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Sickle cell disease (SCD) and transfusion-dependent β-thalassemia (TDT) are the most prevalent monogenic disorders worldwide. Trial HGB-205 ( NCT02151526 ) aimed at evaluating gene therapy by autologous CD34

Musculoskeletal disorders (MSD) remain the most common occupational disease in the Western countries. Beside the effects on workers themselves, MSDs may lead to high costs to enterprises and the society as a whole. Among the painful or tiring positions encountered in healthcare settings, caregivers often cite the filling of portable infusion pumps (PIP). This context was conducive to achievement of the goal of the following project: the development of a motorized apparatus designed to minimize the risk of MSDs caused by repeated gestures of filling PIPs.: The development of the tool followed eleven steps including: (a) a market analysis, (b) a SWOT analysis, (c) definition of the specifications of the apparatus, and (d) a concurrent engineering methodology.: In approximately 18 months, a dynamic project group supported by a concurrent engineering methodology has designed and developed a compact and lightweight motorized apparatus able to suppress the risk of MSDs caused by manual filling of PIPs. After a mandatory step of familiarization, all involved operators have unanimously endorsed the new tool.: We believe that this new motorized apparatus may now simply and substantially alleviate occupational MSDs caused by the manually filling of PIPs. Another major benefit of this research program is the clear improvement of the intrinsically quality of therapeutic objects (TO) through the creation of a more accurate and reproducible method of PIP filling.

β-globin gene disorders are the most prevalent inherited diseases worldwide and result from abnormal β-globin synthesis or structure. Novel therapeutic approaches are being developed in an effort to move beyond palliative management. Gene therapy, by ex vivo lentiviral transfer of a therapeutic β-globin gene derivative (β(AT87Q)-globin) to hematopoietic stem cells, driven by cis-regulatory elements that confer high, erythroid-specific expression, has been evaluated in human clinical trials over the past 8 years. β(AT87Q)-globin is used both as a strong inhibitor of HbS polymerization and as a biomarker. While long-term studies are underway in multiple centers in Europe and in the United States, proof-of-principle of efficacy and safety has already been obtained in multiple patients with β-thalassemia and sickle cell disease.

International audience; BACKGROUND Donor availability and transplantation-related risks limit the broad use of allogeneic hematopoietic-cell transplantation in patients with transfusion-dependent beta-thalassemia. After previously establishing that lentiviral transfer of a marked beta-globin (beta(A-T87Q)) gene could substitute for long-term red-cell transfusions in a patient with beta-thalassemia, we wanted to evaluate the safety and efficacy of such gene therapy in patients with transfusion-dependent beta-thalassemia. METHODS In two phase 1-2 studies, we obtained mobilized autologous CD34+ cells from 22 patients (12 to 35 years of age) with transfusion-dependent beta-thalassemia and transduced the cells ex vivo with LentiGlobin BB305 vector, which encodes adult hemoglobin (HbA) with a T87Q amino acid substitution (HbA(T87Q)). The cells were then reinfused after the patients had undergone myeloablative busulfan conditioning. We subsequently monitored adverse events, vector integration, and levels of replication-competent lentivirus. Efficacy assessments included levels of total hemoglobin and HbA(T87Q), transfusion requirements, and average vector copy number. RESULTS At a median of 26 months (range, 15 to 42) after infusion of the gene-modified cells, all but 1 of the 13 patients who had a non-beta(0)/beta(0) genotype had stopped receiving red-cell transfusions; the levels of HbA(T87Q) ranged from 3.4 to 10.0 g per deciliter, and the levels of total hemoglobin ranged from 8.2 to 13.7 g per deciliter. Correction of biologic markers of dyserythropoiesis was achieved in evaluated patients with hemoglobin levels near normal ranges. In 9 patients with a beta(0)/beta(0) genotype or two copies of the IVS1-110 mutation, the median annualized transfusion volume was decreased by 73%, and red-cell transfusions were discontinued in 3 patients. Treatment-related adverse events were typical of those associated with autologous stem-cell transplantation. No clonal dominance related to vector integration was observed. CONCLUSIONS Gene therapy with autologous CD34+ cells transduced with the BB305 vector reduced or eliminated the need for long-term red-cell transfusions in 22 patients with severe beta-thalassemia without serious adverse events related to the drug product. (Funded by Bluebird Bio and others; HGB-204 and HGB-205 ClinicalTrials. gov numbers, NCT01745120 and NCT02151526.)

Sickle cell disease results from a homozygous missense mutation in the ß-globin gene that causes polymerization of hemoglobin S. Gene therapy for patients with this disorder is complicated by the complex cellular abnormalities and challenges in achieving effective, persistent inhibition of polymerization of hemoglobin S. We describe our first patient treated with lentiviral vector-mediated addition of an antisickling ß-globin gene into autologous hematopoietic stem cells. Adverse events were consistent with busulfan conditioning. Fifteen months after treatment, the level of therapeutic antisickling ß-globin remained high (approximately 50% of ß-like-globin chains) without recurrence of sickle crises and with correction of the biologic hallmarks of the disease. (Funded by Bluebird Bio and others; HGB-205 ClinicalTrials.gov number, NCT02151526 .).

Background LentiGlobin gene therapy contains autologous CD34+ hematopoietic stem cells (HSCs) transduced with the BB305 lentiviral vector (LVV), encoding human β-globin with a T87Q substitution. This substitution confers anti-sickling properties to the gene therapy-derived hemoglobin (HbAT87Q) and allows for its quantification in transduced HSCs. The proof of concept for LentiGlobin gene therapy in patients with transfusion-dependent β-thalassemia (TDT) and sickle cell disease (SCD) was established in the recently completed HGB-205 study (NCT02151526). Herein, we provide the safety and efficacy outcomes and long-term follow-up data for all 7 treated patients, 4 with TDT and 3 with SCD. Methods Patients 5−35 years old with TDT (≥ 100 mL/kg of packed red blood cells [pRBCs]/year) or severe SCD (e.g., ≥ 2 acute chest syndromes [ACS] or ≥ 2 vaso-occlusive crises in the preceding year or the year before regular transfusions) were enrolled. CD34+ HSCs were obtained by mobilization and apheresis in patients with TDT or by bone marrow harvest in patients with SCD. Following collection, cells were transduced with the BB305 LVV. Patients underwent busulfan myeloablative conditioning and were infused with transduced cells. Patients were monitored for engraftment, adverse events (AEs), HbAT87Q levels, and other hematologic and clinical parameters. After 2 years in HGB-205, patients transitioned into the long-term follow-up study, LTF-303 (NCT02633943). Summary statistics are shown as median (min-max). Results As of June 2019, patients with TDT (n=4) and SCD (n=3) had a median follow-up of 49.6 (40.5-60.6) and 28.5 (25.5-52.5) months, respectively. Table 1 shows patient and drug product characteristics and several key efficacy outcomes. All patients achieved HSC engraftment. LentiGlobin safety profile was consistent with busulfan myeloablative conditioning and, in case of SCD, with the underlying disease state. The most common non-hematologic Grade ≥ 3 AEs post-LentiGlobin gene therapy (≥ 2 patients) for patients with TDT were stomatitis (n=4) and increased aspartate aminotransferase (n=2), and for patients with SCD were ACS (n=2) and vaso-occlusive pain (n=2). In all 4 patients with TDT, total Hb and HbAT87Q levels remained generally stable up to 5 years post-LentiGlobin infusion. Three of 4 patients achieved transfusion independence (TI; defined as weighted average Hb ≥ 9g/dL without pRBC transfusions for ≥ 12 months), for an ongoing duration of 56.3 (38.2-57.6) months. Weighted average total Hb during TI was 11.4 (10.5-13.0) g/dL. One patient has been off transfusions for 37.5 months and had total Hb of 7.7 g/dL, which was below the ≥ 9 g/dL requirement to meet the protocol definition of TI. At last visit, HbAT87Q levels in these 4 patients ranged from 6.2-11.2 g/dL, which contributed 73.8-86.8% of the total Hb. The first patient treated with LentiGlobin for SCD experienced one vaso-occlusive pain episode, which developed at 30 months after LentiGlobin gene therapy following a case of acute gastroenteritis with fever and dehydration. The second SCD patient had 2 serious AEs (SAEs) of ACS approximately 6 and 8 months after LentiGlobin gene therapy. The patient resumed chronic pRBC transfusions and hydroxyurea treatment and subsequently experienced 2 SAEs of vaso-occlusive pain; no additional SAEs of vaso-occlusive pain or ACS were reported during the last 16 months of follow-up after LentiGlobin infusion. The third SCD patient had no episodes of vaso-occlusive pain or ACS during 25.5 months of follow-up post-LentiGlobin gene therapy as of the data cut-off. Two patients with SCD who have been off chronic pRBC transfusions, showed improvement in hemolysis markers post-LentiGlobin treatment and stabilization of HbAT87Q expression at approximately 6 months post-LentiGlobin infusion. Total Hb levels for patients with SCD at last visit were 13.0 g/dL (patient 1), 9.4 g/dL (patient 2), and 9.8 g/dL (patient 3), with corresponding HbAT87Q contributions of 47.9%, 7.9%, and 25.8%, respectively. Summary With up to 5 years of follow-up, treatment with LentiGlobin gene therapy was well tolerated and resulted in improvement in hematologic parameters and disease-related symptoms. Further results from the completed study will be presented. Disclosures Hermine: Celgene: Research Funding; Novartis: Research Funding; AB science: Consultancy, Equity Ownership, Honoraria, Research Funding. Brousse:bluebird bio, Inc: Consultancy; AddMedica: Consultancy. El Nemer:Hemanext: Other: Other. Bartolucci:Novartis: Membership on an entity's Board of Directors or advisory committees; AddMedica: Honoraria, Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; HEMANEXT: Membership on an entity's Board of Directors or advisory committees. Asmal:bluebird bio, Inc: Employment, Equity Ownership. Whitney:bluebird bio, Inc: Employment, Equity Ownership. Gayron:bluebird bio, Inc: Employment, Equity Ownership. Huang:bluebird bio, Inc.: Employment, Equity Ownership. de Montalembert:AddMedica: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; bluebird bio, Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Ribeil:bluebird bio, Inc: Employment, Equity Ownership. Cavazzana:SmartImmune: Other: Founder.

International audience; This study compares the performance of a reference method of HPLC to Raman spectroscopy (RS) for the analytical quality control (AQC) of complex therapeutic objects. We assessed a model consisting of a widely used anticancer drug, i.e., 5-fluorouracil, which was compounded in a complex medical device, i.e., an elastomeric portable infusion pump. In view of the main objective, the two methods provided excellent results for the analytical validation key criteria, i.e., trueness, precision and accuracy, ranging from 7.5 to 50mg/mL and in either isotonic sodium or 5% dextrose. The Spearman and Kendall correlation tests (p-value

Purpose: Activating KRAS mutations are frequent (>90%) in pancreatic ductal adenocarcinoma (PDAC) and drive downstream deregulation of both MAPK and PI3K-mTOR pathways. MEK inhibitors (MEKi) are under clinical evaluation in PDAC, in combination with other agents including PI3K-mTOR inhibitors. While RAS and BRAF mutations, EMT, PI3K-mTOR activation, and pERK inhibition have been suggested as predictive markers for MEKi efficacy, they are not validated in PDAC. We aimed to explore the cellular and molecular effects of MEKi GSK1120212 (GSK212) alone or in combination with PI3K-mTOR inhibitors, in PDAC cell lines and on an innovative ex vivo system. Methods: GSK212 is an allosteric non-ATP competitive MEKi, everolimus (Ev) a mTORC1 inhibitor, and BKM120 a pan-class PI3K inhibitor. Effects on proliferation were evaluated by MTT assay. Combinations were analyzed by the Chou-Talalay method. Protein expression was assessed by Western blot. Ex vivo drug assays were performed at different drug concentrations on cultures of fresh tumor tissue slices prepared from patient surgical specimens. Apoptosis and proliferation were assessed by cleaved caspase 3 (Cas-3) and MIB-1 (Ki67) immunostainings, respectively; MEKi sensitivity was defined as cleaved Cas-3 expression in 30% or more of cancer cells. Correlations between protein expressions were explored using linear regression and Pearson’s R2 calculation. Samples will be classified into PDAC subtypes according to Collisson’s, Moffit’s and Bailey’s transcriptomic signatures using non-negative matrix factorization and correlation methods. Results: MIAPaCa-2 and PANC-1 are two mesenchymal KRAS mutated/BRAF wild-type PDAC cell lines with very different response to GSK212, MIAPaCa-2 being sensitive (72h-IC50=0.009µM) and PANC-1 resistant (72h-IC50=33µM). MIAPaCa-2 was more sensitive than PANC-1 to Ev (IC50=23.3µM vs 47.0µM) and BKM120 (IC50=4.19µM vs 31.6µM). Combination of GSK212 and Ev or BKM120 for 72h resulted in synergistic effects (CI0.50 discriminated between sensitive and resistant tumors. There was no correlation with RAS/RAF mutation status. Predictive value of transcriptomic signatures for MEKi response will be presented. Conclusion: Response to combined MEK/mTOR pathway inhibition was not correlated with known biomarkers of response to MEKi. Besides the therapeutic potential of MEK/mTOR pathway inhibition in PDAC, this work provides the first evidence of feasibility of pharmacodynamic biomarker monitoring on fresh PDAC tissue slices. Citation Format: Cindy Neuzillet, Annemilaï Tijeras-Raballand, Anguraj Sadanandam, Pierre Bourgoin, Philippe Bourget, Pawan Poudel, Maria Serova, Armand De Gramont, Philippe Ruszniewski, Valérie Paradis, Eric Raymond, Jérôme Cros, Pascal Hammel.{Authors}. Effects of MEK inhibition alone or in combination with PI3K-mTOR pathway inhibitors in pancreatic ductal adenocarcinoma in vitro and on an innovative ex vivo fresh tumor tissue culture model. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B57.

Introduction: β-globin gene transfer into hematopoietic stem cells (HSCs) has the potential to reduce or eliminate the symptoms of severe sickle cell disease (SCD) and reduce or eliminate transfusion requirements in transfusion-dependent β-thalassemia (TDT). LentiGlobin Drug Product (DP) contains autologous CD34+ cells transduced with the BB305 lentiviral vector, which encodes a human β-globin gene containing a single point mutation (AT87Q) designed to confer anti-sickling properties similar to those observed with γ-globin. We previously reported proof of concept for LentiGlobin DP treatment in severe SCD and early data from 4 treated patients with TDT. We now report 18 months of follow-up for the patient with SCD and between 9 and 30 months of follow-up for the 4 patients with TDT. Patients (5-35 years of age) with severe SCD (e.g. ≥2 acute chest syndrome episodes or ≥2 vaso-occlusive crises [VOC] in preceding year/in year prior to regular transfusions) or TDT (≥100mL/kg of packed red blood cells [RBCs] per year) were enrolled. Following mobilization and apheresis (for TDT) or bone marrow harvest (for SCD), autologous CD34+ cells were transduced with the BB305 lentiviral vector. Patients underwent myeloablative conditioning with busulfan prior to infusion of the transduced cells. After infusion, patients were monitored for hematologic engraftment, vector copy number (VCN), and HbAT87Q expression. Disease-specific assessments included transfusion requirements for TDT, or VOCs and hospitalizations for SCD. Safety assessments included adverse events (AEs) and integration site analysis. Results: As of July 2016, 1 patient with severe SCD (male; 13 years old) and 4 patients with TDT (2 male, 2 female; 16-19 years old) have received LentiGlobin DP in Study HGB-205. The median LentiGlobin DP cell dose was 8.9 (range 5.6-13.6) x 106 CD34+ cells/kg with a DP VCN of 1.2 (range: 0.8-2.0) vector copies/diploid genome. Median post-infusion follow-up as of July 6, 2016 is 20.8 months (range 9.5-31.3). All subjects successfully engrafted after receiving LentiGlobin DP, with a median time to neutrophil engraftment of 17 days (range 14-38 days). VCN in peripheral blood has remained generally consistent from Month 3 in all patients with a range of 0.2 to 3.4 at last measurement. The toxicity profile observed from start of conditioning to latest follow-up remains consistent with myeloablative conditioning with single-agent busulfan, with no DP-related ≥Grade 3 AEs or serious AEs and no evidence of clonal dominance reported to date. Three patients with TDT have β0/βE genotypes and 1 is homozygous for the severe β+ mutation IVS1 nt 110 G>A. The 2 patients who have completed the 2-year primary follow-up period (both β0/βE) have not required RBC transfusions for 31 and 28 months, with total Hb of 10.9 and 13.5 g/dL, and HbAT87Q expression of 7.7 and 10.1 g/dL, respectively, at most recent study visit. Iron chelation has been discontinued and phlebotomy initiated for 1 of the patients. The remaining patient with β0/βE genotype has 9 months of follow-up and has not required transfusions since 4 days post-LentiGlobin DP infusion, achieving a total Hb of 11.3 g/dL at last study visit. The patient with the severe IVS1 genotype has 12 months of follow-up and has been free of transfusions for 9 months, with a total Hb of 8.3 g/dL at last study visit. The patient with severe SCD, who prior to study enrollment received regular RBC transfusions, has experienced no clinical symptoms or complications of SCD in the 18 months since treatment, despite discontinuing transfusions 3 months after LentiGlobin DP infusion. Total Hb in this patient was 12.5 g/dL, with 6.6 g/dL HbAT87Q (53%) and 5.7 g/dL HbS (45%) at last study visit. Compared with values at screening, unconjugated bilirubin had dropped 78% (50 to 11 μmol/L), lactate dehydrogenase had dropped 54% (626 to 287 U/L), and reticulocyte count had dropped 45% (238x109/L to 132x109/L) by Month 18. Conclusions: Data from this ongoing Phase 1/2 clinical study suggest that treatment with LentiGlobin DP can result in sustained production of therapeutic HbAT87Q, which ameliorates the clinical and biochemical effects of severe SCD and TDT, with an acceptable safety profile. Gene therapy presents a potentially promising therapy for patients with severe hemoglobinopathies. Further follow-up and additional data from patients are needed to confirm the encouraging results seen to date in this study. Disclosures Ribeil: Bluebirdbio: Consultancy; Addmedica: Research Funding. Payen:bluebird bio: Patents & Royalties. Hermine:Alexion: Research Funding; Celgene: Research Funding; Novartis: Research Funding; AB science: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding, Speakers Bureau. Asmal:bluebird bio: Employment, Equity Ownership. Joseney-Antoine:bluebird bio: Employment, Equity Ownership. De Montalembert:Addmedica: Research Funding; Novartis: Research Funding, Speakers Bureau. Leboulch:bluebird bio, Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Objective To determine the optimal dose and route of methotrexate (MTX) in the treatment of ectopic pregnancy (EP). Design Prospective randomized study. Setting Department of Obstetrics and Gynecology (A. Beclere Public Hospital, Clamart, France, Paris-Sud University). Patients and Methods Forty-eight patients with unruptured EP clearly visualized by ultrasound were randomly allocated into four groups of treatment (12 patients in each group): group 1,1mg/kg injected locally in the ectopic gestational sac and 1mg/kg by IM administration 48hours later; group 2, 1mg/kg locally; group 3, 0.5mg/kg locally; group 4, 1mg/kg by IM administration. Inclusion criteria used a pretherapeutic score ≤12. Blood samples were collected at time 0.25, 0.5, 1, 2, 6, 12, 24, 36, and 48hours after MTX administration. Pharmacokinetics of MTX plasma levels were measured by fluorescence polarization immunoassay. Kinetic parameters were compared by Wilcoxon test and Mann-Whitney test. Plasma hCG concentrations were assessed on days 2, 5, and 10 and then weekly until they returned to undetectable levels. Results Success rate was 12 of 12, 11 of 12, 10 of 12, and 10 of 12 in groups 1, 2, 3, and 4, respectively. Six patients in group 3 required an additional MTX IM injection because of an inadequate decrease of hCG plasma levels. Five patients underwent surgery for abdominal pain or inadequate decrease of hCG. Area under the curve decreased more rapidly after injection in the gestational sac alone than after IM injection and was similar in groups 1 and 2 after local injection and lower in group 3. Terminal half-life and mean residence time remained similar in the four groups. Systemic side effects of MTX therapy occurred in three cases in groups 1 and 4. The regression curve of hCG plasma levels appeared similar in the four groups with a decrease to pretreatment values between days 6 and 8 after an initial rise after MTX was given. Conclusion Area under the curve found after injection in the ectopic sac may be related to a decrease in bioavailability of MTX that links to trophoblastic cells. Patients in group 3 were clearly undertreated by 0.5mg/kg MTX and required additional therapy. Residual values of MTX plasma levels were always below the limit of detection of our assay and confirmed that citrovarum factor rescue is unnecessary. Injection of 1mg/kg of MTX in the ectopic sac appears as effective as systemic (IM) injection with less side effects for the patients.

Gérard Guiffant1, Jean-Jacques Durussel1, Patrice Flaud1, Jean-Pierre Vigier1, Christian Dupont2, Philippe Bourget3, Jacques Merckx11University Paris Diderot, Paris, France; 2University Teaching Hospital Cochin, Paris, France; 3University Teaching Hospital Necker-Enfants Malades, Paris, FranceAbstract: The use of elastomeric diffusers (EDs) has grown together with the expansion of home care. In these devices, the fill volume of the drug reservoir and the flow rate are preset and cannot be modified. The elastomer, which makes up the reservoir walls, is what makes the infusate flow due to the pressure it exerts. The purpose of this work was to quantify, under standardized experimental conditions and following recommended conditions of use, the mechanical performances of the 2 commonly used elastomers (silicone and polyisoprene) and their impact on infusion flow rate consistency. Results show that they exhibit different mechanical performances which leads to concerns regarding the use of these devices for some intravenous (IV) therapies.Keywords: elastomeric diffusers, perfusion, drug administration rate, infusion flow, infusion devices

Context The use of patient-controlled analgesia (PCA) allows patients to be managed at home and may increase the quality of life of patients with regard to drug administration. To ensure that intact drug is delivered to the patient in this setting, it is important to study its microbiological and physicochemical stability. Although these factors have been widely studied for many parenteral opioids, very few authors have investigated oxycodone stability associated with long-duration infusion in cancer patients. Objectives The aim of this study was to assess the microbiological and physicochemical stability of oxycodone hydrochloride solution in PCA devices and thereby to determine the feasibility of extending the expiration dates after mixing. Methods Sixteen CADD® reservoirs and 32 Rythmic® reservoirs were filled aseptically with pure (10mg/mL) and diluted (1mg/mL) oxycodone solution. Three different vehicles (0.9% sodium chloride, water for injection, and 5% dextrose) were used for dilution. Among the PCA systems stored over 28 days at room temperature, 16 Rythmic® reservoirs were protected from light. Microbiological stability was assessed by performing sterility tests. The physicochemical study was performed by determining aspect, pH, osmolality evolution, and weight. Drug concentrations were determined using the stability-indicating high performance liquid chromatography combined to ultraviolet detection technique. Results There was no significant change in pH, weight, and osmolality values of any solutions. No precipitation or change in color was observed in any of the sample solutions. There was no significant loss of oxycodone, and no trace of degradation products was detected. Conclusion This study indicates that pure and diluted oxycodone solutions in the PCA systems are stable for 28 days at room temperature when prepared aseptically.

Background: In patients with hemoglobinopathies, hematopoietic stem cell (HSC) gene therapy has the potential to induce production of functional β-globin in the red blood cell lineage with the aim of reducing or eliminating the symptoms of disease. Previous results from 1 subject with severe sickle cell disease (SCD; 6 months follow-up) and 2 subjects with β0/βE-thalassemia major (up to 15 months follow-up) treated in clinical study HGB-205 suggested that transplantation with autologous CD34+ cells transduced with the LentiGlobin BB305 lentiviral vector containing an engineered βA-T87Q-globin gene (LentiGlobin BB305 Drug Product) resulted in near-normal levels of total hemoglobin (Hb) and rapid clinical improvement. Here we provide data on a new subject enrolled and additional follow-up data on the 3 subjects previously presented in Study HGB-205. Subjects and Methods: Subjects with severe SCD underwent HSC collection via bone marrow harvest, while subjects with β-thalassemia major underwent HSC collection via peripheral blood apheresis following mobilization. CD34+ cells were selected and transduced with LentiGlobin BB305 lentiviral vector to produce the drug product. Subjects underwent myeloablation with intravenous busulfan, followed by infusion of drug product. Subjects were monitored for hematological engraftment, vector copy number, βA-T87Q-globin expression, adverse events and transfusion requirements. Integration site analysis (ISA) and replication-competent lentivirus (RCL) assays were performed. Prophylactic RBC transfusions were continued in subjects with SCD who were on chronic transfusion pre-transplant to maintain HbS Results: As of 31 July 2015, 1 subject with severe SCD (Subject 1204, βS/βS with multiple vaso-occlusive crises, silent infarct, acute chest syndrome, and on prophylactic transfusions) and 3 subjects withβ-thalassemia major (Subjects 1201, 1202 and 1203) have been infused with the LentiGlobin BB305 Drug Product. The outcome of these subjects to date is shown in Table 1. No subject has experienced a drug product-related adverse event, and ISA analyses demonstrate highly polyclonal reconstitution without clonal dominance. The subject with severe SCD is producing approximately 51.5% of anti-sickling hemoglobin (48% HbAT87Q, 1.8% HbF, 1.7% HbA2) at 9 months post-infusion. This subject has not had a post-infusion hospitalization for a SCD-related event despite stopping chronic transfusions at Day +88. Both subjects with β0/βE-thalassemia major have remained transfusion-free for at least 15 months post-infusion, with a consistent expression of βA-T87Q-globin; the subject with β0/β0-thalassemia major has only had 1 month follow-up post-drug product infusion to date. Conclusion: The subject with severe SCD is producing approximately 51.5% anti-sickling globins with HbS of 48.5% and remains free of SCD-related events despite stopping chronic transfusion therapy. Two subjects with β0/βE-thalassemia major remain transfusion-free for at least 15 months post infusion of LentiGlobin BB305 Drug Product. Gene therapy using autologous HSC transduced with LentiGlobin BB305 lentiviral vector is a promising approach for the treatment of patients with hemoglobinopathies. Table 1. Demographics and Transplantation Outcomes Subject Age (years)/ Sex (M/F) Genotype BB305 Drug Product Day of Neutrophil Engraftment Drug Product- related Adverse Events Day of Last pRBC Transfusion Last Study Visit (Months) Hb at Last Visit (g/dL) VCNa CD34+ cell dose (x106 per kg) Subject with severe sickle cell disease HbAT87Q/HbF/ HbS/Total Hb 1204 13/ M βS/βS 1.2 / 1.0 5.6 Day +37 None Day +88 9M 5.5/0.2/5.5/11.4 Subjects with β-thalassemia major Hb AT87Q/ Total Hb 1201 18/ F β0/βE 1.5 8.9 Day +13 None Day +10 18M 7.8/10.7 1202 16/ M β0/βE 2.1 13.6 Day +15 None Day +12 15M 9.7/12.8 1203 19/ M β0/β0 0.8 8.8 Day +28 None Day +15 1M Pending/9.2 As of 31 July 2015 aVCN, vector copy number; F=female; M= Male for gender, and months for day of last follow-up Disclosures Payen: bluebrid bio: Consultancy. Beuzard:bluebird bio Inc: Consultancy, Equity Ownership. von Kalle:bluebird bio, Inc.: Consultancy. Sandler:bluebird bio, Inc.: Employment, Equity Ownership. Soni:bluebird bio, Inc.: Employment, Equity Ownership. De Montalembert:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Leboulch:bluebird bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Introduction: Busulfan (Bu) is recognized worldwide as the cornerstone for HSCT conditioning regimens (CR). It has a narrow therapeutic window (TW), and graft rejection or toxicity are related to Bu exposure. In very young children, Bu exhibits large pharmacokinetic (PK) variability, and its metabolic clearance is non-linearly related to body weight (BW). In this setting, Bu dosage is stratified according to BW and commonly monitored once after the 1st of 16 total 2-hr infusions and finally, 96-hr exposure to this major alkylating agent. Given the narrow TW of Bu in relation to clinical outcomes and to better understand its PK/PD relationship, it is important to attempt to optimize both the duration and the intensity of exposure to Bu in children undergoing HSCT, whatever their BW and pathologies. Study objective: To optimize Bu duration/intensity exposure in children undergoing HSCT, we studied the possible contribution of double TDM of IV Bu dosing in children receiving Bu-based CR for HSCT, by comparing the expected targeted values of exposure after performing 1 PK (1st dose, PK1) and finally 2 PK (1st dose, PK1, and 9th dose, PK2). Patients and methods: In this single-centre* prospective observational study (05/2012-07/2015), the PK of Bu was performed using the NONMEM® software; a one-compartment PK model suitably fitted the concentrations vs. time data. The PK of Bu was assessed on 6 plasma samples (3 samples/PK) using a validated LC-MS/MS method, with area under the concentration-time curve (AUC) calculation from the 1st and 9th doses. AUC calculated after the 1st dose and extrapolated to the 16th one were compared intraindividually by the non-parametric Wilcoxon signed-rank test to the sum of AUC1-6 with TDM1 applied from the 7th dose with or without TDM2 applied from the 13th or 14th dose; p≤0.025 was considered statistically significant. 63 patients (Pts) undergoing HSCT were included with median follow up of 14 months [1-39]. Median age was 17 months [1-193] and BW 11 kg [3-59]. Most Pts had non-malignant diseases, received allogenic HSCT and Bu-based CR in combination with Flu (morphometric, disease and CR details will be provided). *one of the 14 French Pediatric BMT units, involved in the therapeutic monitoring of IV Bu dosing in children undergoing HSCT. Results: 63 Pts (37 males (59%) and 26 females (41%)) were included. According to the EBMT-ESID recommendations, a median Bu posology of 1 mg/kg 4x/day for 4 days [0.6-1.3 mg/kg] was given. We demonstrate that: (a) estimated total AUC obtained from 2 PK differ significantly from those calculated after none (p=8.6455 E-7) or PK1 alone (p=7.2157 E-8), (b) double TDM allows achieving no difference between the expected AUC [20,706-23,180 mmol/min] desired by the medical staff in view of diseases vs. estimated AUC (p=0.5285). In 2/63 (3.2%) Pts, PK analysis did not lead to change Bu dosage. In 11/63 (18%) and 15/63(24%) Pts, dosage was modified after PK1 (TDM1 Group) or PK2 alone, respectively. In 35/63 (56%) Pts, changes were required twice, after the 2 PK (Double-TDM Group). The mean total dose of Bu were as follows: (a) a theoretical value of 220.80 mg (20.11 mg/kg) would have been given through 16 consecutive infusions without any TDM, (b) 199.35 mg (18.08 mg/kg) were administered in the TDM1 Group (16 doses), (c) 246.00 mg (19.46 mg/kg) were administered in the Double-TDM Group, avoiding the infusion of 24.00 mg of Bu vs. PK1 alone in these Pts. In 16/63 (25%) Pts, a decision of discontinuation of Bu exposure was taken. Indeed, the value that was desired by the medical staff was achieved after 13 (6 Pts) or 14 (10 Pts) doses; the mean total amounts of Bu were 273.22 mg (14.96 mg/kg) and 225.88 mg (16.16 mg/kg), respectively, vs. 336.27 mg (18.45 mg/kg) and 262.56 mg (18.60 mg/kg) theoretically after TDM1 alone. Conclusion: In this large paediatric cohort, the double TDM of Bu is a relevant and feasible option to better control Bu exposure and to potentially minimize the risk of overexposure. Correlations with toxicities (VOD, aGVH), CR, OS are under analysis. Based on these data, the double-TDM procedure is routinely applied in our centre. Disclosures Petain: Laboratoires Pierre Fabre: Employment. Nguyen:Laboratoires Pierre Fabre: Employment.

The aim of this study was to assess the microbiological and physicochemical stability of opioid solutions containing fentanyl or sufentanil and thereby determine the feasibility of extending the expiration dates after mixing. Five systems containing fentanyl or sufentanil solutions at 50 microg/mL in portable patient-controlled analgesia (PCA) systems were filled and stored at room temperature for 14 days. They were sampled immediately after preparation, at day 3, and each day of the following weeks. Microbiological stability was assessed by performing sterility tests. The physicochemical study was performed by determining aspect, pH, and osmolality evolution. All samples were tested for appearance, change in color, and loss of concentration using an analytical method. There was no significant change in pH and osmolality values of any solutions. No precipitation or change in color was observed in any of the sample solutions. There was no significant loss of fentanyl or sufentanil over 14 days (4.3% and 4.1%, respectively). This study indicates that both drug solutions in the PCA systems are stable for a minimum of 14 days at room temperature.

Background The purpose of this study was to report the pharmacokinetics (PK) and tolerance profile of intraoperative intraperitoneal chemo-hyperthermia (IPCH) with oxaliplatin and irinotecan. Patients and methods Thirty-nine patients with peritoneal carcinomatosis (PC) of either gastrointestinal or peritoneal origin underwent complete cytoreductive surgery followed by IPCH with a stable dose of oxaliplatin (460 mg/m2), plus one among seven escalating doses of irinotecan (from 300 to 700 mg/m2). IPCH was carried out with the abdomen open, for 30 min at 43°C, with 2 l/m2 of a 5% dextrose instillation in a closed continuous circuit. Patients received intravenous leucovorin (20 mg/m2) and 5-fluorouracil (400 mg/m2) just before IPCH to maximize the effect of oxaliplatin and irinotecan. Results Irinotecan concentration in tumoral tissue increased until 400 mg/m2 and then remained stable despite dose escalations. It was 16–23 times higher than in non-bathed tissues. Increasing doses of intraperitoneal irinotecan did not modify the PK of intraperitoneal oxaliplatin, and the drug concentration ratio was 17.8 higher in tumoral tissue (bathed) than in non-bathed tissues. The hospital mortality rate was 2.5% and the non-hematological complication rate was 25%. However, grade 3–4 hematological toxicity rate was 58%. Conclusion Intraperitoneal heated oxaliplatin (460 mg/m2) plus irinotecan (400 mg/m2) presented an advantageous PK profile and was tolerated by patients, despite a high hematological toxicity rate.

Chronic renal failure is a common pathology. The high frequency of this disease in the general US population has been assessed in the NHANES III study. However, the frequency of chronic renal insufficiency among cancer patients remains unclear.316 cancer patients were in a one-month study, included regardless of their pathology, treatment (antineoplastic drugs used or programmed to be used, pretreated, or no treatment), or any other criteria.Among the patients, 287 (90.8%) had normal serum creatinine levels (110 micromol/l), i.e. a frequency of 9.2% for renal insufficiency in our population. However, when renal function was estimated by calculating creatinine clearance using the Cockcroft and Gault formula, 33% of the patients had an estimated GFR of less than 80 ml/min. Among these, 28% had a creatinine clearance ranging form 80 to 50 ml/min and 5% had a creatinine clearance of less than 50 ml/min. Renal insufficiency is frequent in cancer patients since almost one third of the patients present renal insufficiency. Furthermore, among patients with normal serum creatinine levels, one patient out of five has asymptomatic renal insufficiency.Therefore, it is of major importance that renal function be assessed by calculation of creatinine clearance using the Cockcroft and Gault formula in every patient, even when serum creatinine is within the normal range.

Background Advanced Systemic Mastocytosis (adSM) still have very poor prognosis. No standard of care is yet available. Midostaurin (PKC412) is a powerful TKI inhibit of VEGFR, FLT3 and most importantly the mutated CKITD816V and D816Y. Preliminarily results from an international phase II trial (Gotlib et al., Blood 2012) reported interesting results in this subgroup of patients. We report herein on the French experience of the compassionate use of PKC412 for the treatment of adSM and compare their outcome to matched adSM historical control patients (pts) who did not receive PKC412 (control group). Methods Over a period of 12 months (from August 2012, date of PKC412 Temporary Approval for Use in France), 22 pts received PKC412 and could be analysed. PKC412 100 mg twice daily was administered orally as continuous 28-day cycles until progression or unacceptable toxicity. Pts were evaluated according to the response criteria used in the recent phase 2 trial on midostaurin (Gotlib et al.). The overall survival (OS) of these 22 patients was compared to the OS of 42 age and WHO subcategories matched control adSM patients who were treated before PKC412 era. Results 2 female and 20 male pts with a median age of 65 years [35-84] received midostaurin. Overall, 21 pts (95%) with adSM (5 ASM; 2 MCL; 1 MCS; 12 ASM-AHNMD; 1 SSM-AHNMD,1) and 1 pt progressive SSM. Median number of C-Findings was 3 (0-4). Hepato- and/or splenomegaly were present in 20 (91%) and 21 (95%) pts. Median tryptase level was 204 ng/l (85-2000). Median haemoglobin level, platelet and PMN counts were respectively 10.1g/dl (6.8-13.9), 117G/L (22-289) and 2.8G/L (0.3-17). Twenty (91%) pts had cKIT D816V mutation, 2 pts had cKITWT. ASXL1 and TET2 were mutated in 3/7 and 2/7 tested, respectively. Steroids, 2-chlorodeoxyadenosine and Interferon were administered in 9, 4 and 2 pts prior to midostaurin. After a median exposure time of 6.9 [1.6-27.5] months, the overall response rate (ORR) was 77.2% including, major responses (n=13; 59%) (incomplete major in 8 pts, pure clinical in 5); partial response (n=5, 23%) (good partial in 3, minor partial in 2). Three patients were rapidly progressive ( The control group consisted in 42 pts with a median age of 65.5 years [23-84]. There were 17 ASM-AHNMD, 21 ISM-AHNMD, 2 SSM-AHNMD, 1 CM-AHNMD and 1 MCL patients. Median tryptase level was 107ng/l [19-501]. Median haemoglobin level, platelet and PMN counts were respectively 12.1g/dl [8-15], 200G/L [10-1036] and 4.6G/L [0.5-24.3]. Thirty-three (79%) pts had cKIT D816V mutation, 7 pts were cKITWT. ASXL1 and TET2 were mutated in 6/29 (21%) and 7/29 (24%) of pts tested, respectively. Steroids, 2-CdA, interferon, imatinib, thalidomide have been used in 19, 21, 6, 4, and 6 pts respectively. Survival distributions were estimated using the adjusted Kaplan-Meier method from the date of diagnosis to the last follow-up for the PKC412 treated and the control groups. Median survival time for PKC412 treated patients was significantly longer than control patients (p=0.04). Conclusion PKC412 is active in advanced SM. Despite the absence of complete remission, PKC412 gives survival advantage for patients with adSM over pts who did not receive the drug. Disclosures No relevant conflicts of interest to declare.

Aim: Beyond platinum-based first line chemotherapy for advanced cholangiocarcinoma (CK), second line 5FU-based treatments yield median progression-free survival (PFS) of Methods: This multicenter phase 2 study enrolled pts with locally advanced intrahepatic CK failing prior first-line platinum-based chemotherapy, ECOG PS 0-1, and adequate liver function with bilirubin 6.3 months (primary objective) other endpoints being PFS, response (RECIST 1.1 & Choi criteria), safety, pharmacokinetics (PK), and pharmacodynamic (PD) biomarkers (VEGFA, VEGFC, sKIT, HGF, SDF1, and osteopontin). Results: Among 51 pts yet enrolled in this ongoing trial, 34 are currently evaluable for safety and efficacy. Median age was 62 years (range 36–80), 19 females/15 males were included, and ECOG PS was 0/1 in 23/11 pts. With a median follow-up of 15.4 months and a median duration of treatment of 3.8 months, the median OS was 9.6 months [95%CI: 5.8-13.1], exceeding the primary objective. Five pts (15%) had partial responses and 24 stable diseases (71%) by RECIST (disease control rate 85%) with 10 pts having disease control > 6 months (range 6-14 months). Among 12 evaluable patients for Choi criteria, 11 (92%) had objective responses. Median PFS was 5.2 months. Frequently reported adverse events were grade (Gr) 1-2 asthenia (80% of pts), mucositis (80%), diarrhea (60%), and hand-foot syndrome (43%). Gr 3/4 asymptomatic hematological toxicity occurred in 24% of patients (neutropenia 8 pts, thrombocytopenia 7 pt), Gr 3 hypertension was observed in 7 pts, and Gr 3 asthenia in 4 pts. PK & PD data will be updated at the meeting. Conclusions: Sunitinib (continuous daily dose of 37.5 mg) is well tolerated and shows promising activity with 9.6 months OS in second line therapy in pts with intrahepatic advanced CK. Disclosure: J. Seitz: Honoraria Support from: Merck Serono, Novartis, Amgen, Sanofi-Aventis, Pfizer, Amgen SAS, Bayer-Schering-Pharma, Lilly; L. Fartoux: Honoraria Support from: Bayer and Bristol Myer Squibb; D. Malka: Grant Support from: Amgen, Roche, Merck Serono, Sanofi-Aventis Honoraria Support from: Merck Serono, Ipsen, Celgene, Novartis, Amgen, Roche, Sanofi-Aventis, Imclone, Teva, Keocyt, Boehringer-Ingelheim; M. Bouattour: Honoraria Support from: Bayer; E. Raymond: Honoraria Support from: Bayer, Pfizer, Novartis; S. Faivre: Honoraria Support from: Bayer, Pfizer, Novartis; All other authors have declared no conflicts of interest.

A global postproduction quality program was developed to secure chemotherapy infusion at the Gustave Roussy Institute. Despite rigorous procedures and computerized prescriptions, an analytical check was necessary to improve the quality of ready-to-use solutions of cytotoxic drugs in our Centralized Antineoplastics Reconstitution Unit. High-performance, thin-layer chromatography was selected as the analytical tool to assay 12 anticancer drugs. One of the analytical methods can separate 4 antimetabolite substances, i.e., fludarabine (FDB), cytarabine (CTB), gemcitabine (GTB), and fluorouracil (5 FU). For all infusion bags manufactured, up to 26 samples could be assayed per series using a double standard calibration (GTB and 5 FU).

The concentrations of teicoplanin in the sera and mediastinal and heart tissues of 23 patients undergoing cardiac surgery were measured after two regimens of teicoplanin administration. Intraoperative pharmacokinetic parameters were also obtained. Patients were randomized into two groups. Those in group 1 were given teicoplanin at 6 mg x kg(-1) intravenously at the time of induction of anesthesia. Patients in group 2 were given teicoplanin at 12 mg x kg(-1) during the same period. The maximum concentration in serum (71 +/- 20 and 131 +/- 44 mg x l(-1)), the minimum concentration in serum (3.6 +/- 1.3 and 6.8 +/- 2.1 mg x l(-1)), the area under the concentration-time curve (AUC) from 0 to 12 h (108 +/- 20 and 217 +/- 38 microg x h x ml(-1)), and the AUC from 0 h to infinity (154 +/- 36 and 292 +/- 77 microg x h x ml(-1)) were twice as high after 12-mg x kg(-1) injections as after 6-mg x kg(-1) injections. No differences in mean residence time (9.7 +/- 4.9 and 8.4 +/- 2.7 h) or terminal half-life (8.5 +/- 3.8 and 7.5 +/- 2.3 h) were observed. Teicoplanin penetrated mediastinal and heart tissues but not sternal bone, where the antibiotic was detectable in only 1 of 13 patients in group 1 and 2 of 10 patients in group 2. In group 1, 7 of 13 patients had teicoplanin concentrations in tissue that were lower than the MIC for 90% of the strains of potential pathogens tested (MIC90) that cause infection after cardiac surgery. All of the patients in group 2 but one had teicoplanin concentrations in tissue (other than in sternal bone) far in excess of the MIC90 for the potential pathogens. In conclusion, the 12-mg x kg(-1) regimen of teicoplanin is followed by a significant increase in teicoplanin concentrations in heart and mediastinal tissues and should be preferred to the 6-mg x kg(-1) regimen if teicoplanin is selected for antimicrobial prophylaxis in open heart surgery.

Abstract 4913 Background: Among tyrosine kinase inhibitors, midostaurin (PKC412) takes place in the treatment of patients presenting with advanced systemic mastocytosis and, specifically aggressive systemic mastocytosis (ASM) or mast cell leukemia (MCL) with or without associated clonal hematological non-mast cell lineage disease (AHNMD). Our knowledge of the pharmacokinetic (PK) behavior of this major drug remains unfortunately sporadic. We developed a powerful liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay, which is able to sensitively detect PKC412 as its two major metabolites CGP52421 and CGP62221 in human plasma. The method was validated in clinical situation and, especially in the aim to monitor plasma concentrations of PKC412 in 11 patients, already enrolled in a Phase II clinical trial (Novartis Lab.). In addition, we also determined the PK profile of PKC412 at steady-state for one specific patient. Methods: A sensitive LC-MS/MS assay was developed for the simultaneous quantification of the product PKC412 and its two major metabolites in human plasma. Midostaurin-D5 was used as internal standard. The method was then validated in accordance with international guidelines and within the bioanalytical ±15% acceptance limits. In any case, PKC412 regimen was 100 mg bid. A total of 61 blood samples were collected from 11 patients and, between 15 min to 12 hrs following oral administration. Sampling took place over a period of 1 to 924 days following the beginning of treatment. PKC412 residual concentration at steady-state, range and mean concentration and, intra- and inter-individual variability coefficients were also calculated. On the other hand, a model independent method was used to determine the PK profile of PKC412 for a 12th patient (using 22 samples collected within 24 hrs): maximal concentration post-dosing (Cmax – ng/mL), time to Cmax (Tmax - hrs) were experimental values; terminal half-life (t1/2lz - hrs) and AUC0–12 were calculated during each 12 hrs interval post-dosing. Data are expressed as means ± SD. Results: This LC-MS/MS method is satisfactory for the key validation criteria i. e. trueness, bias, precision (repeatability and intermediate precision), accuracy and, linearity, over a range of concentrations of 75 to 2500 ng/mL for both PKC412, CGP52421 and, CGP62221. Patients monitored (8/11 males) were 57±14 yrs-old. All except one were treated for ASM±AHNMD (the last one had MCL), 10/11 patients carrying the ckit D816V mutation. The main characteristics at inclusion were as followed: body weight = 65±11 kg, BMI = 22±3 kg/m2, albumin = 31±6 g/L, GFR = 64±12 mL/min, bilirubin = 14±6 mg/L, GGT = 103±78 UI/L, ALP = 264±158 UI/L, tryptase = 412±411 μg/L. At the last follow-up visit i. e. between 54 to 924 day after initiation therapy, 8/11 patients were either in major or partial response with the following clinical picture: body weight 74±16 kg, BMI 24±4 kg/m2, albumin 40±2 g/L, DFG 59±11 mL/min, bilirubin 12±8 mg/L, GGT 56±57 UI/L, ALP 128±59 UI/L, tryptase 256±264 μg/L. It is important to point out that the tolerability profile was very good. The PKC412 residual concentrations value determined at steady-state were between 487 to 3254 ng/mL (1333±867 ng/mL). The intra- and inter-individual coefficients of variations were 18 and 65% respectively. About our 12th subject, major PK parameters were: Cmax = 3692±447 ng/mL, Tmax = 2, 5±2 hrs, t1/2lz = 95±88 hrs, AUC0–12= 34326±2197 ng/mL. hrs. Conclusions: The LC-MS/MS method is efficient and enables a sensitive and discriminative analysis of PKC412 and its major metabolites in human plasma. These first results highlight the high intra- and inter-individual variability of the PK behavior of the drug and, suggest the interest of its routine therapeutic monitoring vs. both adherence to the treatment and, its tolerability profile. Further, it has been shown that the 2 major metabolites of PK412 are active; under these conditions, we are working to the simultaneous therapeutic drug monitoring of the 3 compounds. Disclosures: No relevant conflicts of interest to declare.