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Background:In several malignancies, gender-based survival differences after specific therapeutic interventions have been demonstrated. It is not known whether such differences exist after plaque brachytherapy of uveal melanoma. Methods:All patients who received brachytherapy for uveal melanoma at St. Erik Eye Hospital from November 1, 1979 through November 20, 2017 were included (n= 1,541). Retrospective data were retrieved including baseline patient and tumor characteristics, brachytherapy nuclide (ruthenium-106 or iodine-125), radiation dose, treatment duration, tumor relapses, date of metastasis, and cause of death. Results:A total of 775 men and 766 women were treated with plaque brachytherapy. There were no significant differences between the genders in baseline characteristics, treatment, or follow-up. Men and women had similar rates of tumor relapses, hazard for repeated brachytherapy (men vs. women 0.8, p= 0.47), enucleation-free survival, and survival after detection of metastasis. Five-, 10-, and 15-year melanoma-related mortality was 14, 24, and 27% for men and 15, 26, and 32% for women, respectively. There were no significant differences in hazard for melanoma-related mortality (men vs. women 0.9, p= 0.32), median Kaplan-Meier disease-specific survival (men 18.2 years, women 15.5 years, p= 0.22), or median overall survival (men 13.5 years, women 12.6 years, p= 0.60). Conclusion:There are no relevant differences between men and women in ocular or patient survival after brachytherapy for uveal melanoma.

The aim of this study is to assess the clinical burden of silent coronary artery disease (CAD) in cirrhotic candidates for liver transplantation (LT), and to evaluate the usefulness of a CAD screening approach. Between July 1999 and January 2006, we evaluated 627 LT candidates. All of them underwent a detailed clinical history. Sixteen had a previous diagnosis of CAD or symptoms suggestive (2.5%). The remaining 611 underwent further tests according to a predefined protocol, including EKG, echocardiogram and, on the basis of CAD risk factors, heart stress tests. Selective coronary angiography (SCA) was performed in the 30 patients with positive heart stress test: in only 2 did SCA show any CAD, and in both it was subcritical disease requiring neither intervention nor contraindicating LT. The 611 screened patients continued their follow-up until study closure or death. No coronary events occurred in the study population in a mean follow-up of 32.50 months (+/−23.67 DS). No perioperative mortality related to CAD occurred in the 233 transplanted patients. In conclusion, no prognostic advantage was achieved by following a strict CAD screening protocol, leading us to believe that the cost-effectiveness of a similar screening can be unacceptably high in our setting.

Al fine di ampliare le possibilità di rotazione nell'uso di molecole erbicide si è voluto saggiare l'efficacia di azione di una nuova molecola, il pinoxaden, rispetto a quella di altri erbicidi comunemente impiegati per il diserbo del frumento duro.

Introduction:Decitabine has been recently approved in Europe for treatment of AML patients (pts) aged more than 65 years and unfit to receive standard chemotherapy. However data on its efficacy and tolerability derive mainly from clinical trials performed in selected pts. Herein we report on a population based series of AML pts treated with decitabine and registered in observational prospective studies.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Martinelli: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.

Myeloproliferative diseases (MPDs) are a group of haematological disorders characterized by the hyper proliferation of different blood cells in peripheral blood and other hematopoietic organs. The clinical heterogenity of these neoplasms reflects the different gene pathways involved, most of which are only partially known. Given the genetic homology and the physiological similarity to mammals, zebrafish has emerged as an ideal model to study human normal and malignant haematopoiesis. In the last decade several oncogenes involved in the development of hematopoietic neoplasms have been used to model leukemia in zebrafish with the aim to discovery new molecular pathways involved in malignant transformation. Despite the first encouraging results these experimental models failed to fully recapitulate human myeloproliferative disorders.We took advantage of the Gal4/UAS binary system to induce the expression of human oncogenic HRASV12G in the zebrafish hematopoietic compartment. We used a specific transgenic line that drives oncogene expression in zebrafish early hematopoietic progenitors under control of the FLI.1 (Friend Leukemia virus Integration 1) promoter.We observed the development of a myelo-erythroid proliferative disease in few days in zebrafish transgenic larva. The pathological phenotype is characterized by the expansion of the hematopoietic tissue, an increased expression of myelo-erythroid specific genes (PU.1, gata1, mpx, c-mpl) associated with a slight increase of staminality markers (lmo2, scl, c-myb, runx.1), and a higher number of l-plastin expressing cells. Moreover blood smear of pathological larva displayed leukemic blasts and the arrest of erythrocyte differentiation whereas kidney marrow of juvenile fish displayed abnormal myelopoiesis characterized by the increase of erythro-myeloid progenitors.We found that the pathological phenotype is associated with a down regulation of the Notch pathway as shown by the decreased gene expression of notch pathways target genes (notch1, notch3, her6). Furthermore we discovered a novel set of genes involved in neoplastic transformation induced by HRASV12 expression through RNA-Seq analysis of pathological larva.The expansion of the zebrafish hematopoietic compartment characterized by the hyper-proliferation of the myelo-erythroid progenitors that we found in this model reproduces some of the pathological features of human myeloproliferative disorders. This study showed that forcing oncogene expression in the hemogenic endothelial cells induces the transdifferentiation of the early hemogenic pluripotent stem cells into abnormal myeloerythoid progenitors by repressing the Notch pathways. Transcriptome analysis identified a number of potential effectors of this transformation.No relevant conflicts of interest to declare.

Azacitidine (AZA) is a DNA methyltransferase inhibitor currently approved for the treatment of high-risk MDS patients, which has been demonstrated to be feasible and effective also in low-risk MDS (Fenaux P et al, Lancet Oncol 2009; Musto P et al, Cancer 2010). However, at least 4 or 6 cycles of therapy are required for assessing the hematologic response, and predictive markers of responsiveness are still lacking. PI-PLCbeta1 plays a role in the MDS progression to AML and is a specific target for AZA therapy (Follo MY et al, PNAS 2009). Indeed, PI-PLCbeta1 has been demonstrated to be a dynamic marker for responsiveness to demethylating therapy, in that PI-PLCbeta1 mRNA increase or decrease could be associated with favourable response or failure, respectively. Stemming from these data, in this study we further investigated the role of PI-PLCbeta1 in MDS patients during AZA therapy.The study included 60 patients, 22 low-risk MDS (WHO: RA, RARS, RCMD, RAEB-1, and IPSS risk Low or Int-1), and 38 high-risk MDS (WHO: RCMD, RAEB-1, RAEB-2, and IPSS risk Int-1 or High). All the patients received a minimum of 6 cycles, in the absence of disease progression or unacceptable toxicity. Hematologic response was defined according to the revised IWG criteria (Cheson et al, Blood 2006). Positive clinical responses were defined as: Complete Remission (CR), Partial Remission (PR) or Hematologic Improvement (HI). At a molecular level, for each patient we quantified the amount of PI-PLCbeta1 mRNA at baseline and before each cycle of AZA therapy. PI-PLCbeta1 ratio was calculated as the mean expression of PI-PLCbeta1 at cycles 1 to 3, as compared with the baseline level within the same subject. In case the mean value of PI-PLCbeta1 gene expression during the cycles 1 to 3 was above the baseline level, we defined it as a “PI-PLCbeta1 early increase”. On the contrary, a “stable PI-PLCbeta1” expression was observed when subjects did not show any increase during the first three cycles of therapy, as compared with baseline.Patients' median age was 69 years (range 37–85) and the median follow-up was 23 months (range 1–103). The median number of AZA cycles was 11 (range 3–59) for high-risk MDS, and 8 (range 1–8) for low-risk MDS. Positive clinical responses were observed in 37/60 (62%) of the MDS patients (7 CR, 1 PR, 29 HI). In particular, 13/22 (59%) of our low-risk MDS and 24/38 (63%) of our high-risk MDS patients showed a positive clinical response to AZA, with 4 CR, 1 PR, and 19 HI in high-risk MDS, and 3 CR and 10 HI in low-risk MDS. Overall survival (OS), Progression-Free Survival (PFS), and Overall Response Rate (ORR) were analyzed using a Kaplan-Meier method, considering p-values<0.05 as statistically significant. No differences in OS nor in PFS were noted between patients with early increased or stable PI-PLCbeta1 (OS: 36 vs. 30 months, p=0.45; PFS: 28 vs. 24 months, p=0.06). However, PI-PLCbeta1 early increase was significantly associated with ORR (increase: 25/38 (65%) vs. stable: 4/22 (18%); p<0.05). The predictive value of PI-PLCbeta1 was also analyzed: PI-PLCbeta1 early increase was significantly associated with duration of AZA response (increase vs. stable: 26 vs. 12 months; p<0.05), showing that an early increase of PI-PLCbeta1 was associated not only with a positive clinical response, but also with a higher probability of a longer response.Taken together, our data confirm the role of PI-PLCbeta1 as a dynamic marker of response to AZA and show that the detection of an increase in PI-PLCbeta1 gene expression within the first three cycles of AZA therapy is associated with a better clinical outcome and a longer hematological response. Further analyses are needed to confirm in a larger group of patients the predictive role of PI-PLCbeta1 mRNA detection during AZA therapy.No relevant conflicts of interest to declare.

β-herpesviruses, such as CMV and HHV6, are important pathogen in transplanted patients. The morbidity because of CMV reactivation after allogeneic stem cell transplantation (SCT) has led to the monitoring of this virus and to introduction of preemptive therapy. However, CMV infection is still one of the most challenging complications, because CMV disease may occur as life-threatening pneumonitis, and may increase the risk of opportunistic infections. HHV6 reactivation has been demonstrated after SCT and this virus is recognized as important pathogen, either by direct infection or via interaction with CMV. Innate and adaptive immune response against these viruses involves the activation of Toll-like receptors (TLRs). TLRs belong to type I transmembrane glycoprotein receptor family and recognize pathogen-associated molecular patterns (PAMPs). Viral nucleic acids and viral structural proteins, such as glycoproteins, are considered as PAMPs. Endosomal TLRs (TLR3, 7, 8 and 9) recognize viral nucleic acids and some surface TLRs may be involved in the detection of structural proteins. Some clinical and experimental evidences indicate that CMV and HHV-6 can modulate the immune system and influence the immune reconstitution after SCT. However, the role of TLRs in this complex interplay remains unclear, especially in the setting of allogeneic SCT.The aim of this study was to evaluate the expression of TLRs on lymphocytes and monocytes in relation to CMV and HHV6 reactivation in the early period after allogeneic SCT.CMV and HHV6 reactivation was monitored weekly by quantitative real-time PCR until the second month after SCT. The expression of TLRs on lymphocytes and monocytes was analysed by flow cytometry as mean fluorescence intensity at day +30 and in any case before CMV or HHV6 reactivation. Functional data were obtained by ELISA assay after TLRs activation. The cell supernatants were collected and assayed for TNF-alpha, IFN-gamma and MCP-1. Relative induction of these cytokines was calculated in relation with unstimulated controls.CMV reactivation within 2 months after transplantation was observed in 13 out of 33 patients. CMV pneumonitis was observed in 1 patient. HHV-6 reactivation was detected in 1 patient. Median age was 45 years (range, 22–64) and 21 patients were male. TLRs expression and function did not significantly differ in controls and patients without CMV. Lymphocytes of patients with CMV reactivation showed an increased expression of TLR5 (4,1±2,4 vs 2,0±1,7 p=0,008). TLR8 expression was lower on monocytes with CMV reactivation (0,8±0,9 vs 2,0±1,7 p=0,03). MCP-1 relative induction post-stimulation of TLR1 and 8 was significantly decreased in patients with CMV reactivation (p<0,04).Surface TLR2 and intracellular TLR3 and 9 are reported to recognize CMV by some authors. In our study, surface TLR5 and intracellular TLR8 seem to be involved in the interaction between CMV and the immune system of transplanted patients. In particular, TLR8 could play a protective role. MCP-1 production upon TLR1 and 8 activation negatively correlates with CMV reactivation. The defective immune system after SCT could explain these results, which could be confirmed by the assessment of a larger number of patients and the analysis of other possible interfering factors.No relevant conflicts of interest to declare.

Emerging trends emphasize the importance of both innate and adaptive immune system in the response against infections, and in the pathogenesis of autoimmune and graft-versus-host (GVHD) diseases. Pattern recognition receptors such as Toll-like receptors (TLRs) play a key role in the cross-talk between innate and adaptive immune system. TLRs belong to type I transmembrane glycoprotein receptor family and recognize pathogen-associated molecular patterns (PAMPs), such as common protein, carbohydrate or DNA/RNA pattern motifs. TLRs are also receptors for endogenous ligands and damaged tissue, suggesting that both pathogen-derived molecules and products of damaged tissue can trigger signals which are responsible for the regulation of innate and adaptive immune responses. Extracellular ligands are recognized by surface TLRs (TLR1,TLR2,TLR4,TLR5, and TLR6). Intracellular TLRs (TLR3,TLR7,TLR8 and TLR9) bind mainly to foreign nucleic acids and sometimes detect self DNA/RNA.Very little is known about expression and function of TLRs in vivo in patients who underwent allogeneic stem cell transplantation (SCT). The aim of this study was to evaluate the expression of TLRs on lymphocytes and monocytes in relation to the onset of acute GVHD.The expression of TLRs on lymphocytes and monocytes was analysed by flow cytometry as mean fluorescence intensity at day +30 and at the onset of GVHD. Functional data were obtained by ELISA assay after TLRs activation. The cell supernatants were collected and assayed for TNF-alpha, IFN-gamma and MCP-1. Relative induction of these cytokines was calculated in relation with unstimulated controls.We analyzed 17 healthy donors and 34 patients. Median age was 46 years (range, 22–64) and 22 patients were male. Acute GVHD developed in 19 patients (12 with grade >=2). Clinical and transplant characteristics did not differ in patients with and without GVHD. Lymphocytes and monocytes of patients with acute GVHD showed higher levels of TLR5 (3,5±2,3 vs1,9±1,6 p=0,03; 25,8±25,9 vs 9,0±5,0 p=0,02) and a decreased expression of TLR1 (2,5±2,8 vs 4,3±2,8 p=0,02; 21,4±21,9 vs 54,9±37,4 p=0,005) and TLR9 (63,8±30,4 vs 111,1±62,9 p=0,03; 85,3±73,9 vs 164,2±90,6 p=0,01). IFN-gamma relative induction post-stimulation of TLR2,3,4 and 9 was significantly decreased in patients with acute GVHD (p< 0,04).TLRs show a different profile of expression in patients with acute GVHD in comparison with patients without it. These results suggest that the innate immune response via TLRs activation could be involved in the development of GVHD. In particular, a decreased expression of TLR-9 (receptor of hypomethylated DNA) on lymphocytes and monocytes can promote TLR-7 activation, inducing type I interferons and other pro-inflammatory cytokines. TLR-1 and −5, which are ligands for bacterial cell wall, could also be involved in the pathogenesis of GVHD. Moreover, acute GVHD negatively correlates with IFN-gamma production upon TLR2,3,4 and 9 activation. The assessment of a larger number of patients could be useful to understand the complex interplay among pathogens, self or non-self DNA and RNA, and the immune system.No relevant conflicts of interest to declare.

Abstract 4711

Abstract 4704

No relevant conflicts of interest to declare.

Azacitidine (AZA) at a dose of 75 mg/mq/day subcutaneously for 7 days, every 28 days, induces high hematologic response rates and prolongation of survival in high-risk MDS patients (pts) (Fenaux, 2009). However few data are hitherto available concerning the efficacy and safety of Aza in lower risk MDS. A lower dose regimen, AZA 5 (75 mg/mq daily, subcutaneously, for 5 consecutive days every 4 weeks) have shown to induce response rates consistent with the currently approved schedule (Lyons, 2009), however in this study pts were not classified according to IPSS risk.The use of AZA in the earlier phases of disease could be more effective and useful to control the expansion of MDS clone and disease progression. In our phase II, prospective, multicentric trial, AZA 5 regimen was administered to IPSS low-or-intermediate-1 risk pts, for a total of 8 courses, in order to evaluate its efficacy and safety. Furthermore pharmacogenomic studies (GEP, SNP) cytokine network and PI-PLC-beta1 methylation and gene expression, before and at the end of 4th and 8th course of Aza treatment, were planned to identify new biological markers to predict the response.From September 2008 to February 2010, 34 patients (24 males, 10 females), with a median age of 71 (56-84) yrs, with symptomatic transfusion-dependent anemia, previously unresponsive to erythropoietin (EPO) or not expected to respond to EPO, or with severe neutropenia or thrombocytopenia, were enrolled into the study. According to WHO classification, 15 pts had RA, 6 RARS, 7 RCMD and 6 RAEB-1.At present time 30/34 pts are evaluable: 23/30 pts (77%) completed the treatment plan (8 courses), 3/30 pts (10%) are ongoing and 4/30 (13%) died during the treatment period. According to the 2006 International Working Group criteria, overall response rate (ORR) was 60,9 % (14/23 pts): 5 pts (21,7%) achieved complete remission (CR), while 9 pts (39,1%) showed an hematologic improvement (HI) (7 erythroid responses, 1 erythroid/platelet response and 1 neutrophil/platelet response). 9/23 pts (39%) maintained a stable disease (SD). Generally the drug was very well tolerated. The most commonly reported hematologic toxicities were neutropenia (55%) and thrombocytopenia (19%). 4 pts (11,7%) died during treatment (2 pts after the 1th cycle and 2 pts after the 4th course) because of septic shock, gastrointestinal hemorrage, pneumonia, and respiratory distress, respectively. The median duration of response was 3,5 months (range 1–14 months). Surprisingly, 3/14 patients (2 CR and 1 HI erythroid) showed a long duration of response (11, 13 and 14 months, respectively), still ongoing, after discontinuation of AZA. Preliminary data on the lipid signalling pathways suggested a direct correlation between the demethylating effect on PI-PLC-beta1 and responsiveness to treatment.Our study shows that AZA low-dose schedule may be a feasible and effective treatment for low-risk MDS pts and may induce durable responses. Despite AZA safety, extreme caution is needed in pts with age-related comorbidities and/or with severe neutropenia or thrombocytopenia, especially in low-risk MDS. Furthermore, PI-PLC–β1 demethylation and gene expression could represent a new biological marker to predict the clinical response to AZAOff Label Use: In Italy the use of Azicitidine for Low-Risk Myelodysplastic patients is off-label. The use of azacitidine in our study is part of a Phase II clinical trial. Finelli:Celgene: Consultancy.

The inhibition of the c-Kit signal transduction pathway by imatinib mesylate (STI571) and the inhibition of the post-translational modification of the N-K Ras proteins by farnesyl transferase inhibitors (FTIs) have shown potential efficacy in treating acute myeloid leukemias. We investigated the activity of (STI571) and of two distinct FTIs, R115777 and SCH66336, on two pairs of acute leukemia (AL) human tumor cell lines, each pair consisting of the parental cell line, HL60 and CCRF-CEM, and of its drug-selected multidrug resistant (MDR) Pgp-positive subline, HL60-DNR and CEM-VLB. The effectiveness of STI571, R115777 and SCH66336 in inhibiting cell proliferation of AL cell lines have been evaluated by colorimetric MTT assay. Cell growth was evaluated after a 7-day incubation at 37°C and 5% CO2 by using 50 microl per well of the MTT solution (5 mg/mL). The inhibition dose 50 (ID50) was defined as the drug dose that inhibited cell growth to 50% of the control. The results were correlated with the MDR phenotype and c-Kit expression. The toxic effect of STI 571 on all the acute leukemias derived cell lines was very low for both parental and MDR-Pgp+ sublines, ranging from 0.9 and more than 5 microM. STI571 activity was influenced by the c-Kit (CD117) expression and not by the MDR expression of cell lines. In fact, among these acute leukemia derived cell lines the higher toxic effect (ID50=0.9 microM) was obtained in HL60 DNR, the only cell line with a CD117 positivity. R115777 was more toxic than SCH66336 on all the tested AL cell lines. The Inhibition Dose 50 (ID50) of the two farnesyl transferase inhibitors ranged from 0.0065 to 0.21 microM for R115777 and from 0.3 to 6.5 microM for SCH66336. These results suggest that a potential synergistic effects of STI571 and R155777 combination could be explored in c-Kit positive acute myeloid leukemias.