ALBERT

Description: Gene transfer to airway epithelial cells is hampered by extracellular (mainly mucus) and cellular (tight junctions) barriers. Magnetofection has been used to increase retention time of lentiviral vectors (LV) on the cellular surface. In this study, magnetofection was investigated in airway epithelial cell models mimicking extracellular and cellular barriers. Bronchiolar epithelial cells (H441 line) were evaluated for LV-mediated transduction after polarization onto filters and dexamethasone (dex) treatment, which induced hemicyst formation, with or without magnetofection. Sputum from cystic fibrosis (CF) patients was overlaid onto cells, and LV-mediated transduction was evaluated in the absence or presence of magnetofection. Magnetofection of unpolarized H441 cells increased the transduction with 50 MOI (multiplicity of infection, i.e., transducing units/cell) up to the transduction obtained with 500 MOI in the absence of magnetofection. Magnetofection well-enhanced LV-mediated transduction in mucus-layered cells by 20.3-fold. LV-mediated transduction efficiency decreased in dex-induced hemicysts in a time-dependent fashion. In dome-forming cells, zonula occludens-1 (ZO-1) localization at the cell borders was increased by dex treatment. Under these experimental conditions, magnetofection significantly increased LV transduction by 5.3-fold. In conclusion, these results show that magnetofection can enhance LV-mediated gene transfer into airway epithelial cells in the presence of extracellular (sputum) and cellular (tight junctions) barriers, representing CF-like conditions.

Description: Abstract 1221 Poster Board I-243 Introduction A subset of Multiple Sclerosis (MS) patients shows a clinical trend to a fast deterioration of disability despite the use of approved drugs. New immunosuppressive agents are currently employed only in the early phase of the disease but in almost 10% of patients they either do not show any clinical/ radiological improvement or have to be halted for different reasons. Autologous HSCT has been reported as a promising approach for MS patients unresponsive to the available therapies but long term clinical and laboratory follow-up with a stringent MRI monitoring are not yet available. We report here the long term follow-up of a prospective phase II multicenter trial of the Italian GITMO-Neuro cooperative network. Method 21 MS patients (4 Relapsing Remitting and 17 Secondary Progressive) were enrolled between 1999 and 2004 in a prospective trial, aimed to monitor both clinical outcome and MRI imaging. Patients were shown to be refractory to conventional treatments, had a EDSS between 5.0 and 6.5 and at least 1 Gadolinium (Gd) enhancing area in brain MRI. PBSC were mobilized with Cyclophosphamide (4g/m2 ) and Filgrastim; patients were conditioned with BEAM plus rabbit ATG (Thymoglobulin®, Genzyme) and infused with unmanipulated graft. The effect of HSCT was evaluated with serial monthly Gd-enhanced brain MRI for a pretreatment period of 3 months and compared with serial monthly Gd-enhanced MRI imaging for the following 6 months. Subsequently, MRI scans were carried out at, +9, +12, +18 + 24 and at the last follow-up. Clinical outcome was evaluated by both EDSS assessment and number of clinical relapses after the transplant. The same MRI scanning protocol was used at each neuroradiological examination: T1 and T2 total lesion load, new T2 lesions, new hypointense lesions, Gd enhancing activity and progression of brain atrophy were evaluated. Results All patients showed a sustained engraftment with modest early side effects, as previously reported (Saccardi R. et al. Blood 2005 105:2601-07). At a median follow up of 7 years (range 4-10 years) no late effects were reported. Two patients had a clinical relapse 5 years after the HSCT, spontaneously recovered. Nine patients showed a clinical progression at a median of 30 months (16 – 66) after transplant, while the others are either stable (7) or improved (5), as compared with baseline. No patients received any immunosuppressive treatment after HSCT. At MRI examination total lesion load assessed by T2 and T1 sequences remained stable through the follow-up, and only 5 new T2 lesions appeared after HSCT. No Gd enhancing activity was ever observed after transplantation in the examined cases. Brain atrophy progression was higher in the first 2 years after transplantation and then decreased significantly in the following years Conclusions The clinical and MRI results of this prospective study are extremely positive, considering that the majority of cases remained stable at a median of 7 years after transplantation. MRI activity, as evaluated with Gd enhancing areas and the appearance of new lesions, was absent or negligible. Also brain atrophy, rapidly progressing early after the transplant probably due to the suppression of the inflammation, was thereafter shown to be in the range of MS patients. The duration of the follow-up and the stringent MRI methodology provide an evidence of the efficacy of this procedure in this subset of aggressive MS patients. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2322 New onset of AD have been reported after autologous and allogeneic HSCT and AD patients (pt) treated by HSCT may be at higher risk for developing a secondary AD. A single US centre study showed that more profound T cell depleting conditioning with antithymocyte globulin (ATG) or alemtuzumab enhanced the risk of secondary AD after HSCT for primary AD. We therefore aimed at specifying the incidence, nature and risk factors for secondary AD after autologous or allogeneic HSCT for a primary AD. Methods: Retrospective analysis of AD pts treated by HSCT as reported to the EBMT data management system ProMISe until November 2009. Each EBMT participating centre was asked to identify all pts having developed at least one AD and those not having developed AD after HSCT as controls with complete detailed information on HSCT and outcome of original disease and treatment and outcome of secondary AD for cases. Cumulative incidence curves were used to estimate incidence of AD considering death as a competing event. Associations of patients and graft characteristics with secondary AD were evaluated by multivariate analyses, using Cox proportional hazards. All tests were two-sided. The type I error rate was fixed at 0.05 to determinate factors associated with time to event outcomes. Results (median, extremes): Out of 338 patients from 26 centres in 12 European countries, 33 developed at least one secondary AD within 569 (19-1489) days after HSCT, 305 pts without secondary AD served as controls. Characteristics of both groups are in table 1. The cumulative incidence of secondary AD after HSCT for primary AD was 6.5 (±1) % after 3 years and 11.1 (±2) % after 5 years. Diagnoses of secondary AD were thyroiditis (n=13), autoimmune hemolytic anemia (AIHA) (n=4), autoimmune thrombopenia (n=3), acquired hemophilia (n=3), Graves' disease, rheumatoid arthritis, sarcoidosis, antiphospholipid syndrome and psoriatic arthritis in 2 pts each and myasthenia gravis and vasculitis in 1 pt each. Two pts developed two secondary AD. After multivariate analysis age younger than 33 years p=0.016 (overall median age at HSCT), primary systemic lupus erythematosus (SLE) p= 0.003, ex vivo manipulation p= 0.015 and HSCT performed after the year 2002 p=0.02 remained as risk factors for secondary AD. At last follow-up within 5.9 years (0.25-15 years) after HSCT, 28/33 pts with secondary AD were alive and 257/305 nonAD pts. 0ne Systemic Sclerosis pt and one Multiple Sclerosis transplanted pt died from secondary embolic antiphospholipid syndrome and acquired hemophilia respectively. 26 primary AD pts received specific therapy after HSCT for their secondary AD. The pts with acquired hemophilia received steroids plus cyclophosphamide and additional Rituximab or ivIG or plasmapheresis, those with immune thrombocytopenia responded to steroids. One pt with thrombocytopenia followed by AIHA needed additional immunosuppression and Rituximab. Two pts with AIHA received steroids, cyclophosphamide and additional Rituximab, and one case also ivIG and ultimately allogeneic HSCT for secondary AID. At last follow-up, 12 pts remained in remission of secondary AD without treatment, 15 pts needed ongoing therapy, 3 pts had persistent secondary AD without therapy. This first multicenter study after HSCT for primary AD showed that secondary AD occurred in 10% of 338 pts after HSCT and led to death in 2 pts. Younger age and SLE as primary AD were significant risk factors for secondary AD, but contrary to previous single centre US experience, conditioning with ATG or alemtuzumab did not increase the secondary AD risk. Secondary AD after HSCT should be added to the post transplant follow up clinical parameters. Disclosures: Lenhoff: Celgene: Honoraria.

Description: Abstract 334 Background: Multiple Sclerosis (MS) is a chronic inflammatory demyelinating disease believed to be mediated by autoreactive lymphocytes that invade the Central Nervous System and cause oligodendrocyte, axonal and neuronal damage as well as glial scarring.to and resulting in demyelination, neuronal death and brain atrophy. Hematopoietic Stem Cell Transplantation (HSCT) has been tried in the last 15 years as a therapeutic option in patients with a poor prognosis autoimmune disease not responding to conventional treatments. Worldwide ≥600 patients with MS have been treated with HSCT, most of them having been recruited in small, single center, phase 1–2 uncontrolled trials. Clinical and MRI outcomes from case series reports or Registry-based analyses suggest that a major response is achieved in most patients; quality and duration of response are better in patients transplanted earlier in the relapsing-remitting phase. The intensity of the immunosuppression from transplant treatment may be determined by several factors, such as the use of chemotherapy in the mobilization regimen, the intensity of the conditioning regimen and ex-vivo T-cell depletion. Methods: We report here the Italian multi-center experience on 74 MS patients treated with AHSCT between 1996 and 2008, all mobilized with Cyclophosphamide/G-CSF and conditioned with BEAM and rabbit ATG. Clinical and MRI outcomes were reported to the Italian Registry; the median follow-up is 48.3 (range 30–210) months. All patients clinically deteriorated in the year prior to HSCT, with an increase of Extended Disability Scale (EDSS) of at least one point (average EDSS change=1.5 points, range=1–9), Results: Two patients (3.3%) died for transplant-related causes. At 5 years after the transplant, 66% of patients remained stable or improved. Progression free survival (PFS) was slightly better in relapsing-remitting (RR) (5 years PFS=71%) than in secondary-progressive (SP) forms (5 years PFS=62%, p=0.28). Amongst patients with a follow up longer than 1 year, 8 out of 25 RR subjects (31%) had a 6–12 months confirmed EDSS improvement 〉 1 point as compared to 1 out of 36 (3%) SP patients (p=0.009), Figure 1. Out of 18 cases with a follow up longer than 7 years, 8 (44%) remained stable or had a sustained improvement whilst 10 (56%), after an initial period of stabilization or improvement with a median duration of 3.5 years, showed a slow progression of disability. Conclusions: This study shows in a large cohort of patients with a long follow-up that AHSCT with BEAM/ATG conditioning regimen has a profound effect in suppressing disease progression in aggressive MS cases, unresponsive to conventional therapies. Indeed it results in a sustained improvement of the disability, free of immunosuppression, in a significant amount of RR patients. Clinical improvement is scarcely reported in MS literature as it is rather infrequent with conventional treatments; nevertheless it results in a stable increase of the quality of the life in this subset of young patients and should be considered in the choice of a therapeutic strategy in the early phase of MS. Disclosures: Cuneo: Roche: Consultancy, Speakers Bureau.

Description: To specify the incidence and risk factors for secondary autoimmune diseases (ADs) after HSCT for a primary AD, we retrospectively analyzed AD patients treated by HSCT reported to EBMT from 1995 to 2009 with at least 1 secondary AD (cases) and those without (controls). After autologous HSCT, 29 of 347 patients developed at least 1 secondary AD within 21.9 (0.6-49) months and after allogeneic HSCT, 3 of 16 patients. The observed secondary ADs included: autoimmune hemolytic anemia (n = 3), acquired hemophilia (n = 3), autoimmune thrombocytopenia (n = 3), antiphospholipid syndrome (n = 2), thyroiditis (n = 12), blocking thyroid-stimulating hormone receptor antibody (n = 1), Graves disease (n = 2), myasthenia gravis (n = 1), rheumatoid arthritis (n = 2), sarcoidosis (n = 2), vasculitis (n = 1), psoriasis (n = 1), and psoriatic arthritis (n = 1). After autologous HSCT for primary AD, the cumulative incidence of secondary AD was 9.8% ± 2% at 5 years. Lupus erythematosus as primary AD, and antithymocyte globulin use plus CD34+ graft selection were important risk factors for secondary AD by multivariate analysis. With a median follow-up of 6.2 (0.54-11) years after autologous HSCT, 26 of 29 patients with secondary AD were alive, 2 died during their secondary AD (antiphospholipid syndrome, hemophilia), and 1 death was HSCT-related. This European multicenter study underlines the need for careful management and follow-up for secondary AD after HSCT.