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BACKGROUND: Accumulation of galactosphingolipids is a general characteristic of Fabry disease, a lysosomal storage disorder caused by the deficient activity of α-galactosidase A encoded by the GLA gene. Although many polymorphic GLA haplotypes have been described, it is still unclear whether some of these variants are causative of disease symptoms. We report the study of an inheritance of a complex intronic haplotype (CIH) (c.-10C > T, c.369 + 990C > A, c.370-81_370-77delCAGCC, c.640-16A > G, c.1000-22C > T) within the GLA gene associated with Fabry-like symptoms and galactosphingolipid accumulation. We analysed α-Gal A activity in plasma, leukocytes and skin fibroblasts in patients, and measured accumulation of galactosphingolipids by enzymatic methods and immunofluorescence techniques. Additionally, we evaluated GLA expression using quantitative PCR, EMSA, and cDNA cloning. RESULTS: CIH carriers had an altered GLA expression pattern, although most of the carriers had high residual enzyme activity in plasma, leukocytes and in skin fibroblasts. Nonetheless, CIH carriers had significant galactosphingolipid accumulation in fibroblasts in comparison with controls, and also glycolipid deposits in renal tubules and glomeruli. EMSA assays indicated that the c.-10C > T variant in the promoter affected a nuclear protein binding site. CONCLUSIONS: Thus, inheritance of the CIH caused an mRNA deregulation altering the GLA expression pattern, producing a tissue glycolipid storage. Acknowledgements. The authors gratefully thank Cesar Vallejo for technical support; Dr. Erika Fernandez-Vizarra for critical revision of the manuscript and Dr. Gracia Mendoza for immunofluorescence support. This study was supported by grant FIS (PI 09/02556) and by the Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), an initiative of the ISCIII.

It has been suggested that interleukin-17 (IL-17) plays a crucial role in the development of several autoimmune diseases. However, there are no data about the relationship between myasthenia gravis and IL-17. The aim of this study was to measure the concentration of IL-17 and determine whether levels depend on the severity of MG. Serum IL-17 concentrations were measured in 25 patients. IL-17 concentrations were higher in generalized MG compared with controls and correlated with anti-acetylcholinesterase receptor antibody titers.

Gaucher disease induces some metabolic abnormalities so increased serum ferritin appears in more than 60% at diagnosis. The storage of glucosylceramide in macrophages produces an inflammatory response with iron recycling deregulation and release of cytokines. Iron homeostasis is controlled by the circulating peptide hepcidin and its production is influenced by inflammatory cytokines. Iron damages cells by excess of catalyzing reactive oxygen species, removal of the excess iron has a positive influence on the response to treatment and survival in patients with iron overload. We have analyzed some inflammatory biomarkers of macrophage activation and related to the iron profile, including hepcidin and liver iron deposits determined by MRI, in 8 type 1 GD patients with hyperferritinemia. We have explored the changes in this profile after 4 months under therapy with two different iron chelators, deferoxamine or deferasirox, by evaluating response, adverse events and quality of life. We observed a significant reduction in serum ferritin and hepcidin levels and in liver iron deposits. No differences were observed in chitotriosidase activity, CCL18/PARC concentration and IL-4, IL-6, IL-7, IL-10, IL-13, MIP-1α, MIP-1β,TNF-α cytokine levels. After two years on follow-up, clinical and analytical data were improved and stable ferritin levels maintained less than 700 ng/dL.

It has been suggested that interleukin-17 (IL-17) plays a crucial role in the development of several autoimmune diseases. However, there are no data about the relationship between myasthenia gravis and IL-17. The aim of this study was to measure the concentration of IL-17 and determine whether levels depend on the severity of MG. Serum IL-17 concentrations were measured in 25 patients. IL-17 concentrations were higher in generalized MG compared with controls and correlated with anti-acetylcholinesterase receptor antibody titers.

Gaucher disase(GD), the most studied lysosomal rare disorder, is characterized by an inflammatory status involved in the high incidence of complications, immune impairment and comorbidities (infections, bone, pulmonary involvement, gammapathies and parkinsonism). The role of cytokines in this inflammatory state is partially known, and the modifications in this profile in GD patients under enzymatic replacement therapy have not been reported and neither included in clinical trial. Our group has reported some changes on the cytokine profile in patients with severe bone involvement and some inflammatory biomarkers of macrophage activation related to the iron profile. (Gervas 2015, Medrano 2015). However there are not specific studies related to different therapies. Here we are presenting the final outcomes of a multicentre prospective non interventional study to explore the changes in the biomarkers of immune response in a cohort of Spanish type 1 Gaucher disease patients after one year on Velaglucerase alfa therapy. Patients and Methods: A total of 17 type 1 GD from 15 centers, were included in a prospective protocol following these criteria: symptomatic patients of both sexes, aged older than 4 years, naïve or previously treated but without ERT at least one month previous to be included. The study was approval by ethical committees and designed according Helsinki declaration rules; every one patient signed the informed consent and commitment to complete all study. Current recommendation for ERT with Velaglucerase alfa were followed in every centre. The study included hematological parameters, goals of therapy assessment, bone disease assessment were carried out in every visit and bone marrow MRI evaluated by Spanish-MRI score (Roca et al 2004) and densitometry were performed at baseline and in the final visit according the availability in each centre. GD biomarkers (Chitotriosidase, CCL18/PARC), proinflammatory biomarkers (ferritin level, serum protein electrophoresis and gammapathy profile including free light chain analysis) and the following cytokine profile: IL-10, IL-13, IL-4, IL-6, IL-7, Mip1a, Mip1b,TNFa, performed at baseline and 12 months after therapy. Results: General characteristics: 9 males, 8 females, mean age: 37.5 years (9-72). 3 splenectomized patients (17.6%); genotype: 3 N370S homozygous one heterozygous for N370S/L444P and the rest heterozygous for N370S/other. Seven patients (41.2%) have previous history of bone disease complications. All patients received velaglucerase alfa (30U/kg-60U/kg) iv every two weeks for 1 year in every day clinical practice, and achieving an bjective response on goals of disease (hemoglobine, platelets count, spleen reduction and clinically asymptomatic),bone marrow burden decrease and stabilization were registered by MRI in 14 patients, bone mineral density improvement were reported during the year of therapy in those patients. No infusion reactions were reported, neither antibodies against velaglucerase alfa; concerning to biomarkers, a reduction or stabilization of CT activity and CCL/18PARC concentration were significantly reduced (p=0.011; p=0.041 respectively), no modification in the ferritin concentration were registered, no monoclonal gammopathy were found but polyclonal gammopathy were observed in the majority of patients, a tendency of normalization were detected. The cytokine profile showed a decrease in all inflammatory cytokines tested however for Mip1a (p=0.017) and TNFa (p=0.023) a significant reduction were achieved. Table 1 Conclusion: Velaglucerase alfa is a well-tolerated therapy in every day clinical practice, with a positive impact in the immune response with a significant decrease on the inflammatory state reflected through the cytokine reduction and preventing development of bone complications in this cohort independently of previous therapies. Acknowledgments: This work was partially supported by a grant from Shire and FIS: PS12/01219 Table 1. Comparative reduction of cytokines between baseline and 12 months IL10 IL13 IL4 IL6 IL7 Mip 1a Mip 1b TNF a Chi X2 3.415 0.000 2.668 1.606 1.545 8.159 3.308 4.263 grade 2 2 2 2 2 2 2 2 significance 0.181 1.000 0.263 0.448 0.462 0.017 0.191 0.023 A significant decrease on Mip1a (p=0.017) and TNFa (p=0.023) and the end of study Disclosures No relevant conflicts of interest to declare.

Background: For molecular assessment in CML, NCCN and European LeukemiaNet had defined the cut-off values. Until now the gold standard for BCR-ABL quantification is the real time quantitative polymerase chain reaction (RQ-PCR), and this technique has a limited sensibility to MR4.0 or MR4.5 in the majority of laboratories worldwide. There are several discontinuation studies for CP-CML patients under imatinib therapy who achieved and sustained at least MR4.0 during 2 years or more, and the fact of achieve a deeper response and the time during this response had been the major factors for good outcomes in these studies, now situated in around 40% of success. The droplet digital PCR (ddPCR), and other kind of digital PCR as the nanofluidic ddPCR, had showed more sensitivity for detect minimal amounts of BCR-ABL transcripts in patients with undetectable BCR-ABL transcripts by RQ-PCR, leading a window for investigation in this field. Aims: to evaluate the usefulness of the ddPCR in detect the presence of transcripts of BCR-ABL in patients with CP-CML under TKI therapy compared with RQ-PCR. Material & methods: A total of 54 samples from 32 patients with CP-CML under TKI therapy were analyzed. Sampling was performing obtaining one sample in EDTA for RQ-PCR and other in a TempusTM tube for ddPCR. The RNA was obtained from peripheral blood using TempusTM Spin RNA isolation kit according manufacturer recommendations. The analysis by ddPCR for each sample was done using 5 μg of the obtained RNA and processed in Qx100 Droplet Digital PCR, Biorad; using primers and probes described by Gabert et al., 2003 using ABL1 for control; the results was analyzed using QuantaLife Biorad software and expressed in absolute copies of BCR-ABL and in ratio BCR-ABL/ABL1. Only experiments with >32,000 copies of ABL1 were considered as valid for ddPCR to consider at least MR4.5. The use of a dual-determination in the same experiment with the ddPCR; one for BCR-ABL and other for ABL1 permit us to relativize the experiment and create results expressed in ratios easy to compare with the results obtained during the analysis by RQ-PCR for the same samples. Period of Study: August-2013 to Jun 2014. Results: The final analysis shows that 26 of the 54 analyzed samples presents values of BCR-ABL/ABL1 ratio within MR4.0 or more in the RQ-PCR test, of them 15 with MR4.5 values and 11 with MR4.0. However only 13 (50%) shows ratio values according to MR4.5/4.0 in the ddPCR analysis. The other 13 samples were compatible to MR3.0 or inferior. The samples that for RQ-PCR obtained values of MR4.5 or more (15), in only 6 (40%) a MR3.0 or inferior value of ratio were obtained by ddPCR, however in samples with values of MR4.0 by RQ-PCR (11), the proportion of samples with MR3.0 or inferior was higher, 63.6% (7 samples). See Table 1. The rest of the samples (28) the molecular results were within MR3.0 or inferior response values by RQ-PCR and for ddPCR this was confirmed, except in 3 samples in with ddPCR show a MR4.0 or superior response value. This led us to hypothesize that the use of ddPCR for confirms the response of the patients with MR4.0 or superior would improve the follow-up of the patients, and the success ratio for discontinuation. Comments: The ddPCR offers a new way to assess CP-CML patients, in especially in patients with >MR4.0 molecular response, and would offer a more accurately tool to select patient for discontinuation trials. Actually around 60% of patients who discontinues therapy lost MR4.0 or MR4.5 at 6 months, in our cohort, 50% of samples classified as MR4.0 for RQ-PCR had MR3.0 or less by ddPCR and would not be suitable for discontinuation trials; maybe if we use ddPCR as the standard for select patient to discontinuation less patients will lost the response. Table 1. Molecular response correlation between RQ-PCR and ddPCR ddPCR(RM4.0) Total RQ-PCR (RM4.0) 7 (63.6%) 4 (36.4%) 11 RQ-PCR (RM4.5) 6 (40.0%) 9 (60.0%) 15 Total 13 13 26 Disclosures No relevant conflicts of interest to declare.

Gaucher disease (GD) is characterized by a latent chronic inflammatory macrophage activation expressed by an imbalance of pro-inflammatory cytokines, hyperferritinemia, hypergammaglobulinemia, altered calcium homeostasis and metabolic syndrome. The incidence of Monoclonal Gammopathy of Uncertain Significance (MGUS) and multiple myeloma (MM) is increased in GD1 patients (3.5-12.5). The hypothesis is that deregulation of immune system resulting from glucocerebroside accumulation may influence the development of hematological malignancies. However, the pathological mechanisms that influence in MM development in GD1 patients are unknown. Cytokines produced secondary to glucocerebroside accumulation, may be a critical factor to explain the pathogenesis of MM in GD. This cytokine environment could trigger lymphocyte expansion and ultimately transformation. Objective: To analyze and compare the proinflammatory cytokines profile among MGUS, MM and GD1patients. Methods: Between February-May 2014 we have analyzed a panel of eight cytokines (IL4, IL6, IL7, IL10, IL13, MIP1α, MIP1β and TNF) by Luminex®100 platform and Millipore cytokine kits. The analysis was performed in plasma samples stored at diagnosis in Aragon Biobank. We have studied four cohorts: 71 healthy controls, 38 GD1 patients (36.8% women, mean age 39.2 years, range: 17-79), 36 MGUS (58.3% women, mean age: 75 years, range 53-89), 11 IgGk; 6 IgGL; 5 IgAk; 8 IgAL; 3 IgMk; 3 doble IgG+IgA and 49 MM (42% women, mean age 68 years, range 39-89), 20 IgGk; 5 IgGL; 8 IgAk; 5 IgAL, 8 ligth chain disease and 3 doble IgG+IgA. Concerning ISS classification 44.0% MM patients had a score 1, 24.3% score 2 and 31.7% score 3. All patients with MGUS or MM had been diagnosed consecutively during 2009 and followed until now. Clinical and analytical data of MGUS and MM were obtained from the records of the Hematology Department; and the data of GD1 patients from the Spanish Registry (FEETEG). Data were analyzed using non-parametric tests (Mann-Whitney-U and Kruskall-Wallis). Results: After 5 years in follow-up 21 MM patients had died (42.8%) and 8 in the MGUS group (22.2%), 4 MGUS developed a MM (11.1%); and 8 another neoplasia (22.2%); in MM patients 7 developed a second tumor (14.2%). Between GD1 patients one developed a MM and two a solid tumor (7.9%). Concerning the cytokine analysis, a significant difference (p Conclusions: These results support that GD1 and MM patients share similar proinflammatory profiles. The chronic cytokines increase in GD patients would contribute to the development of malignancies in these patients. GD1 and MM share skeletal complications, MIP-1α as osteoclast-activating factor could be a good biomarker during follow-up of GD1 or MGUS to detect patients in risk to develop bone complications. MIP-1α is described as significantly elevated in osteopenic MGUS patients(1). Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Abstract 3270 Introduction: Many of the mutations of the lysosomal acid b-glucosidase (b-glucocerebrosidase) associated with Gaucher disease (GD) translate into enzymes that retain partial catalytic activity in vitro but exhibit impaired cellular trafficking as a consequence of aberrant folding. Current investigational therapeutic strategies for include the development of ligands of the enzyme capable of promoting those conformational changes that are required for efficient folding, restoring trafficking. Although somewhat counter intuitive, competitive inhibitors of this b-glucocerebrosidase, at subinhibitory concentrations, can increase steady-state lysosomal levels of active enzyme through this rescuing mechanism, acting as “pharmacological chaperones”. At the massive lysosomal substrate concentration, the inhibitor would be replaced from the active site of the enzyme and the metabolic activity recovered. However, most of the pharmacological chaperones under study are iminosugars that behave as broad spectrum inhibitors, inhibiting simultaneously several glucosidases, which represents a serious inconvenient for clinical applications. An additional problem is that iminosugars and their derivatives are not active as pharmacological chaperones for glucocerebrosidase mutations located outside the domain containing the active site and are associated with neurological involvement, as the L444P mutation. Aim: The aim of this work is to present molecules with a high binding specificity towards b-glucocerebrosidase, with a high ratio of chaperone versus inhibitor activity and capable of producing an increased in the levels of mutant enzymes associated with Gaucher disease, including mutations located outside the catalytic domain. Methods: Different bicyclic derivatives of L-idonojirimycin were designed and chemically synthesized from D-glucose after in silico structural analysis and identification of the most favorable molecular features to interact with the active site of glucocerebrosidase. The chaperone potential of these compounds was evaluated at different concentrations in vitro using a cell model of GDcarrying the more frequent mutations in Gaucher disease, namely N370S and L444P. (P201230804). Results: The obtained results showed an increase in b-glucocerebrosidase activity at various chaperone concentrations, ranging from 1.96 to 4.98 folds for the L444P mutant and from 2.01 to 3.06 folds for the N370S mutation. Comments: The bicyclic derivatives of L-idonojirimycin could be considered as a therapeutic alternative for GD, mainly in patients with mutations located outside the active site of the enzyme and associated with neurologic involvement. Disclosures: Giraldo: Actelion: Membership on an entity's Board of Directors or advisory committees; Genzyme: Research Funding; Shire: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract 4913 Bone disease is one of the most frequent complications in Multiple Myeloma (MM), and as consequence produce irreversible and invaliding lesions. The pathogenesis of bone disease is not completely known. There are several publications about the influence of different cytokines in the bone remodelling, by break the balance between osteoclastic/osteoblastic activities. Aims of the study: to analyze a cytokine panel in a group of consecutive patients diagnosed as Monoclonal Gammopathy according the International Myeloma Working Group, 2003 criteria and to compare the results between MGUS and MM and patients with an without overt bone disease. Patients and methods Between June 2008-June 2009, a total of 87 patients were studied (46.0% females) in Miguel Servet University Hospital; mean age: 71.5 (range: 69.1-73.9), distributed in two groups: a) MGUS: 46; mean age: 72.8 (range: 69.7-75.8); b) MM: 41; mean age: 70.2 (range: 66.3-74.0). According subtypes distribution: a) IgG: 18(39.1%); IgA 16(34.8%), IgM: 9 (19.6%), double MC 3 (6.5%). b) Light Chain: 6 (15.0%) IgG: 21 (52.5%); IgA 11(27.5%), double MC 2 (5.0%); In b group the ISS(1: 9, 22.5%, 2: 21 52.5%, 3: 10 25.0%). Four different degrees of bone disease was established: 0: normal (68.5%); 1 osteopenia (9.6%); 2 osteoporosis (2.7%); 3 Lytic lesions (11.0%); 4 fractures (8.2%). The cytokine assay was performed in serum samples stored at -80°C at diagnosis, using Millipore human cytokines Kit according the established protocol by Luminex®100 platform. The profile cytokine panel used were: IL-4, IL6, IL-7, IL-10, IL-13, MIP-1a, MIP-1b, TNF-a. Previously we had compared the cytokine profile with a group of healthy population matched by age and gender. The results of the cytokine panel were compared between groups MGUS/MM, bone disease, immunochemical subtypes and ISS stage, haemoglobine, b2 microglobuline concentration, free light chain ratio in the MM group. Descriptive analysis and non parametric comparative test was performed using STATA SE v.10. Results For the global serie: IL10 concentration showed significant difference between patients with normal vs abnormal bone. The cytokine profile of MGUS vs MM was significant different for IL-4 (p=0.02), MIP-1a(p=0.03) and TNFa(p=0.003). The comparative analysis of profiles according ISS classification showed significant differences for MIP-1a(p=0.04), IL-13(p=0.02) and IL-6(p=0.07) and b2microglobuline vs TNFa (p=0.002). No significant differences were observed for immunochemicals subtypes, free k/l ratio and hemoglobine. Comments In our experience Luminex®100 technology is a sensible and accurate technique useful to determine cytokine profile in serum. A different significant cytokine profile was observed in patients with MGUS vs MM and different bone affectation, b2microglobuline and ISS. It is necessary to perform more studies with more patients in order to confirm these results. This work has been partially sponsored by a grant from CIBERER Disclosures No relevant conflicts of interest to declare.