ALBERT

Description: Background and rationale. Recent studies show that the risk of VTE in NHL pts is similar to that observed in high risk solid tumors (i.e. pancreatic, ovarian cancer). VTE in NHL occurs in most cases within three months from diagnosis and can have substantial impact on treatment delivery and outcome as well as on quality of life. However few data are available on potential predictors. To better clarify the epidemiology of early (within six months from treatment start) VTE in NHL we conducted a pooled data analysis of 12 clinical trials from FIL. Our analysis included basic demographic features, lymphoma-related characteristics as well the Khorana score (based on histology, BMI, platelets WBC and HB counts) which is extensively used in solid tumors to predict VTE risk. Patients and methods. From Jan. 2010 to Dec. 2014, all pts with B-cell NHL enrolled in prospective clinical trials from FIL for frontline treatment were included. For 9 studies study period included the entire trial population was included. The analyses were conducted based on CRFs as well as pharmacovigilance reports. VTE definition and grading was stated according to standard criteria of toxicity (CTCAE V4.0). Cumulative incidence of VTE from the study enrollment was estimated using the method described by Gooley et al. accounting for death from any causes as a competing risk. The Fine & Gray survival model was used to identify predictors of VTE among NHL pts. Factors predicting the grade of VTE were investigated using an ordinal logistic regression model. This pooled data analysis was approved by local IRB. Results. Overall,1717 patients belonging to 12 studies were evaluated. Eight were phase I/II or II (25% of pts) and 4 phase III (75% of pts). M/F ratio was 1.41, Median age was 57, (IQ range (IQR) 49-66). Histologies were: DLCL-B 34%, FL 41%, MCL 18%, other 6%. Median BMI was 25 (IQR 22-28). Median Hb, WBC and platelets counts were 13g/dl) (IQR 11.5-14.2), 7,1*10^9/l (IQR 5.6-10.3), 224*10^9/l (IQR 169-298), respectively. 1189 pts were evaluable Khorana score: 58% low risk, 30% intermediate risk, 12% were high risk. Human erytropoetin support was given to 9% of patients. All pts received Rituximab. Planned therapeutic programs included ASCT in 27% of pts, conventional chemotherapy in 67% a conventional chemotherapy plus lenalidomide in 6%. Overall 59 any grade VTE episodes occurred in 51 pts (2.9%), including 21 grade III-IV VTE (18 pts). None was fatal. Median time from study enrolment to VTE was 63 days (IQR: 35-110). Considering death as a competitive event the 6 months cumulative incidence of VTE was 2,2% in low risk Khorana score, 4.5% (95%IC: 2.3-6.7) in intermediate and 6.6% (95%IC: 2.4-10.8) in high risk (p=0.012) (figure 1). Khorana score was predictive also for grade III-IV events as they were 0,7% (95%IC:0.1-1.4) in low risk and 2.0% (95%IC:0.8-3.3) in intermediate-high risk (p=0.048). The result were similar also after excluding lenalidomide containing studies. The Fine and Gray multivariate analyses, adjusted for age and stage, showed that Khorana score (intermediate risk adjHR=1.96; 95%IC: 0.84-4.56 and high risk adjHR=3.81; 95%IC: 1.51-9.58) and DLCL-B histotype (adjHR=2.58; 95%IC: 1.01-6.55) were independently associated to an increased risk of VTE. Moreover an ordinal logistical regression model indicated that the increase of one point in the Khorana score resulted in an increased risk of VTE (OR=1.85; 95%CI: 1.23-2.79). Conclusions: our results suggest that DLCL-B hystotype and Khorana score are predictors of VTE in NHL. The latter might become a simple and effective tool to assess the risk of VTE in NHL. Prospective validation including also patients not eligible for clinical trials is needed. Figure 1. Figure 1. Disclosures Off Label Use: This is a metaanalysis on 12 prospective trials which employed several different experimental agents. All experimental agent will be disclosed to the audience. Luminari:Roche: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Teva: Membership on an entity's Board of Directors or advisory committees.

Description: Introduction: Genetic modification of autologous hematopoietic stem and progenitor cells (HSPC) is a promising clinical intervention to cure inherited monogenic diseases. Successful gene therapy trials have already been conducted using CD34+ cells from bone marrow and from mobilized peripheral blood. In this regard, cord blood (CB) represents an attractive source of HSCs due to its high concentration of high proliferative HSPC and increased susceptibility to be transduced by lentiviral vectors. Unfortunately, the major disadvantage is the limited number of HSC in the CB collection. Consequently, ex-vivo expansion of CB-HSC is desirable to extend clinical applications. Purposes: To investigate the ability of UCB-cd34+ cells to be expanded in serum-free media supplemented with the early acting hematopoietic cytokines SCF,TPO and Flt-3 ligant (STF) and to characterize CD34+ cells subtypes, clonogenic capacity and gene expression profile during expansion. We also wanted to investigate the susceptibility of the expanded cd34+ cells to be transduced by a GFP-lentiviral vector (LV-GFP) Material and Methods: CD34+ immunoselected cells from 10 UCB were grown for 8 days in customized serum-free medium formulated for HSC expansion, supplemented with STF cytokines. Numbers end frequency of CD34+cells and co-expression of the primitive surface antigens (CD38, CD133, CD90) was evaluated during expansion. Colonies developed in methylcellulose were scored for enumeration ad typing. LV-GFP transduction efficiency was evaluated in CD34+ cells cultured for 4 days in expansion medium plus STF and for 24 hrs in X-vivo10 medium with STF±IL-3 cytokines; the last condition slightly expands CD34+ cells (1.3 fold) and are currently used for HSPC-lentivector transduction in gene therapy clinical trials. The transduction efficiency was evaluated by measuring the percentage of GFP+ cells in the bulk and in colonies developed in methylcellulose and the VCN/cell by Q-PCR. Gene expression profiles were analyzed by human whole genome Agilent microarray Technology to detect differentially expressed genes between expanded, ex-vivo medium cultured and un-cultured cells. Results: We found an average of 8 fold-increase CD34+cells at day 4 and of 22 fold- increase at day 8 of culture. The frequency of CD34+ was maintained at day 4 and declined of about 50% at day 8. CD34+/CD38- early progenitors doublet as early as day 4, differences in CD34+/CD133+ and CD34+/CD90+cells were not significant. The number of CFU slightly increased during expansion while the relative frequency of colonies type did not significantly changed. Four days expanded CD34+ cells were transduced more efficiently than those grown in ex-vivo medium even in presence of IL-3 added to the STF cytokine cocktail. Comprehensive gene expression profile analysis highlighted about 4000 genes differentially expressed in CD34+ cells expanded for 4 and for 8 days compared to that of the un-cultured cells. Conversely, the expression profiles analysis did not show any clear separation between different cell culture methods (expansion vs ex-vivo medium). Specifically, the number of differentially expressed genes in common between the different culture conditions compared with the un-cultured cells was statistically significant. Unsurprisingly, the common up-regulated genes were related to the cell cycle. The likeness between the gene expression profiles of the different culture conditions was also validated by the identification of a significantly small number of differentially expressed genes between them. Conclusions: UCB-CD34+ cells can be efficiently expanded and transduced in serum free conditions. The expanded cells exhibited phenotypic marchers typical of early progenitors and developed colonies in number and in type similar to the unmanipulated cells and exhibited whole gene expression profile that is consisted with that of CD34+ cells exposed for the short term culture conditions currently used in gene therapy trial mediated by lentiviral vectors. Results from this study open a window on the future possibility of using homologous UCB-HSC as target for gene correction in patients diagnosed for a genetic disorder in prenatal time. The genetically modified cells would be stored and used for gene therapy in the same individual in pediatric age. This work was funded by the F and P Cutino Foundation - Project RiMedRi CUP G73F12000150004 Disclosures No relevant conflicts of interest to declare.

Description: Abstract 4371 Background/Aim: Using of a software for venous thromboprophylaxis prescription in hospitalized patients. Evaluation of the prescriptive mode in relation to the risk profile of the patient. Materials and Methods: Has been developed a software, which guides the physician to the choice of prophylaxis of VTE on the basis of a thromboembolic risk score (The Padua Prediction Score), renal function and the type of admission. We analyzed data derived from prescriptions over a period of 14 consecutive months. Results: 3125 patients were included. 68% of the patients were “high-risk (HR)” and 32% at “low risk (LR)”. Were treated with drug therapy, 89% of HR patients and 40% of LR. 11% of HR patients were not treated for severe renal failure or high risk of bleeding. Conclusion: All HR patients were considered for thromboprophilaxis. 89 % of HR patients and 40% of LR were treated. In our initial experience the software is an effective tool to identify patients at high risk and more effective prophylactic strategy. Furthermore, the evaluation of creatinine clearance allows to assess the bleeding risk associated with prophylactic treatment. Disclosures: No relevant conflicts of interest to declare.

Description: Hematopoietic stem cell engineering is a promising therapy to cure b-thalassemia, in particular for patients who lack a suitable BM donor for allogeneic transplantation. Since the engrafted gene-corrected stem cells will not have any selective advantage over the unmodified ones, the effectiveness of the therapy in this setting largely depends on the infusion of high numbers of gene-modified cells and on the conditioning regimen. The quality of the infused cells is also crucial for the clinical outcome and the duration of the therapeutic effect. HSPCs mobilization, particularly when G-CSF and plerixafor are used in combination, has been proved to be the optimal approach to harvest a large number of CD34+cells in patients with hematological malignancies and in healthy volunteers. However adult heavily-transfused thalassemia patients have intrinsic characteristics that may adversely affect both the safety and the efficacy of mobilization. We conducted a clinical trial to investigate the safety and effectiveness of mobilizing HSPCs with G-CSF+plerixafor in adult patients affected by β-thalassemia major with the aim to reach a cell dose of ≥8x106 CD34+cells/Kg. We studied the kinetic of CD34+cells during mobilization and performed a comprehensive characterization of their molecular and functional properties. All patients completed the mobilization according to the protocol (G-CSF 10 μg/kg/day for four days, followed by plerixafor 240 μg/kg in the evening on day 4) and no serious adverse events occurred. Leukapheresis was done 10-12 hours after plerixafor (on day 5). Three of the four patients reached the target cell dose or more in single-apheresis collections, even one patient where a significant dose reduction of G-CSF was halved due to early hyperleukocytosis. For one patients the number of cells collected in the first apheresis was slightly below the established target and therefore, according to the protocol, she was subjected to a second apheresis on day 6, after an additional dose of plerixafor. The total yield from the combined apheresis in this patient was 13.0 CD34+cells /Kg. CD34+ cell yields per single apheresis in our patients were comparable to those in healthy donors (12 pts) mobilized in our hospital with G-CSF alone. A significant increase in the mean peripheral blood CD34+ cells (12.1± 8.2 fold), was unanimously observed after plerixafor addiction. The frequencies of the more primitive CD34+cell subtypes (CD34+CD38- and CD34+CD38-133+) as well as the clonogenic capacity tested in short term in vitro assay were found significantly increased too. Comprehensive microarray analysis of genes expressed in the CD34+ cells purified from the same patient upon mobilization with G-CSF alone (G/CD34+cell) and with G-CSF+plerixafor (G+pl/CD34+cell) highlighted a different HSCs repertoire. According to the mechanism of plerixafor mobilization, CXCR-4 gene expression was found 5-fold higher in G+pl/CD34+cells. CXCR-4 gene is known to be expressed on the surface of more primitive CD34+ HSCs with long-term repopulating potential and plays a central role in the regulation of adhesion of them to native niche in the BM. A substantial number of genes with previously shown implication in mechanisms of homing and engraftment (CXCR4, CD82, DPP4, ROBO4), or genes linked to stress resistance (CXCL4, SOD2, IL8, PPBP) as well as several chemokines genes involved in cell mobility (CXCL2, CXC3, CXCR2) were also found to be up-regulated in G+pl/CD34+cells. Overall, the yields, the primitive signatures of CD34+cells indicate the G-CSF+plerixafor mobilized peripheral blood as optimal graft that should favor HSPCs engraftment after transplantation. This findings has therapeutic implications not only for b-thalassemia but also for other hematopoietic stem cell gene therapy applications. This work was funded by the F and P Cutino Foundation - Project RiMedRi CUP G73F1200015000. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Acquired and inherited thrombophilias are associated with unfavourable pregnancy outcome. Low-molecular-weight heparin (LMWH) has demonstrated utility in the prevention and treatment of thrombosis during the pregnancy. There is no consensus on the dose of LMWH for prophylaxis, between fixed dose and “adjusted” dose according to anti-Xa levels. We report our experience the management of heparin-therapy during pregnancy in thrombophilic women. Methods We performed a retrospective analysis of patients with high risk of venous thromboembolism (VTE) and obstetric complications during pregnancy. Table 1 shows the risk factors of VTE in pregnancy. The obstetric complications considered were: preeclampsia, fetal growth restriction, fetal death and recurrent miscarriages. All patients were prescribed enoxaparin 70-100 IU /Kg SC once daily. To determine the dose of LMWH we evaluated levels of anti-factor Xa (chromogenic test) and the utero-placental circulation with color Doppler ultrasound. Dose adjustment was performed if the activity of factor Xa was suboptimal (prophylaxis range 〉 0.4IU/ml and 〈 0.8 IU/ml anti-Xa value) or were detected alteration in the utero-placental circulation. Outcome Measures Time of delivery, birth weights and Apgar-index. Results 70 pregnancies for 68 women were recruited between January 2008 and December 2012. The median age of patients was 35 years (range 25- 43years) and 7 (10%) were over 40. Twenty-four patients had a previous VTE and thirty-six had an obstetric complications. No one patients reported thrombotic and/or hemorrhagic complications during heparin treatment. In 97 % of cases the time of delivery occurred after 36th weeks of pregnancy, with a birth weight greater than 2600 grams and Apgar score index higher than 7. The dose of LMWH had to be adjusted at least once during the course of the pregnancy and the mean daily dose was increased from 4.000UI to 8.000UI between the 12th and 32th week of gestation. Conclusion In pregnancy LMWH thromboprophylaxis enables the modulation of systemic haemostatic parameters by the inhibition of factor Xa and by increasing TFPI levels. In the literature-in some high risk pregnancy- the weight-based dosing of LMWH could seem to fail achieving a prophylactic anticoagulation. In our experience adjusted doses prevent unfavourable events and improve fetal growth. °Carriage of defects of antithrombin, protein C or S, factor V Leiden, G20210A prothrombin mutation, antiphospholipid syndrom Disclosures: No relevant conflicts of interest to declare.

Description: Background A Virtual Screening algorithm has to adapt to the different stages of this process. Early screening needs to ensure that all bioactive compounds are ranked in the first positions despite of the number of false positives, while a second screening round is aimed at increasing the prediction accuracy. Results A novel CNN architecture is presented to this aim, which predicts bioactivity of candidate compounds on CDK1 using a combination of molecular fingerprints as their vector representation, and has been trained suitably to achieve good results as regards both enrichment factor and accuracy in different screening modes (98.55% accuracy in active-only selection, and 98.88% in high precision discrimination). Conclusion The proposed architecture outperforms state-of-the-art ML approaches, and some interesting insights on molecular fingerprints are devised.