/NCKX-activity were significantly enhanced in klothohm DCs. The [Ca²⁺]i increase upon acute application of LPS (1 μg/ml) was significantly lower in klothohm DCs than in klotho+/+ DCs, a difference reversed by the NCKX blocker 3',4'-dichlorobenzamyl (DBZ; 10 μM). CCL21-dependent migration was significantly less in klothohm DCs than in klotho+/+ DCs but could be restored by DBZ. NCKX activity was enhanced by pretreatment of klotho+/+ DC precursors with 1,25(OH)₂D₃ the first 2 days after isolation from bone marrow. Feeding klothohm mice a vitamin D-deficient diet decreased NCKX activity, augmented LPS-induced increase of [Ca²⁺]i, and enhanced migration of klothohm DCs, thus dissipating the differences between klothohm DCs and klotho+/+ DCs. In conclusion, Klotho deficiency upregulates NCKX1 membrane abundance and Na⁺/Ca²⁺-exchange activity, thus blunting the increase of [Ca²⁺]i following LPS exposure and CCL21-mediated migration. The effects are in large part due to excessive 1,25(OH)₂D₃ formation.

There have been few reports of a response to dasatinib or nilotinib after failure of two prior sequential tyrosine kinase inhibitors. We report the outcome of 82 chronic phase patients who received nilotinib or dasatinib as third-line alternative tyrosine kinase inhibitor therapy. Thirty-four patients failed to respond to nilotinib and were started on dasatinib as third-line tyrosine kinase inhibitor therapy while 48 patients were switched to nilotinib after dasatinib failure. Overall, we obtained a cytogenetic response in 32 of 82 patients and major molecular response in 13 patients; disease progression occurred in 12 patients. At last follow up, 70 patients (85.4%) were alive with a median overall survival of 46 months. Our results show that third-line tyrosine kinase inhibitor therapy in chronic myeloid leukemia patients after failure of two prior sequential tyrosine kinase inhibitors may induce a response that, in some instances, could prolong overall survival and affect event-free survival.

Mammalian genomes are replicated under a flexible program, with random use of origins and variable fork rates, and many details of the process must be still unraveled. Molecular combing provides a set of direct data regarding the replication profile of eukaryotic cells: fork rates; organization of the replication clusters; proportion of unidirectional forks; and fork dynamics. In this study the replication profiles of different primary and immortalized non-cancer human cells (lymphocytes, lymphoblastoid cells, fibroblasts) were evaluated at the whole-genome level or within reference genomic regions harboring coding genes. It emerged that these different cell types are characterized by specific replication profiles. In primary fibroblasts, a remarkable fraction of the mammalian genome was found to be replicated by unidirectional forks, and interestingly, the proportion of unidirectional forks further increased in the replicating genome along the population divisions. A second difference concerned in the proportion of paused replication forks, again more frequent in primary fibroblasts than in PBL/lymphoblastoid cells. We concluded that these patterns, whose relevance could escape when genomic methods are applied, represent normal replication features. In single-locus analyses, unidirectional and paused replication forks were highly represented in all genomic regions considered with respect to the average estimates referring to the whole-genome. In addition, fork rates were significantly lower than whole-genome estimates. Instead, when considering the specificities of each genomic region investigated (early to late replication, normal or fragile site) no further differentiating features of replication profiles were detected. These data, representing the integration of genome-wide and single-locus analyses, highlight a large heterogeneity of replication profiles among cell types and within the genome, which should be considered for the correct use of replication datasets.

A protocol for the simultaneous visualization of minor and major satellite DNA by primed in situ DNA synthesis (PRINS) was developed in cytokinesis-blocked murine splenocytes. After individuation of optimal experimental conditions, a micronucleus (MN) test was carried out by treating splenocytes in vitro with the clastogenic agent mitomycin C and the aneugenic compound Colcemid. It was found that PRINS gives highly reproducible results, also comparable with the literature on MN results obtained by fluorescent in situ hybridization (FISH). Therefore the PRINS methodology may be proposed as a fast alternative to FISH for the characterization of induced MN.

The cytokinesis-block micronucleus assay (MN) on murine splenocytes was used for the estimation of chromosome damage in a resting cell population in vivo that can be induced to proliferate in vitro. Mitomycin C at different doses (10(-8), 6 x 10(-8), 10(-7), 6 x 10(-7) and 10(-6)M) was used to induce cytogenetic damage in resting and cycling splenocytes. Antikinetochore antibodies (CREST) and two-colour fluorescence in situ hybridization (FISH) with minor and major satellite DNA were applied. These approaches allowed the detailed characterization of the mechanisms by which MN originates, since it was possible to identify breaks induced in pericentric heterochromatic (resulting in MN containing the major but not the minor satellite DNA) or detachment/disruption of kinetochore (resulting in different frequencies of MN containing kinetochore or both probes). Based on the evidence that resting and cycling mouse splenocytes are characterized by different spatial distribution of centromeric regions, the hypothesis was tested that the damage induced by mutagens at centromeres is influenced by the phase of the cell cycle in which the cells are treated. Data presented here show that resting and cycling splenocytes are both sensitive to mitomycin C action, and indicate that this compound has an aneugenic potential, besides its strong clastogenic activity. In particular, results obtained after CREST and FISH characterization of MN differed when cells were treated during proliferation, suggesting a disruption/detachment of kinetochores induced by mitomycin C at this cell stage. Furthermore, under the same treatment condition the proportion of MN containing the major satellite DNA only was greater than expected on the basis of random breakage at this site. Treatment of resting cells produced aneugenic damage, but without evidence of disruption/detachment of kinetochores or preferential breakage at the centromere. These results indicate that the amount and type of chromosome damage induced by mitomycin C in mouse splenocytes differ in relation to the proliferative status of treated cells.

Background. The fusion protein encoded by the BCR-ABL1 fusion gene may differ in size, but the great majority of chronic myeloid leukemia (CML) patients have a e13a2 (b2a2) or a e14a2 (b3a2) junction. In CML patients treated frontline with imatinib, the e14a2 transcript has been recently associated to faster and deeper molecular responses; in some studies a better outcome has been also reported. Very limited information on the prognostic impact of the BCR-ABL1 transcript type in CML patients treated frontline with second generation tyrosine kinase inhibitors (TKIs) is still available: a study from MDACC reported lower molecular response rates and a trend for inferior event-free survival in e13a2 patients. Aim. To evaluate if the BCR-ABL1 transcript type (e14a2 vs e13a2) affect the response and the clinical outcome in newly diagnosed adult CML patients treated frontline with nilotinib (NIL). Methods. An analysis of 345 CML patients in early chronic phase (ECP) enrolled within 3 multicentric prospective studies of the GIMEMA CML Working Party (ClinicalTrials.gov NCT00481052, NCT00769327, NCT01535391) was performed. The initial treatment was NIL 300 mg BID or NIL 400 mg BID. Definitions: major molecular response (MMR), BCR-ABL1IS ratio < 0.1%; deep molecular response (MR4.0), BCR-ABL1IS ratio < 0.01% with > 10,000 ABL1 copies; progression, transformation to advanced phases; death, at any time and for any reason. Cumulative incidences of response were estimated under consideration of competing risks (progression, death) and compared by Gray test. Progression-free survival (PFS) and overall survival (OS) were estimated using the Kaplan-Meier method and compared by log-rank test. Results. Seven patients expressing rare transcripts (e1a2 or e19a2) and 10 patients with unknown transcript type were excluded: 328 out of 345 patients were evaluable, 124 (38%) with e13a2 transcript, 174 (53%) with e14a2 transcript and 30 (9%) expressing both transcripts. The baseline characteristics of patients with e13a2 or e14a2 transcripts were comparable: no significant differences in age, gender, Sokal or EUTOS long-term survival score distribution, presence of clonal chromosomal abnormalities in Ph+ cells, NIL dose were observed; the only difference was a higher platelet count in patients with e14a2 transcript (median 374 vs 313 x 103/µl, p=0.006). The median follow-up was 60 months in both groups (range 24-82 months). The response rates and the survival probabilities were uniformly lower in patients with e13a2 transcript (N=124) compared to patients with e14a2 transcript (N=174), but the differences were not significant: MMR by 12 months, 66% vs 72%, p=0.244; MR4.0 by 36 months, 56% vs 66%, p=0.067; estimated cumulative incidence of MMR, 82% vs 88%, p=0.135; estimated cumulative incidence of MR4.0, 60% vs 69%, p=0.101; estimated PFS, 88% vs 93%, p=0.547; estimated OS, 89% vs 94%, p=0.436 (Figure 1). The responses and the survival probabilities of patients co-expressing the e13a2 and the e14a2 transcripts (N=30) were similar to or even better than the ones of e14a2 patients. Grouping together the patients with e14a2 transcript alone and the patients with co-expression of both transcripts (N=174+30=204), and comparing them to patients with e13a2 transcript alone (N=124), the response differences became significant (cumulative incidence of MMR and MR4.0, p=0.050 and p=0.036, respectively), but no outcome differences emerged (PFS and OS, p=0.340 and p=0.276, respectively). Conclusions. Despite a trend for lower response rates and inferior outcome in patients with e13a2 transcript, the observed differences were small and mostly not significant. Further studies in larger patient cohorts are required to clarify whether nilotinib and other second generation TKIs are able to overcome the adverse prognostic impact of transcript type, potentially affecting the speed and the depth of molecular response, the probability of achieving a treatment-free remission and the patient outcome. Figure 1 Figure 1. Disclosures Castagnetti: Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; ARIAD Pharmaceuticals: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Gugliotta:Bristol Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Breccia:Ariad: Honoraria; Bristol Myers Squibb: Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Honoraria; Celgene: Honoraria. Carella:Millenium: Speakers Bureau; Genentech: Speakers Bureau. Tiribelli:Ariad Pharmaceuticals: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Speakers Bureau. Bocchia:Janssen: Honoraria; Novartis: Honoraria; Bristol-Myers Squibb: Honoraria. Soverini:Ariad: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy. Cavo:Bristol-Myers Squibb: Consultancy, Honoraria; Millennium: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Martinelli:ARIAD: Consultancy; Roche: Consultancy; MSD: Consultancy; Novartis: Speakers Bureau; Genentech: Consultancy; Amgen: Consultancy; BMS: Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; ARIAD: Consultancy; Roche: Consultancy; MSD: Consultancy; Amgen: Consultancy; Genentech: Consultancy. Rosti:Roche: Honoraria, Research Funding, Speakers Bureau; Incyte: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau.

Median age of CML patients at diagnosis is reported to be around 66 years. Few data about the characteristics and outcome of patients younger than 40 years are available, and the clinical trials of dasatinib and nilotinib as first line treatment were not stratified by age. We retrospectively analyzed 251 young patients with CML in chronic phase from 12 different Italian Institutions, diagnosed between October 2001 and October 2016. Up to 2011 all patients were treated frontline with imatinib while from January 2011 onwards with imatinib or a second generation TKI (nilotinib or dasatinib), based on clinical judgment. At diagnosis median age was 32,6 years [interquartile range (IQR) 27,6- 36,9]; 143 (57%) were male. Splenomegaly was found in 129 out of 233 evaluable patients, in 29,2% of the cases spleen was palpable >5 cm below the costal margin. The risk score was low in most of the cases (low risk Sokal 71%, low risk Eutos 91,3% vs high risk Sokal 9,8%, high risk Eutos 8,7%). Clinical features of the patients in treatment with different inhibitors are summarized in table 1. There were two main statistically significant differences in the group of patients treated with dasatinib: a higher proportion of high risk (30,8%) according to Eutos score and a lower median Hb level at diagnosis (8,5 gr/dL). These features probably reflected the trend of using dasatinib in patients with a more aggressive disease, as this drug was shown to be more potent since the first in vitro studies. Out of 251 patients 179 were treated with imatinib, 57 (22%) with nilotinib, 15 (5,9%) with dasatinib. Median follow up of the whole cohort was 76,5 months (IQR 41- 116); as expected, the follow up was longer in the imatinib group compared to the nilotinib and dasatinib groups (100 months vs 39,6 and 23,0 respectively). In the whole cohort, the cumulative incidence of Complete Cytogenetic Response (CCyR) and Major Molecular Response (MMolR) were 90,4% and 75,7% respectively; a deep molecular response (negative nested PCR, MR4.0, MR4,5) was achieved by 52.2% of patients, without differences among the 3 groups. Primary resistance occurred in 12,4% of patients, without differences among the 3 groups: secondary resistance occurred in 15.9% of patients, with a higher rate in the imatinib group (19,5%) as compared with nilotinib (7,0%) and dasatinib (6,7%) (p=0.047). Treatment discontinuation due to toxicity was observed in 6.0% of patients, without differences among the 3 groups. Blast transformation occurred in 7 out of 251 patients (2,8%), all in the imatinib group, after a median time from the diagnosis of 31 months (range 4-110). The 4-year cumulative Event-Free Survival (EFS) and Overall Survival (OS) were 72,4% (95%CI 66,7 - 78,5) and 98,1% (95%CI 96,4 - 99,8) respectively, without differences among the 3 groups. All deaths were related to blast transformation in imatinib group. In conclusion, as expected in a younger population, response to treatment and OS were excellent. However, the 4-year EFS was lower than expected, in spite of the presence of patients with predominantly low risk score. Furthermore, while the higher rate of secondary resistance in the imatinib group probably reflects the longer follow-up, it is worth of note that no statistically significant difference was observed between imatinib and 2nd generation TKI groups in terms of OS and EFS. Disclosures Breccia: Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Honoraria; Celgene: Honoraria; Ariad: Honoraria; Pfizer: Honoraria.

It is well known that DNA replication affects the stability of several trinucleotide repeats, but whether replication profiles of human loci carrying an expanded repeat differ from those of normal alleles is poorly understood in the endogenous context. We investigated this issue using cell lines from Friedreich’s ataxia patients, homozygous for a GAA-repeat expansion in intron 1 of the Frataxin gene. By interphase, FISH we found that in comparison to the normal Frataxin sequence the replication of expanded alleles is slowed or delayed. According to molecular combing, origins never fired within the normal Frataxin allele. In contrast, in mutant alleles dormant origins are recruited within the gene, causing a switch of the prevalent fork direction through the expanded repeat. Furthermore, a global modification of the replication profile, involving origin choice and a differential distribution of unidirectional forks, was observed in the surrounding 850 kb region. These data provide a wide-view of the interplay of events occurring during replication of genes carrying an expanded repeat., Author Summary The expansion of trinucleotide repeats (TNR) is associated with a large number of human neurodegenerative and neuromuscular diseases, among which the most known are Friedreich's ataxia (GAA/TTC), Huntington's disease (CAG/CTG), and myotonic dystrophy (CTG/CAG). TNR are among the most unstable DNA regions in the genome, and an important step in their expansion is the attainment of a threshold-length. This process occurs during paternal or maternal gametogenesis, leading to an earlier onset of the disease in the next generation. The severity of the disease is strictly related to the TNR length. The repeat instability results from non-B DNA secondary structures formed during DNA replication, repair, recombination and gene transcription. However, the pathways leading to expansion remain poorly understood. Here, we describe the effects of the GAA-expansion on the DNA replication of Frataxin gene. By analyzing the replication profile of mutated and normal genotypes, we have found that the replication of the expanded gene is slowed or delayed in comparison with the non-expanded condition. Interestingly, this observation accords to a global modification of the replication profile affecting the usage of both replication forks and origins, which can be referred as the functional units of any replication program.

In January 1935, at the Teatro Cómico in Madrid, Eduardo Marquina premieres La Dorotea. Comedia en verso, en tres jornadas, inspirada en la famosa obra de Lope de Vega. This study analyzes the Catalan author’s drama and its intertextual relationships with the Fénix’s text, as well as the reception of the play in the context of the tricentenary of Lope’s death., En enero de 1935, en el Teatro Cómico de Madrid, Eduardo Marquina estrena La Dorotea. Comedia en verso, en tres jornadas, inspirada en la famosa obra de Lope de Vega. El presente estudio pretende analizar la pieza del dramaturgo catalán, sus relaciones intertextuales con el texto del Fénix, así como su significado y recepción en el contexto del tricentenario de la muerte de Lope.

Dendritic cells (DCs) are antigen-presenting cells linking innate and adaptive immunity. DC maturation and migration are governed by alterations of cytosolic Ca(2+) concentrations ([Ca(2+)](i)). Ca(2+) entry is in part accomplished by store-operated Ca(2+) (SOC) channels consisting of the membrane pore-forming subunit Orai and the ER Ca(2+) sensing subunit STIM. Moreover, DC functions are under powerful regulation of the phosphatidylinositol-3-kinase (PI3K) pathway, which suppresses proinflammatory cytokine production but supports DC migration. Downstream targets of PI3K include serum- and glucocorticoid-inducible kinase isoform SGK3. The present study explored, whether SGK3 participates in the regulation of [Ca(2+)](i) and Ca(2+)-dependent functions of DCs, such as maturation and migration.Experiments were performed with bone marrow derived DCs from gene targeted mice lacking SGK3 (sgk3(-/-)) and DCs from their wild type littermates (sgk3(+/+)). Maturation, phagocytosis and cytokine production were similar in sgk3(-/-) and sgk3(+/+) DCs. However, SOC entry triggered by intracellular Ca(2+) store depletion with the endosomal Ca(2+) ATPase inhibitor thapsigargin (1 µM) was significantly reduced in sgk3(-/-) compared to sgk3(+/+) DCs. Similarly, bacterial lipopolysaccharide (LPS, 1 µg/ml)- and chemokine CXCL12 (300 ng/ml)- induced increase in [Ca(2+)](i) was impaired in sgk3(-/-) DCs. Moreover, currents through SOC channels were reduced in sgk3(-/-) DCs. STIM2 transcript levels and protein abundance were significantly lower in sgk3(-/-) DCs than in sgk3(+/+) DCs, whereas Orai1, Orai2, STIM1 and TRPC1 transcript levels and/or protein abundance were similar in sgk3-/- and sgk3(+/+) DCs. Migration of both, immature DCs towards CXCL12 and LPS-matured DCs towards CCL21 was reduced in sgk3(-/-) as compared to sgk3(+/+) DCs. Migration of sgk3(+/+) DCs was further sensitive to SOC channel inhibitor 2-APB (50 µM) and to STIM1/STIM2 knock-down.SGK3 contributes to the regulation of store-operated Ca(2+) entry into and migration of dendritic cells, effects at least partially mediated through SGK3-dependent upregulation of STIM2 expression.

Ultrasound investigation is more and more useful instrumental investigation, for early detection of rising signs of bowel occlusion, if compared to conventional abdominal x-ray.A personal collection of 25 intestinal occlusions is reported, comparing the conventional radiographic and ultrasonographic essays.The static x-ray of abdomen didn't reveal adynamic condition in 11/15 patients, confirmed by US supporting clinical examination; 4/25 has been even treated conservatively, completely restored, though radiological diagnosis of occlusion, relying on clinical and sonographic reports. In 9/25 subjects x-ray has not been performed, relying on US only to achieve instrumental diagnosis.The most important advantage of echography, such as dynamic evaluation, allows the study of potential mechanical peristaltic disorders, revealing the stratification of liquid and gas enteric contents, one of the most peculiar sign of intestinal occlusion. Supporting clinical suspect, in the reported collection, it recruited on one hand early surgical solution for the most of them and conservative approach for five patients on the other.

Early identification of spontaneous pneumomediastinum can be achieved by thoracic ultrasound in emergency.A spontaneous pneumomediastinum in a 24 -year old man is reported.The patient, who refused hospitalization, returned 12 hours later, complaining a massive subcutaneous emphysema, with a mild dyspnea. Thoracic CT confirmed suspected pneumomediastinum and the young man has been transferred to specialized DEA, with a good outcome by conservative treatment.Revaluation of the patient has been useful to critically analyze the unusual echographic report too. The major sensitivity and specificity of thoracic US, compared to conventional supine X-ray, is more and more appreciated, regarding occult pneumothoraces. Furthermore the recruitment of training to detect penumomediastinum is needed.Spontaneous pneumomediastinum is an extremely rare benign complications of chronic asthma in young population, caused by increased alveolar pressure, instrumental investigation may be supported by US in emergency too, associated to conventional X-ray and CT.

BACKGROUND: Optimal adherence to imatinib therapy is of paramount importance to maximise treatment effectiveness in patients with chronic myeloid leukaemia (CML). The main objective of this study was to investigate patient-reported personal factors associated with adherence behaviour. METHODS: Analysis was conducted on 413 CML patients receiving long-term therapy with imatinib. Adherence behaviour was measured with the Morisky Medication Adherence Scale and personal factors investigated included: quality of life, perceived social support, fatigue, symptom burden, psychological wellbeing and desire for additional information. Key socio-demographic and treatment-related factors were also taken into account. Univariate and multivariate logistic regression analyses were used to investigate factors associated with optimal adherence to therapy. RESULTS: In all, 53% of patients reported an optimal adherence behaviour. The final multivariate model retained the following variables as independent predictors of optimal adherence to therapy: desire for more information (ref. no), odds ratio (OR)=0.43 (95% confidence interval (CI), 0.29-0.66; P

Tissue transglutaminase (TG2), a multifunctional enzyme implicated in cellular proliferation and differentiation processes, plays a modulatory role in the cell response to stressors. Herein, we used olfactory ensheathing cells (OECs), representing an unusual population of glial cells to promote axonal regeneration and to provide trophic support, as well as to assess whether the effect of some Growth Factors (GFs), NGF, bFGF or GDNF, on TG2 overexpression induced by stress conditions, such as glutamate or lipopolysaccaride (LPS). Glial Fibrillary Acidic Protein (GFAP) and vimentin were used as markers of astroglial differentiation and cytoskeleton component, respectively. Glutamate or LPS treatment induced a particular increase of TG2 expression. A pre-treatment of the cells with the GFs restored the levels of the protein to that of untreated ones. Our results demonstrate that the treatment of OECs with the GFs was able to restore the OECs oxidative status as modified by stress, also counteracting TG2 overexpression. It suggests that, in OECs, TG2 modulation or inhibition induced by GFs might represent a therapeutic target to control the excitotoxicity and/or inflammation, which are involved in several acute and chronic brain diseases.

In vivo methods are described to detect clastogenic and aneugenic effects of chemical agents in male and female germ cells in vivo. The knowledge of stages of germ cell development and their duration for a given test animal is essential for these experiments. Commonly, mice or rats are employed. Structural chromosome aberrations can be analyzed microscopically in mitotic cell divisions of differentiating spermatogonia, zygotes, or early embryos as well as in first meiotic cell divisions of spermatocytes and oocytes. Numerical chromosome aberrations are scorable during second meiotic divisions of spermatocytes and oocytes. The micronucleus test is applicable to early round spermatids and to first cleavage embryos, and as in somatic cells, it assesses structural as well as numerical chromosome aberrations. In contrast to the somatic micronucleus assay, the timing of cell sampling determines whether the micronuclei scored in round spermatids were formed from structural or numerical aberrations, i.e. with short treatment-sampling intervals the micronuclei are formed by exposed meiotic divisions and represent induced non-disjunction. On the -contrary, after longer intervals of 12-14 days micronuclei are formed from induced unstable structural aberrations in differentiating spermatogonia or during the last round of DNA-synthesis in early spermatocytes. Furthermore, labelling with fluorescent DNA-probes can be used to confirm these theoretical expectations. The mouse sperm-FISH assay is totally based on scoring colour spots from individual chromosomes (e.g. X, Y, and 8) hybridized with specific DNA-probes. The most animal demanding assay described here is the dominant lethal test. It is commonly performed with treated male laboratory rodents and allows the determination of the most sensitive developmental stage of spermatogenesis to a particular chemical under test. Theoretically, unstable structural chromosome aberrations in sperm will lead to foetal deaths after fertilization at around the time of implantation in the uterus wall. These can be scored as deciduomata or early dead foetuses in the uterus wall of the females at mid-pregnancy. None of the tests described in this chapter provide data for a quantitative estimate of the genetic risk to progeny from exposed germ cells. The only tests on which such calculations can be based, the heritable translocation assay and the specific locus test, are so animal and time-consuming that they can no more be performed anywhere in the world and thus are not described here.

In vivo methods are described to detect clastogenic and aneugenic effects of chemical agents in male and female germ cells in vivo. The knowledge of stages of germ cell development and their duration for a given test animal is essential for these experiments. Commonly, mice or rats are employed. Structural chromosome aberrations can be analyzed microscopically in mitotic cell divisions of differentiating spermatogonia, zygotes, or early embryos as well as in first meiotic cell divisions of spermatocytes and oocytes. Numerical chromosome aberrations are scorable during second meiotic divisions of spermatocytes and oocytes. The micronucleus test is applicable to early round spermatids and to first cleavage embryos, and as in somatic cells, it assesses structural as well as numerical chromosome aberrations. In contrast to the somatic micronucleus assay, the timing of cell sampling determines whether the micronuclei scored in round spermatids were formed from structural or numerical aberrations, i.e. with short treatment-sampling intervals the micronuclei are formed by exposed meiotic divisions and represent induced non-disjunction. On the -contrary, after longer intervals of 12-14 days micronuclei are formed from induced unstable structural aberrations in differentiating spermatogonia or during the last round of DNA-synthesis in early spermatocytes. Furthermore, labelling with fluorescent DNA-probes can be used to confirm these theoretical expectations. The mouse sperm-FISH assay is totally based on scoring colour spots from individual chromosomes (e.g. X, Y, and 8) hybridized with specific DNA-probes. The most animal demanding assay described here is the dominant lethal test. It is commonly performed with treated male laboratory rodents and allows the determination of the most sensitive developmental stage of spermatogenesis to a particular chemical under test. Theoretically, unstable structural chromosome aberrations in sperm will lead to foetal deaths after fertilization at around the time of implantation in the uterus wall. These can be scored as deciduomata or early dead foetuses in the uterus wall of the females at mid-pregnancy. None of the tests described in this chapter provide data for a quantitative estimate of the genetic risk to progeny from exposed germ cells. The only tests on which such calculations can be based, the heritable translocation assay and the specific locus test, are so animal and time-consuming that they can no more be performed anywhere in the world and thus are not described here.

To focus the attention on critical limb ischemia, high risk condition not for the limb only, but involving the survey of the patient itself.Etiology, diagnosis and therapy of arterial occlusive critical pathology (AOCP). Genetics and life style prophylaxis are shortly treated.AOCP is often asymptomatic pathology, underestimated by physicians themselves, though long-term death rate is the same between subjects affected by occlusive arteriopathy and cardiological failure.Interventional therapy, ranging from endovascular procedures (PTCA, stenting) to open surgery (by-passes, TEA, prostheses), remains the only effective weapon against critical acute limb ischemia, offering excellent revascularization rates (80-90%). Conservative treatment, comprehending thrombolysis, prostanoids and anticoagulant drugs recruit the role of invasive methods or are limited, alone, to ineligible patients. Genetics is still far from certain and durable results.AOCP is a long-term severe condition; multifactoriality and asymptomatology in a great number of patients, often justify misdiagnosed cases. In emergency the acute ischemia involves also demolitive surgery, with short and long-term poor prognosis.

To highlight dasatinib role in the elderly, 125 unselected patients with CP-CML aged >60 years resistant/intolerant to imatinib were retrospectively evaluated. Grade 3-4 haematological and extra-haematological toxicities were reported in 39 (31.2%) and 34 (27.2%) patients; grade 3-4 haematological toxicity was higher in patients with 140 mg starting dose (50.0% vs 19.6%, p=0.001). Grade 3-4 pleuro-pericardial effusions occurred in 10 patients (8.0%). Dose reductions were more common in patients with 140 mg (88.4% vs 26.7%, p

Abstract 2751 Comorbidities have been identified as significant determinants of response to therapy in elderly patients with acute myeloid leukemia, breast cancer, head and neck, and lung cancer. Charlson comorbidity index (CCI) is a list of comorbidities with a weight assigned from 1 to 6 derived from relative risk estimates of a proportional hazard regression model using clinical data. We applied CCI stratification on a large cohort of chronic myeloid leukemia (CML) very elderly patients (> 75 years) treated with imatinib, in order to observe the impact of concomitant diseases on both compliance and outcome. One hundred and eighty-one patients were recruited by 21 Italian centers. There were 95 males and 86 females, median age 78.6 years (range 75–93.6). According to Sokal score, 106 patients were classified as intermediate risk and 55 as high risk (not determined in 20 patients). According to CCI stratification, 71 patients were score 0, 50 patients had a score 1, 37 patients had score 2 and 23 patients had score ≥ 3. Imatinib standard dose was reduced in 68 patients independently from the evaluation of baseline comorbidities but based only on physician judgement: 43.6% of patients with score 0 started with a reduced dose (200–300 mg/day) compared to more than 50% of patients with score ≥ 3. No significant differences were found in terms of further reduction of the dose (39% in patients with score 0 compared to 21% in patients with score ≥ 3) or in terms of discontinuation due to toxicity (58% in patients with score 0 vs 48% in patients with score ≥ 3). We did not find significant differences as regards the occurrence of hematologic side effects, probably due to the initial reduction of the dose: 39% of patients with score 0 experienced grade 3/4 hematologic toxicity (most commonly anemia) compared to 35% of patients with score ≥ 3. Independently from the initial dose, comorbidities again did not have an impact on development of grade 3/4 non-hematologic side effects (most common skin rash, muscle cramps and fluid retention): 62% of patients with score 0 compared to 35% of patients with score ≥ 3. Notwithstanding the reduced dose and the weight of comorbidities we did not find differences in terms of efficacy: 66% of patients with score 0 achieved a CCyR compared to 56.5% of patients with score ≥ 3. Comorbidities appeared to have an impact on EFS (34 months for patients with score 0 vs 23.5 months for patients with score ≥ 3) and influenced the median OS (40.8 months for patients with score 0 vs 10.6 months for patients with score ≥ 3). Our results suggested that treatment of very elderly CML patients might be influenced by personal physician perception: evaluation at baseline of comorbidities according to CCI should improve initial decision-making in this subset of patients. Disclosures: Russo Rossi: Novartis: Honoraria; Bristol Myers Squibb: Honoraria. Rosti:Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Research Funding; Novartis: Honoraria; Bristol Myers Squibb: Honoraria. Alimena:Novartis: Honoraria; Bristol Myers Squibb: Honoraria.

In this study, we confirm the validity of the proposed Hammersmith score, which identifies three risk categories of patients and establish its strength on a large group of 128 chronic myeloid leukemia patients treated with second-generation tyrosine kinase inhibitors (TKIs) after being resistant to imatinib. Sixty-one patients were identified as good risk group, 27 patients as intermediate risk group, and 40 patients as poor risk group. The 1-year cumulative incidence of complete cytogenetic response was 73% in good risk patients, 40% in intermediate risk patients, and 22% in poor risk patients (P = 0.0001). Event-free survival at 3-year was 89% in good risk group, 70% in intermediate group, and 54% in poor risk group (P = 0.0001); the estimated 3-year progression-free survival was 95% in good risk category, 93% in intermediate risk category, and 87% in poor risk category (P=0.05). Kaplan-Meier estimated that the 3-year overall survival was 100% in good risk category, 93% in intermediate risk category, and 82% in poor risk category (P=0.04). In conclusion, some prognostic factors before starting second-generation TKIs might predict cytogenetic response and outcome. The so-called Hammersmith score was not yet validated in large series of patients: we demonstrated that this score is able to discriminate patients at high risk of failure and consequent progression before treatment with second-generation TKIs.

Abstract 4445 Imatinib advent has provided for old CML patients too a chance for an effective treatment, aimed at complete cytogenetic and major molecular response. Indeed, a few studies reported in patients more than 65 year old a response rate comparable to that observed in younger ones. However, fluid retention, a common side effect of imatinib therapy, seems to occur more frequently in elderly patients. Indeed, periorbital and ankle oedema are common patients’ complaints, whereas pleural effusions have not been usually observed in imatinib-treated patients. Conversely, pleural effusions (usually mild to moderate) occur in about 14% of patients treated with the second generation tyrosine-kynase inhibitor (TKI) dasatinib at the optimal dosage of 100 mg by single daily administration. We conducted a retrospective survey of 181 Italian CML patients, above the age of 75, treated with imatinib for chronic phase CML. Among sixty-five patients who were more than 80 year old we observed 5 cases (7.7%) who displayed a severe (grade 3: 4 patients) or moderate (grade 2: 1 patient) pleural effusion. Conversely, such a side effect was not observed in any of the 101 slightly younger (75–80 year old) patients. The 5 patients displaying a pleural effusion were all males, 80.3 – 88.7 year old at the time of imatinib start. One patient was in late chronic phase, and had received hydroxyurea for more than 2 years before imatinib start, whereas the other 4 started imatinib therapy within 2 months from diagnosis. All 5 patients had at least one cardiovascular co-morbidity. Pleural effusion developed after 3–8 months of imatinib therapy. Pericardial effusion and peripheral oedema were also evident in two and one of the 5 patients, respectively. Imatinib was definitely discontinued in 2 patients (one of them had cytogenetically resistant disease) whereas the drug could be resumed, after a few months of interruption, in the other 3, in one case after intolerance to second generation TKI nilotinib. Three patients died: two of myocardial infarction and one for CML blastic phase at 45, 31 and 54 months, respectively, from diagnosis. Two patients are alive, one in major cytogenetic response with resumed imatinib treatment, at 12 and 30 months from diagnosis. Our survey evidenced a significant percentage of very old patients who developed a pleural effusion during imatinib treatment. Although our casistic of patients with pleural effusion is small, some differences are evident, compared to the more common dasatinib-induced pleural effusions. In the case of dasatinib, most of pleural effusions are mild, unrelated to peripheral oedema, and may have an immune-related aetiology. In our imatinib-treated patients pleural effusions were more severe and frequently accompanied by pericardial involvement. Moreover, they were restricted to very old patients and possibly correlated to cardio-vascular co-morbidities. In conclusion, although imatinib still represents the best CML treatment for most of very old patients too, these should be strictly monitored for fluid retention and possible appearance of a pleural and or pericardial effusion. Disclosures: Russo Rossi: Novartis: Honoraria; Bristol Myers Squibb: Honoraria. Rosti:Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Research Funding; Novartis: Honoraria; Bristol Myers Squibb: Honoraria.

The frequency of sister-chromatid exchanges (SCE) was evaluated in mouse bone marrow cells at different time intervals (from 19 h to 10 days) after treatment i.p. with mitomycin C (MMC; 1 and 2 mg/kg body weight). Significantly higher frequencies of SCE were found during the first week after treatment, at both doses tested. This result confirms that chromosomal lesions induced by MMC in the mouse may persist in bone marrow cells, in agreement with previous evidence based on chromosomal aberration analysis in the same cell population. In addition, the observation of a unimodal distribution of SCE/cell frequencies at each time tested indicates that the bone marrow cell population on the whole is affected by increased SCE frequency, i.e., that persistent chromosomal lesions may be transmitted along with cell proliferation.

The frequency of micronuclei (MN) in mouse peripheral blood reticulocytes supravitally stained with acridine orange was assessed at different time intervals after treatment in vivo with two dose levels of mitomycin C (MMC) and benzo[a]pyrene. Increased frequencies were observed for many days after treatment, indicating that MN may be produced as a consequence of chromosomal instability transmitted by proliferating erythroblasts. These results confirm previous evidence of the persistent cytogenetic effects of MMC and suggest that clastogenic agents acting by different primary lesions may have a similar ability to induce this effect.

Background Trigeminal proprioception related to rodent macrovibrissae movements is believed to involve skin receptors on the whisker pad because pad muscles operate without muscle spindles. This study was aimed to investigate in rats whether the trigeminal mesencephalic nucleus (TMnu), which provides proprioceptive feedback for chewing muscles, may be also involved in whisker pad proprioception. Methods Two retrograde tracers, Dil and True Blue Chloride, were injected into the mystacial pad and the masseter muscle on the same side of deeply anesthetized rats to label the respective projecting sensory neurons. This double-labeling technique was used to assess the co-innervation of both structures by the trigeminal mesencephalic nucleus (TMnu). In a separate group of anesthetized animals, the spontaneous electrical activities of TMnu neurons were analyzed by extracellular recordings during spontaneous movements of the macrovibrissae. Mesencephalic neurons (TMne) were previously identified by their responses to masseter muscle stretching. Changes in TMne spontaneous electrical activities, analyzed under baseline conditions and during whisking movements, were statistically evaluated using Student's t-test for paired observations. Results Neuroanatomical experiments revealed different subpopulations of trigeminal mesencephalic neurons: i) those innervating the neuromuscular spindles of the masseter muscle, ii) those innervating the mystacial pad, and iii) those innervating both structures. Extracellular recordings made during spontaneous movements of the macrovibrisae showed that whisking neurons similar to those observed in the trigeminal ganglion were located in the TMnu. These neurons had different patterns of activation, which were dependent on the type of spontaneous macrovibrissae movement. In particular, their spiking activity tonically increased during fan-like movements of the vibrissae and showed phasic bursting during rhythmic whisking. Furthermore, the same neurons may also respond to masseter muscle stretch. Conclusions results strongly support the hypothesis that the TMnu also contains first-order neurons specialized for relaying spatial information related to whisker movement and location to trigeminal-cortical pathways. In fact, the TMnu projects to second-order trigeminal neurons, thus allowing the rat brain to deduce higher-order information regarding executed movements of the vibrissae by combining touch information carried by trigeminal ganglion neurons with proprioceptive information carried by mesencephalic neurons.

To emphasize the role of clinical suspect and echographic approach to heterotopic pregnancy in young women complaining of acute abdominal pain.Tubal pregnancy in a young woman has been reported. Diagnostic clinical suspect, also supported by serum hormonal substances (beta HCG), has been confirmed by US in urgency; transabdominal investigation requires skilled operator, preferably surgically trained, to identify early study of heterotopic pregnancy (fifth week of gestation), which has undergone left salpingectomy in our experience.The early diagnosis of unruptured tubal pregnancy in our experience prevented hemorragic shock and other potential fatal complications, usually occurring in this condition.Ectopic pregnancy is a life threatening condition, rarely occurring during natural cycle; however its incidence is increasing, considering the wider and wider use of assisted reproductive techniques and of gynecological surgical diagnostic and therapeutic procedures. The rupture of ectopic part causes acute abdomen and potentially fatal hemorrhage, whose only solution relies on surgical operation in urgency.Clinical and anamnestic evaluation, ultrasonography and serum gonadotropin concentration are the first choice approach to fertile women suffering from abdominal recurrent pain, when no other causes are suspected. The heterotopic tubal pregnancy described is a very rare condition; in spite of early diagnosis, radical surgery consisting in left salpingectomy has been the only surgical feasible option, patient's life-sparing.

Polytrauma, or the condition of victim affected by two or more lesions compromising vital parameters, is the third cause of death (0.6/l1000/year), and the first one of mortality for subjects 44 year-younger, responsible of 26000 annual deaths, (60% for car accidents).A personal experience concerning four car crash victims contemporary accepted in our Surgical Emergency of a peripheric hospital and an overview of clinical and therapeutical aspects of polytrauma are reported.Topographic distribution of deaths shows this patterns: during the transport to hospital: 4%; peripheric hospital: 10%; Emergency Departments: 13%; during hospitalization: 25%; Intensive Care Units: 46%. The outcome is strictly depending on the quality of the first approach, confirming the importance of golden hour.The complexity of polytrauma is related to the various pathologies affecting the victims, either neurological ones, or thoracic, abdominal, vascular and orthopedic ones, emphasizing the role of diagnostic and therapeutic timely approach.

Abstract 2294 Background. The TKIs Nilotinib and Dasatinib offer additional therapeutic options for patients with CML who are resistant or intolerant to Imatinib. These agents, active against the majority of Imatinib resistant BCR-ABL mutated clones, have a different pattern of kinase target selectivity, pharmacokinetics parameters, cell uptake, efflux properties and adverse events profiles. Preliminary results suggest that some patients may respond to a second TKI used as third line therapy, but little is known about the long term benefit of such an approach.Aim of this collaborative Italian study was to verify the response (rate and duration) and the clinical outcome in patients with CML treated with a third TKI after sequential failure of the previous ones. Methods. We evaluated 66 patients with CML, resistant/intolerant to Imatinib and treated with Dasatinib or Nilotinib, then switched to a third- line TKI after treatment failure. Of these, 29 patients were treated with dasatinib after imatinib/nilotinib failure and 37 with nilotinib after imatinib/dasatinib failure. Patients were monitored with complete blood counts, cytogenetic analysis, bone marrow aspiration RT-PCR and mutational analysis. Results. A total of 66 patients (median age 63 years, range, 33–85 years) were treated with sequential TKIs; 40 (61%) patients had received interferon-a before starting Imatinib; 26 (39%) patients received imatinib as first line therapy. The median time on imatinib therapy was 47.5 months (range 4–101 months). At the start of nilotinib as second line, 27/29 (93%) patients were in CP, 1 (3.5%) in AP, and 1 (3.5%) in BP. 9 patients (31%) had developed mutations before starting treatment. The median time on second line TKI was 8 months (range 2–36 months). In the resistant patients 4 new mutations were identified (F359V in two patients, T315I, Y253H+F359V). At the start of dasatinib as second line, 33/37 (89.2%) patients were in CP, 4 (10.8%) in AP. 7 patients (18.9%) had developed mutations before starting treatment. The median time on second line TKI was 14 months (range 4–59 months).In the resistant patients 5 new mutations were identified (F137L in three pts, M318T, M244V+F317L). At the start of the third TKI, 60/66 (90.9%) patients were in CP, 5 (7.6%) in AP, and 1 (1.5%) in BP. Of these, 7 patients (18.9%) on dasatinib and 7 (24.1%) on nilotinib had mutations before starting treatment. The best response to the third line treatment with TKI was 10 (15.2%) MMR, 10 (15.2%) CCyR, 8 PcyR (12.1%), 5 (7.5%) mCyR, 24 (36.4%) CHR and 9 (13.6%) No Response (NR). In the dasatinib group, 9 (31%) patients discontinued treatment because of toxicity versus 17 (45.9%) patients in the nilotinib group.Two new mutations (F317L, E255V) emerged with dasatinib as third line therapy.After a median follow up of 13 months (range 2–37 months) 50 patients (48 CP, 2 AP) are continuing therapy (33 on nilotinib, 17 on dasatinib).Since the start of the third TKI, 61 patients (92.4%) are still alive for a median overall survival of 110 months (range 15–300) (52 CP, 7 AP, 2 NA); the 5 deaths (7.6%) were caused by disease progression and spread of the gene mutation T315I. Discussion. In our study, about one third of patients derived benefit from the use of three sequential TKIs; patients with better, longer response (28.7%) to third TKI were the same patients with a better response to the Imatinib and 2TKIs therapy. All these patients had taken interferon therapy before the Imatinib. In this subset of patients (good responders: CCyR and MMR) 5 patients developed mutations that were sensitive to the sequential treatment.The lack of a durable cytogenetic remission could be explained by the emergence of new kinase domain mutations as patients are exposed to sequential TKI; a change of therapy resulted in an adequate response. In our series, patients with poor prognosis showed mutations not sensitive to the TKIs treatment. Conclusions. Although allogeneic SCT is the treatment of choice in all patients failing 2 TKIs who are suitable candidates for this approach, alternative strategies are required for ineligible patients. The use of a third TKI after failure of two previous TKIs induces response in some patients. Longer follow up of a larger series of patients is needed to determine the long term impact of the response. Disclosures: No relevant conflicts of interest to declare.

The replication dynamics at common fragile site FRA6E has been evaluated by molecular combing and interphase fluorescent in situ hybridisation (FISH) in primary human lymphocytes cultured under normal or aphidicolin-induced stress conditions. FRA6E is one of the most frequently expressed common fragile sites of the human genome. It harbours several genes, PARK2 being regarded as the most relevant one. According to the results obtained from interphase FISH analysis, FRA6E can be considered a mid-late-replicating sequence characterised by heterogeneous replication timing. Molecular combing did not reveal specific replication parameters at the fragile site: fork rates were highly comparable to those detected at an early replicating locus (LMNB2) used as control and in very good agreement with the whole-genome data obtained in parallel. The same indication applied to the density of initiation zones, the inter-origin distances from adjacent ongoing forks, the frequencies of unidirectional forks, fork arrest events and asynchronous forks. Interestingly, PARK2 appeared embedded in an early/late replication transition zone, corresponding to intron 8 (162 kb) and to the fragility core of FRA6E. In cells exposed to aphidicolin, few forks progressing at a rather slow rate were observed, the majority of them being unidirectional, but again a specific response of the fragile site was not observed. In summary, at FRA6E the replication process is not impaired per se, but chromosome breakages occur preferentially at an early/late replication transition zone. Aphidicolin might increase the occurrence of breakage events at FRA6E by prolonging the time interval separating the replication of early and late replication domains. These results may be of general significance to address the problem of fragile site instability.