ALBERT

Description: Classic Hodgkin Lymphoma (cHL) is a germinal center derived lymphoma with 8,500-9,000 new cases/year diagnosed in the US. Despite 90% stage I cHL patients can respond to current systemic therapy, this drops to 60%, when diagnosed in advanced stages. Furthermore, 20-30% of diagnosed patients, would be refractory or would relapse and have a poor prognosis. Refractory and relapsed disease (RRD) is currently the challenge when treating cHL patients. There is no specific therapy to offer rather than rescue chemotherapy schemes, which fails in 50% of the cases and associates with high risk severe toxicity. This highlights the need to deeper understand the cHL molecular biology, the screening for molecular markers suitable to identify the risk of refractory and relapse disease and specific therapeutic directed-targets. We have previously reported that the alternative NFkB pathway, mediated by Rel-B and NIK (NFkB Inducing Kinase), plays an important role in cHL survival. Its constitutive activation sustains high BCL2 expression levels and seems to be involved in the RRD. BCL2 was found as a specific Rel-B target gene in cHL cells by ChIP-Seq (Chromatin Immunoprecipitation sequencing) and expression arrays. BCL2 exogenous expression was enough to partially rescue the death induce in cHL cells, which highlight the relevance of this alternative NFkB pathway target gene. Since the BCL2 data was obtained in human cHL cell lines established from patients with refractory and relapsed disease, we decided to analyze whether mediators of this pathway and BCL2 could be useful as prognosis markers and would represent potential targetable factors in both refractory and relapsed disease. We analyzed NIK and BCL2 citoplasm expression in Hodgkin Reed-Sternberg cells (HRS) in the lymph node biopsies of 113 cHL naïve of therapy patients by inmunohistochemistry [52 female Md age and (range) 36 (6-88), 61 male 40.7 (9-78)]. The follow-up period range from 6 to 136 months. The univariate analysis showed no correlation between NIK or BCL2 expression and the prognosis clinical and pathological parameters, including the PET Scan indicated at the end of the first line treatment, neither the molecular markers routinely assayed. The statistical significance was maintained in multivariate analysis (Logistic and Cox Regression p=0.01). NIK expression did not associate with prognosis but the BCL2 expression level correlated with lack of response to conventional therapy and both early and late disease progression. The survival analysis, using the Kaplan-Meir curves, showed that patients with ≥60% positive HRS cells had a shorter disease-free survival (DFS) [Log Rank Test (Mantel Cox) p=0.002] and a reduced overall survival (OS) [Log Rank Test (Mantel Cox) p=0.02]. L1236, U-H01, KM-H2, SUPDH1 and L540, human cHL cell lines that express BCL2 protein, were sensitive to venetoclax, a specific BCL2 inhibitor. The drug induced a cell cycle arrest in S-Phase when treated with 1uM each 24 hours during 10 days, as compared to wild type cells and cells treated with the vehicle. In summary, we found that the alternative NFkB pathway plays a role in the refractory and relapsed classic Hodgkin Lymphoma disease, being BCL2 one of its key downstream target genes. BCL2 can be used as a prognosis marker determined by routine immunohistochemistry at diagnosis of the primary disease. BCL2 expression correlated with refractory disease to first line conventional therapy and disease progression. Based on the venetoclax effect in cHL cell lines we believe BCL2 directed-therapy in cHL should be considered in the subgroup of cHL patients that express this protein in ≥60% HRS cells in the lymph node biopsy performed at diagnosis. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: venetoclax used to specifically block BCL2.

Description: INTRODUCTION: A sizable proportion of elderly acute myeloid leukemia (AML) patients receive frontline hypomethylating agents (HMAs), namely azacitidine (AZA) and decitabine (DAC), as they are deemed unfit for intensive chemotherapy (ICT) by their treating physicians. A foreseeable high early death (ED) rate and lack of overall survival (OS) benefit under ICT are the main drivers for this decision. Several groups have published different predictive tools for ED or OS in elderly patients receiving ICT but, since ED in patients treated by HMAs is lower, the research activity has been restricted to OS in this population. METHODS: 415 elderly AML patients (264 M, 152 F) aged 61-90 receiving frontline HMAs (AZA 297, DAC 118), either in daily practice or within clinical trials (AZA 27, DAC 17), with complete relevant clinical information (see Table I) were available from the PETHEMA epidemiologic AML registry (NCT02006004). We analyzed the predictive value for ED (8wk) of the prognostic factors for OS/ED in AML included in the Walter, MRC/LRF, ALFA and ALMA scoring systems, namely age, WBC count, performance status (PS), MRC 2010 cytogenetics, platelet count and secondary disease, as well as the type of HMA. The potential predictors were categorized following previous published models (Walter, MRC/LRF, ALFA, ALMA). Cumulative early death rate at 8 weeks was calculated by the life-time method and the relevant strata were tested for univariate significance by the Wilcoxon test. All significant covariates were included in a Cox multivariate regression model and those significant for death at 8wk were included in a new predictive tool (HMA-EDS). Patients were assigned randomly in a 1:1 ratio to a training cohort (TC) and a validation cohort (VC). The different scoring systems (Walter, MRC/LRF, ALFA, ALMA, HMA-EDS) were checked for their prognostic impact on ED. Finally the 95% CI for the expected death rate at 8wk for the different strata of the new model was calculated for the full patient series. RESULTS: 51 patients out of 415 died and 13 were lost to follow-up before day 56 (cumulative ED rate at 8wk 13%, 95%CI 9-17%). Age, cytogenetics, secondary AML, platelet count and type of HMA were not significantly associated to ED. PS and WBC count strata confirmed their prognostic utility both in univariate and multivariate analysis (Table II). We developed the HMA-EDS by adding WBC (cutoffs 10 and 50, scores 1/2/3) and PS (0-1/2-4, scores 0/1) that classified patients in low-risk (score 1-2/ 84.6% of patients) and high risk (3-4/ 15.4% of patients) strata. When the prognostic utility for ED in the TC and the VC for the different scoring AML systems were checked, only HMA-EDS predicted ED in both cohorts (see Table III). The new EDS discriminates 2 different strata for ED at 8wk in unfit AML patients treated by HMA (see Figure 1 & Table III), namely a lower-risk group (ED rate 10%, 95% CI 6-14) and a high-risk group (ED rate 26%, 95% CI 14-38). CONCLUSIONS: WBC count and PS are the main predictors for ED in unfit AML patients treated by HMAs. A new tool (HMA-EDS) discriminates two different risk groups and supersedes other previously published prognostic systems (Walter's, Wheatley's MRC/LRF, ALFA and ALMA) for this purpose. This score could be useful to select patients for front-line HMA or even HMAs-based combination therapies, given that several cycles are usually needed to achieve a clinical response. We suggest that other patient-related covariates such as geriatric assessment be checked in future studies. Disclosures Ramos: Daiichi Sankyo: Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria; Abbvie: Honoraria. Fernandez:Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Teva: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Description: The recent success of immunotherapy using chimeric antigen receptor modified T cells (CAR T) in B-cell malignancies highlights the potential of these cytotoxic "drugs" for cancer therapy. CAR T therapies generally rely upon manufacturing approaches that include prior T cell activation through engagement of the TCR and costimulatory receptors followed by ex vivo expansion of patient-derived T cells over days to weeks. We previously reported that CD3/CD28 stimulation prior to transduction promotes progressive T cell effector differentiation over time in culture with loss of CAR T cell potency (Ghassemi et al. 2018 PMID: 30030295). Since cell division is not a prerequisite for lentiviral vector-mediated gene delivery, we hypothesized that lentiviral transduction of quiescent T cells without prior activation will enhance engraftment and persistence of CART cells that is associated with long-term leukemia control. Here, we show that functional CD19-specific CAR T cells (CART19) can be generated in as little as 24 hours using lentiviral vectors without the need for prior T cell activation. We showed using a non-optimized process that a mean of 6.5% (range 2%-10%) of freshly isolated quiescent T cells can be transduced using an infrared red fluorescent protein (iRFP)-expressing lentiviral vector with slower kinetics of expression compared with activated T cell transduction (peak at 96 hrs vs. 48 hrs for quiescent and activated T cells, respectively). Although substantially less efficient compared to activated T cells, transduction was detected across all T cells subsets with central memory T cells showing the greatest transduction efficiency with a mean of 4-fold greater transduction compared with naïve T cells. Somewhat unexpectedly, CART19 cells generated from quiescent T cells using a CD19-specific CAR vector showed a 3-5 fold greater transgene expression compared with iRFP vectors transduced at similar MOI. However, we show that CAR expression can occur in quiescent T cells even without reverse transcription or integrase function, so called "pseudotransduction". Importantly, we show that this CAR expression produces T cells with cytolytic activity and effector cytokine production in response to antigen that is similar to activated and transduced CAR T cells. Using the well-characterized Nalm6 model of acute lymphoblastic leukemia, we show that CART19 cells generated by transduction of quiescent T cells for 16 hours followed by washing to remove vector exhibit dose-dependent anti-leukemic activity that is durable with injection of as little as 2x105 total T cells. We estimate the latter to contain ~2x104 T cells with integrated lentiviral vector based upon transduction efficiency determined in studies using non-tumor bearing mice. (Fig 1). In summary, our results support the need for further investigation of CAR T cells that are generated using engineering of quiescent T cells. Taking advantage of the ability of lentiviral vectors to transfer genes to quiescent T cells, the highly abbreviated and simplified manufacturing approach described here has the potential to enhance therapeutic potency while also substantially reducing the materials and labor costs associated with current manufacturing approaches that use activated and expanded T cells. In addition, the rapid nature of this manufacturing has the potential to extend the population of patients that may be treated with these therapies by shortening the interval between aphaeresis collection and re-infusion of CAR T cells, which prevents the treatment of some patients with rapidly progressive disease. Disclosures Ghassemi: Novartis: Patents & Royalties. Milone:Novartis: Patents & Royalties, Research Funding.

Description: Leukemia relapse occurring in donor cells, so called donor cell leukemia (DCL) after allogeneic hematopoietic stem cell transplantation has been previously reported in the literature. Some authors have suggested that the development of DCL is perhaps a more common occurrence than traditionally thought. Donor cell myeloma (DCM) seems to be less frequent than DCL. This 46-year old male when first seen in 2000 was diagnosed with stage IIIa multiple myeloma. A monoclonal IgA kappa spike was recorded at diagnosis. Treatment with melphalan and prednisone was delivered every four to six weeks for a total of 22 courses. Fourty months after the initial diagnosis, an M2 acute myelogenous leukemia was identified. Treatment with chemotherapy resulted in complete remission. Matched UCB cells were localized at the London Cord Blood Bank. The UCB belonged to a male product of a white western European mother and a black Nigerian father who was a carrier of hemoglobin S. Hemoglobins A, F and S were detected in the UCB, consonant with sickle cell trait. The patient was allografted employing the "Mexican" NST conditioning regimen, granulocyte count recovered to more than 0.5 x 109/L on day 14, with the platelet count never dropping below 20 x 109/L. On day +40, the polymorphic microsatellite markers revealed mixed chimerism. The hemoglobin S gene was identified on day +20 and on day +60, full chimerism was shown. Cyclosporine A was stopped on day +350. The patient returned 170 months after the transplant with low back pain and the bone marrow aspiration disclosed 80% abnormal plasma cells, an IgA kappa monoclonal spike of 3.1 gr/dl, and complete chimerism. Malignant plasma cells were sorted by means of flow cytometry before genetic fingerprinting; cells were stained with an admixture of fluorescent monoclonal antibodies and cells co-expressing dim CD45, bright CD38 and CD56 were sorted out to ≥99% purity. Sorted cells were shown to have donor origin (Figure 1). The patient was treated with thalidomide, dexamethasone and bortezomib and the monoclonal spike disappeared; an autologous stem cell transplant is planned. Most people consider that the development of a malignancy in the cells of the donor is a rare event and very few prospective studies have analyzed the real prevalence of this phenomenon. Prospectively, we have found that 7% (95% CI 2.9 to 13.6%) of patients with leukemic activity after an allogeneic graft do have a donor cell-derived leukemia; this figure contrasts with those described elsewhere in non-prospective studies. A major problem in the analysis of donor cell derived malignancies is that demonstration of the donor cell origin of malignant activity. In this case, the demonstration of DNA of the donor in the fluorescence-activated sorted malignant plasma cells is indicative of the origin of the myeloma cells. Interestingly, the immunoglobulin type produced by the initial myeloma cells is the same as that of the donor-cell myeloma; Despite being two myelomas producing the same immunoglobulin subtype, both should be considered as de novomalignancies and as such, treated; we have previously shown that donor cell leukemias do have a response when treated as de novo, non-secondary leukemias. To our best knowledge, this is the second report of DCM following allogeneic HSCT. Prior to this case, Kim et al reported a DCM after an allogeneic transplant in a patient with refractory anemia with ringed sideroblasts. Previously, two cases have been reported of donor-origin MM, but they occurred in patients who underwent solid organ transplantation of the kidney and heart-lung. Kumar et alreported a case of DCM developing after unrelated allogeneic HSCT in the both donor and recipient but they did not conducted a comprehensive molecular cytogenetic study. In the case published by Maestas et al, an abnormal proliferation of plasma cells was identified in the donor, thus making possible that a malignant plasma cell clone was already present in the donor stem cells. In summary, we have clearly shown that this patient has had three different malignancies: 1) De novomultiple myeloma, 2) Secondary acute myelogenous leukemia and 3) De novodonor cell-derived multiple myeloma. The mechanisms involved in these episodes could be useful to better understand tumorigenesis. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: The diagnostic criteria for polycythemia vera (PV) has recently been updated by the World Health Organization (WHO). The criterion for erythrocytosis has been modified downwards: hemoglobin (hb)〉 16.5 g/dL or hematocrit (hto)〉 49% in men and hb〉 16 g/dL or hto〉 48% in women. This reduction increases the potential number of patients that would be test for JAK2 V617F mutation if PV is suspected. The V617F mutation in the JAK2 gene is present in 95% of cases of PV. It is estimated that the prevalence of this mutation in the general population is around 0.2%. Our aims are to determine the prevalence of JAK2 V617F in individuals with erythrocytosis according to WHO2016 criteria and to find prognostic factors that could help to identify patients with PV. Methods: We prospectively studied all hemograms performed in our laboratory during 7 nonconsecutive days. Variables studied were hb, hto, leukocytes, neutrophils, platelets, MCV, MCH, MCHC and RDW. JAK2 V617F mutation was studied in all males that had hb〉 16.5 g/dl or hto〉 49% or females that had hb〉 16 g/dl or hto〉 48%. JAK2 V617F mutation was studied by PCR assay in which an amplification control fragment and the JAK2 mutant allele were simultaneously amplified. All positive samples were confirmed by quantitative real-time PCR in a reference laboratory. Positive results were considered when the JAK2 V617F allele ratio was ≥ 0.7. The variables collected were correlated with the result of the JAK2 test in a univariate way. The T-Student test was used for the quantitative variables and the Chi-square test for the categorical variables. For the cell count variables, the Mann-Whitney U test was used. Results: A total of 15366 HG were analyzed. 1271 (8.3%) met the inclusion criteria for erythrocytosis. JAK2 V617F was performed on 1001 samples (270 samples were not suitable for the PCR assay due to low quality). Twelve samples (1.2%) were positive for JAK2 V617F mutation. However, 5 samples were excluded due to a known diagnosis of myeloproliferative neoplasm. Therefore, finally prevalence of JAK2 V617 mutation in 996 patients that met WHO erythrocytosis criteria was 0.8% (8/996). Medians for all parameter studied for each group are shown in table 1. In order to find out parameters that could increase the incidence probabilities to identify patients with JAK2 V617F we performed an univariate analysis of the variables included, according to JAK2 mutational status. We found that patients with JAK2 V617F had higher levels of leukocytes, neutrophils, platelets and RDW than patients with negative JAK2 (p

Description: Introduction The prognosis of patients with multiple myeloma (MM) has improved in the last years due to the important advances in the knowledge of the biology of the disease, the implementation of new drugs and the incorporation of autologous hematopoietic stem cell transplant (autoHSCT). The allogenic hematopoietic stem cell transplant (alloHSCT) continues to be controversial: it offers a curative potential but with the cost of high toxicity, limiting the procedure to those young patients with a high-risk disease. This procedure shall be performed in expert centers and, whenever possible, in the context of a clinical trial. In the following we describe the experience of our center with alloHSCT in advance multiple myeloma patients. Patients and methods A total of 18 patients were diagnosed with multiple myeloma received an alloHSCT during a 13 year period (1996-2013), with a median age of 46 ± 5.9 years. All of our patients received an allogenic HLA matched sibling donor with reduced-intensity conditioning. The majority of patients were transplanted because of advanced disease, relapse after an autologous transplant or as part of a sequential transplant in patient with a high risk disease. One patient received, in two occasions, an alloHSCT. Around 70% of patients had received more than 3 previous lines of treatment including, in nearly 95%, an autoHSCT. Patient's characteristics can be found on table 1, characteristics of the procedure can be found in table 2.Table 1.Patient«s CharacteristicsN (%)GenderMale Female10 (55,5%) 9 (44,4%)Secreted ProteinIgGκ IgG λ IgA κ BJ Plasmocitoma8 (44,4%) 4 (22,2%) 2 (11,1%) 3 (16,7%) 1 (5,6%)Debut DS stageII-A II-B III-A III-B Plasmocitoma5 (27,8%) 1 (5,6%) 8 (44,4%) 3 (16,7%) 1 (5,6%)Cytogentics at diagnosisMissing Unfavorable Favorable10 (55,5%) 6 (33,3%) 2 (11,1%)Previous lines of treatment²2 3-4 ³56 (33,3%) 10 (55,5%) 2 (11,1%)Previous autoHSCTYes No17 (94,5%) 1 (5,6%)Previous radiotherapyYes No8 (44,4%) 10 (55,6%)Disease status at transplantComplete remission Partial remission Relapse9 (50,0%) 3 (16,7%) 6 (33,3%)Table 2.Treatment characteristicsN (%)Conditioning regimenMyeloablative Reduced-intensity6 (33,3%) 12 (66.7%)Stem cell sourceBone marrow Peripheral blood4 (22.2%) 14 (77.8%)GVHD prophylaxisCsA+MTXCsA+CSCsA+MMF10 (55.6%) 3 (16.7%) 5 (27.8%)InfectionsYes No16 (88.9%) 2 (11.1%)MucositisYes No12 (66.7%) 6 (33.3%)Acute GVHDYes II-IV III-IV No4 (22.3%) 3 (16.7%) 1 (5.6%) 14 (77.8%)Chronic GVHDNo Limited Extensive8 (44.3%) 5 (27.8%) 5 (27.8%) Results: Transplant related mortality (TRM) before day 100th was one case due to a thromboembolic event. Global TRM was 16.6% (3 cases). The incidence of acute graft versus host disease (aGVHD) was 22%, controlled on most cases when corticosteroids were initiated. More than half of the patients developed chronic graft versus host disease (cGVHD), with an equal distribution on either presentation as limited or extensive. (Table 2) The total number of patients eligible for analysis was 17 (one patient was lost on follow-up). With a median follow up of 11 years, the overall survival (OS) was of 8.06 years [IC 95% 4,33-11,78] (figure 1.) and the estimated progression free survival (PFS) was of 25.83 months [IC 95% 8.87-42.79](figure 2). A total of 5 (29,4%) patients are still alive and 2 (11,7%) of them are in complete remission, of these 1 patient did not have a previous autoHSCT with a follow up of almost 15 years. Conclusions: Our results are similar to those reflected on the literature1-2. However we have to point out that our population is homogenous with advanced MM with more than 3 previous lines of treatment including in most cases auto-HSCT. In spite of this, morbility and mortality in our cohort was acceptable with the limitation of a high rate of cGVHD. There is a need of more studies including more patients to evaluate the role of alloHSCT in the era of new drugs for MM. References 1. Rosi-ol L et al. Allogeneic hematopoietic SCT in multiple myeloma: long-term results from a single institution. Bone Marrow Transplant. 2015. 2. Beaussant Y et al. Hematopoietic Stem Cell Transplantation in Multiple Myeloma: A Retrospective Study of the Société Française de Greffe de Moelle et de Thérapie Cellulaire (SFGM-TC). Biol Blood Marrow Transplant. 2015 Disclosures Alegre: Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.

Description: Background: Eltrombopag is an oral thrombopoietin receptor agonist (TPO-RA) drug approved in primary chronic ITP. Lack of clinical trials in secondary ITP avoids a clear demonstration of its potential in terms of safety and efficacy on secondary ITP. Aims: To evaluate the efficacy and safety of eltrombopag in secondary ITP patients in daily clinical practice in Spain. Methods: Ninety-eight secondary ITP patients (aged 18 years or more) from 30 Spanish centers, treated with eltrombopag and included in the Spanish Eltrombopag Registry were retrospectively evaluated. Our study was performed in accordance with the standards of the Helsinki declaration and approved by the Hospital Universitario de Burgos Ethics Committee. Results: Our case series included 98 patients we allocated to four categories: immune disorders (n=47), infections (n=23), lymphoproliferative disorders (n=20), and neoplasms (n=8). The median age of the cohort was 62 (IQR, 40-71) years with 38 men and 60 women. At diagnosis, 34 patients had a Charlson Comorbidity Index score of 2 or more. Median time from ITP diagnosis to eltrombopag initiation was 13 (IQR, 2-66) months. Median number of therapies against thrombocytopenia before eltrombopag was 2 (IQR, 1-3), including rituximab (24), splenectomy (18) and romiplostim (13). Median platelet count when treatment started was 15 x 109/L (IQR, 5-43 x 109/L). Meanwhile, 44 patients had bleeding symptoms. Concomitant therapy was administered to 55 ITP (corticoids in 33) (Table I). Whole cohort eltrombopag response rate was 59% of responses (R; platelet count ≥30 x109/L and at least 2-fold increase the baseline count and absence of bleeding) with 52% of complete responses (CR; platelet count 〉100 x 109/L). Regarding the disease associated to ITP we observed high response rates in immune disorders and infection groups (67% of R, 76 % of R, respectively). Nevertheless, in lymphoproliferative disorders and neoplastic groups efficacy rates were much lower (36 % of R, 37 % of R respectively). The proportion of patients achieving platelet response was quite similar regardless the other studied parameters: age, sex, concomitant treatment, bleeding and platelet count at start of eltrombopag treatment. 30 adverse events were reported with eltrombopag, being 18 of them grade 3-4. 14 deaths were observed but only two were caused by bleeding. The remaining causes of death were: 4 because of bacterial sepsis and another 4 due to progression of basal disease. 2 secondary neoplasms, 1 aspergillosis and one death due to a non-treated severe anemia were also reported (Table II). Conclusion: The use of eltrombopag for treating secondary ITP is effective and safe. To point out, its efficacy in lymphoproliferative disorders and in neoplasm-associated ITP is lower than in benign diseases. Certainly, more studies are needed to confirm usefulness of TPO-RAs in secondary ITP cases. Table 1. Patient characteristics Variable Total(n = 98) Type of disease, n Immune disorders  SLE 13  Evans Syndrome 8  Antiphospholipid Syndrome 6  Sjögren Syndrome 5  Rheumatoid Arthritis 3  Immunodeficiencies 3  Autoimmune Hepatitis 2  Primary Biliary Cirrhosis 2  Psoriatic arthritis 1  Evans Syndrome-Immunodeficiencies 1  Evans Syndrome-HCV 1  Graves-Basedow disease 1  Inflammatory Bowel disease 1 Lymphoproliferative disorders  Lymphoproliferative diseases 16  HCV-Lymphoma 3  HIV-Lymphoma 1 Infections  Hepatitis C Virus 16  HIV 5  HCV-HIV 2 Neoplasms  Myeloid Neoplasms 8 Age, years, median [Q1;Q3] 62[40;71] Men/Women n 38/60 Bleeding at start of eltrombopag treatment, n 44 Concomitant treatment, n 55  Corticoids 33  Immunoglobulins 6  Corticoids and Immunoglobulins 7 Table 2. Adverse events with Eltrombopag Variable n Total, n 30 Serious Adverse Events (Grade 3-4), n 18  Progression of basal disease 4  Severe Bacterial Infections 3  Deep venous thrombosis 3  Stroke 2  Medullary fibrosis 2  Severe Bleeding 1  Aspergillosis 1  Pulmonary Embolism 1  Secondary neoplasms 1  Acute Pancreatitis 1  Acute Myocardial Infarction 1 Deaths, n 14  Bacterial Infections 4  Progression of basal disease 4  Secondary neoplasms 2  Severe Bleeding 2  Aspergillosis 1   Severe Anemia due to negative of patient to transfusion 1 Disclosures Off Label Use: We describe the possibility of using eltrombopag, an oral thrombopoietin receptor analog, for secondary ITP patients..

Description: BACKGROUND: The plasma protein C1-inhibitor (C1-inh), belongs to the serpin superfamily and is the major inhibitor of the proteases of the complement and contact phase pathways. Hereditary or acquired deficiency of functional C1-inh results in angioedema episodes in affected individuals due to uncontrolled contact pathway activation and therapeutic C1-inh products are effective treatment for these patients. Therapeutic C1-inh products have also been shown to attenuate neutrophil activation and infiltration in various inflammatory conditions. This 'novel' anti-inflammatory effect of C1-inh is attributed to its non-serpin N-terminal domain. This domain is thought to express the tetrasaccharide, sialyl Lewisx (SLeX), through which C1-inh can interact with selectins on inflamed endothelium and prevent neutrophil rolling. However, C1-inh products contain small but significant amounts of co-purified proteins, the major one being the glycoprotein α1- antichymotrypsin (ACT), which is also an anti-inflammatory serpin. The potential influence of the glycans of ACT on SLeX - selectin interactions is not clear. METHOD: We investigated the presence of SLeX -like epitopes on C1-inh and ACT from commercially available therapeutic C1-inh preparations using western blotting and mass-spectrometry. The influence of the products and separated C1-inh and ACT on SLeX -selectin interaction was investigated in an a model system where SLeX -beads were rolled on immobilized E-selectin molecules. RESULT: We do not find any evidence of SLeX on C1-inh using either western blotting with anti-SLeX antibodies or by mass spectrometric analysis of C1-inh N- glycans. C1-inh products show modest but significant interference in SLeX -selectin interaction but surprisingly this is not observed for 'pure C1-inh' obtained from gel-filtration of the commercial product. On the contrary, ACT, also from the C1-inh product, shows the presence of SLeX -like epitopes, as detected by the antibody HECA-452 on western blot. In addition, at concentrations present in C1-inh products (20 -150 μg ACT/ mg active C1-inh), ACT can interfere with SLeX -selectin interactions, in a sialic acid dependent manner. These concentrations of ACT can be achieved in vivo with a dose of as low as 2000 U of a C1-inh product, suggesting that ACT can contribute to the anti-inflammatory effects observed in studies with C1-inh products. CONCLUSION: We conclude that the 'novel' anti-inflammatory effects of C1-inh are unlikely due to SLeX and can in fact be partly due to ACT. This fresh evidence challenges a long held assumption and paves the way for development of ACT, alone or in combination with C1-inh, as a new anti-inflammatory therapeutic. Disclosures Engel: ViroPharma Inc.: Research Funding. Nunez:ViroPharma Inc.: Research Funding. Roem:ViroPharma Inc.: Research Funding. van Mierlo:ViroPharma Inc.: Research Funding. Wouters:ViroPharma: Research Funding. Zeerleder:ViroPharma: Other: Receives an unrestricted grant from Viropharma.

Description: Background: Bone marrow stroma provides a favorable microenvironmental niche for ALL cell survival. We and others have demonstrated that bone marrow stromal cells contribute to prevention of apoptosis in ALL cells. Objective: Identify potentially "drug-able" molecules derived from marrow stromal cells that contribute to prevention of ALL cell apoptosis. Methods: We have developed an in vitro system to identify stromal gene products that deliver antiapoptotic signals to ALL cells. Primary human ALL cells are co-cultured with human bone marrow stromal cells. We manipulate stromal cells with siRNA directed against candidate stromal cell genes. Two days later the siRNA is washed out of culture and primary ALL cells are added to the stromal cells. Controls include irrelevant siRNA. Five days later we measure viability and apoptosis in ALL cells by flow cytometry. Results: (1) Knockdown of stroma cell CXCL12 or TGFBI reduces ALL survival. We performed global gene expression analysis upon human marrow stromal cells using RNASeq technology. Using bioinformatic approaches we are selecting some of the expressed stromal genes as candidates for the molecular mechanisms by which stromal cells prevent ALL apoptosis. We present preliminary results for two of our early candidates. (A) CXCL12 is a paracrine chemokine known to have activity in the marrow microenvironment upon hematopoietic cells and we hypothesized it may participate in the effect. Knockdown of CXCL12 with siRNA increased ALL cell death in the co-culture system. As measured by quantitative reverse transcriptase PCR stromal cell CXCL12 mRNA was reduced approximately 75% by siRNA treatment. Figure 1 displays representative results of the impact of CXCL12 knockdown in stromal cell on the survival of ALL cells in the coculture. The magnitude of effect was ~40% increase in ALL cell death. (B) TGFBI (transforming growth factor beta induced) is expressed by stromal cells. The gene is involved in cell-collagen interactions and we hypothesized it played a role. siRNA reduced stromal gene expression by about 90%. Figure 2 displays representative results in which ALL cell death increased by about 50%. (2) Validation of results using inhibitors to CXCL12. The gene knockdown experiments suggested a potential role for CXCL12 in prevention of ALL cell apoptosis. To further test this we tested the effect of plerixafor, a specific inhibitor of CXCL12/CXCR4 interactions, on survival of ALL. ALL cells express CXCR4. In a dose dependent manner (25 - 400 micromolar) we observed a 31-39% reduction in ALL survival in stromal co-cultures including plerixafor. Figure 3 depicts representative results with plerixafor 200 micromolar. We are evaluating small molecules to block TGFBI. (3) Potential augmentation of chemotherapy drug effects on ALL. We hypothesize that interference with stromal cell molecules that prevent apoptosis in ALL cells may increase the effectiveness of conventional antileukemia drugs. In our stromal cell/ALL coculture system we have identified the effective in vitro concentrations of the most commonly used ALL drugs. We measured the impact of combination of low dose plerixafor (LD10) and these individual drugs (used at approximately the LD50 concentrations). Figure 4 demonstrates increased antileukemia effects related to plerixafor for dexamethasone, vincristine, and 6-mercaptopurine. Results are plotted as a percentage of ALL cells surviving in the absence of any drugs. The low dose plerixafor alone control did not produce a statistically significant reduction in ALL survival. Conclusions: Marrow stromal cell-produced CXCL12 may contribute to prevention of apoptosis in human ALL cells. Pharmacological interference with its effect may enhance the effectiveness of some conventional chemotherapy drugs. Marrow stromal cell-produced TGFBI may also contribute to prevention of apoptosis in human ALL cells. Disclosures No relevant conflicts of interest to declare.

Description: Background: Eltrombopag is a thrombopoietin receptor agonist approved for primary chronic ITP patients. Due to the non-existence of clinical trials using eltrombopag in persistent and newly diagnosed ITP, there are no clear data about its usefulness in this setting. Aims: To evaluate efficacy and safety of eltrombopag in persistent, newly diagnosed and chronic ITP in routine clinical practice in Spain. Methods: Two hundred and twenty adult ITP patients from thirty Spanish centers who had been treated with eltrombopag and included in the Spanish Eltrombopag Registry were retrospectively evaluated. This study was performed in accordance with the standards of the Helsinki declaration and approved by the Hospital Universitario de Burgos Ethics Committee. Results: Here we report efficacy and safety results of primary ITP Spanish Eltrombopag Registry cohort. According to the standard definition, patients were allocated to newly diagnosed (n=30), persistent (n=30) and chronic (n=160) ITP groups. Each group is described separately in Table I. There are no statistical significant differences regarding response and duration of response among ITP groups. There is a trend towards a greater efficacy in newly diagnosed ITP with 93.3% of responses (platelet count ≥30 x109/L and at least two-fold increase the baseline count and absence of bleeding) and 86.7% of complete responses (CR; platelet count 〉100 x 109/L). Persistent ITP achieved 83.3% of responses and 80.0% of CR. Similarly 79.4% of responses with 73.1 of CR were observed in chronic ITP. Response rates were similar in all groups regardless all other studied parameters. Another trend towards a longer response duration in persistent ITP was found, with a median of 424 (IQR, 288-664) days. Response durations were similar in chronic ITP (median, 370 days; IQR, 174-624) and in newly diagnosed ITP (median, 378 days; IQR, 154-485). In newly diagnosed ITP, eight adverse events (AEs) with only three grade 3-4 AEs were observed. We reported three deaths; Two of them were due to upper respiratory tract infections in previously diagnosed pulmonary patients. A cerebral hemorrhage was the only death directly related to thrombocytopenia. In persistent ITP, four grade 1-2 AEs and two grade 3-4 AEs (one stroke, one cerebral bleeding) were reported. The only observed death was secondary to the mentioned cerebral hemorrhage. Twenty-one grade 1-2 AEs, ten grade 3-4 AEs and eight deaths (only two caused by bleeding) occurred in chronic ITP. Conclusion: Use of eltrombopag for treating persistent and newly diagnosed ITP is effective and safe. However, more studies are needed to confirm usefulness of TPO-RAs in this setting. Table 1. Patient characteristics Variable Newly-Diagnosed ITP (n = 30) Persistent ITP(n=30) Chronic ITP (n=160) Age, years, median [Q1;Q3] 66[46;79] 66[47;76] 61[47;75] Men/Women n 12/18 15/15 47/113 Charlson comorbidity Index 〉 1, n (%) 7(25.9) 5(17.2) 25(16.7) Months with ITP, median [Q1;Q3] 1[1;2] 6[4;10] 79[30;193] Past ITP treatments, median [Q1;Q3] Rituximab, n (%) Splenectomy, n (%) Romiplostim, n (%) 2[1;3] 3(10.7) 2(7.1) 3(10.7) 2[1;3] 5(17.2) 4(13.8) 4(13.8) 3[2;4] 43(28.3) 47(30.7) 37(24.3) Platelet count at start of eltrombopag treatment, (x109/L), median [Q1;Q3]Bleeding at start of eltrombopag treatment , n (%)Concomitant treatment, n (%) Corticoids Immunoglobulins Corticoids and Immunoglobulins 15[7;29] 13(43.3) 10(33.3) 6(60) 0 2(20) 14[6;26] 10(33.3) 9(30) 7(77.8) 0 2(22.2) 22 [9;38] 50 (31.3) 46 (28.8) 27 (57.4) 10 (21.3) 8 (17) Disclosures Off Label Use: We describe the possibility of using eltrombopag, an oral thrombopoietin receptor analog, for persistent and newly diagnosed ITP patients..