ALBERT

Description: Background: Acute myeloid leukemia (AML) is an aggressive hematological malignancy which has an incidence of 40 children on average per year in the Netherlands and Belgium combined (population of 28 million). Despite the fact that almost all children achieve complete remission (CR), 30-40% of patients eventually relapse. Since survival after relapse is poor (35-40%), the overall survival (OS) of pediatric AML remains relatively low (~70%) compared to the increasing remission rates. The most effective strategy to improve the dismal outcome of AML patients is thus to prevent relapse. Over the last decades accumulating evidence is suggesting that AML develops in a hierarchical structure; originating from hematopoietic stem cells which are transformed to leukemia initiating cells, also referred to as leukemic stem cells (LSC). LSC possess self-renewal capacity and are more resistant to therapy. Therefore, LSC are supposed to be responsible for outgrowth of both the initial leukemia and the relapse. This holds true for adult AML, where the frequency of LSC at diagnosis has shown to be of importance for clinical outcome. However, while it has been shown that a high number of immature cells (CD34+/CD38-/CD45-/low) associates with an increased risk of relapse in pediatric AML, relatively little is known about the prognostic impact of LSC within this compartment. RAEB-t. Flow-cytometric LSC characterization was performed on either bone marrow (BM) or peripheral blood (PB) from AML patients collected at diagnosis. Purified white blood cells (WBC) were obtained after lysing the red blood cells using lysing solution (Pharm Lyse or FACSLysing, BD Biosciences) and were subsequently incubated with monoclonal antibody combinations and analyzed using an 8-color flow cytometer approach. LSC were defined as CD34+ CD38- cells with aberrant expression of CD123, CD7, CD56 or CD2. Results: Data were available from 103 patients and patient characteristics are listed in Table 1. In this cohort relapse-free survival (RFS) was 59.4%. Fortunately 56.4% of relapsed patients achieved a second CR and OS was 85.4%. LSC-load is defined as % of aberrant CD34+ CD38- cells within the CD34+ compartment. In our pediatric cohort, 17 patients (16.5%) had no expression of CD34 on blast cells at all (CD34null) and consequently no aberrant CD34+ CD38- LSC could be detected. Absence of CD34 has often been associated with good prognosis in literature. In our cohort relapse-rate seemed to follow this trend (4 out of the 17 CD34null patients (23.5%) vs. 34 out of the 83 CD34positive patients (41%)) but did not differ significantly (Plogrank = 0.196). ROC curve analysis showed that a cut-off of 17.2% LSC at diagnosis was associated with the occurrence of developing relapse with a specificity of 92%. Kaplan-Meier survival analysis, as depicted in Figure 1, showed a significant association between a high LSC-load and impaired RFS (34% relapses in LSClow vs. 64% relapses in LSChigh) (Plogrank= 0.027). Univariate analysis showed that next to LSC percentage, FLT3 mutation status and WBC count were significantly associated with RFS. After multivariate adjustment (taking into account cytogenetic risk groups and mutational status) LSC frequency was the only independent predictor of relapse or RFS (HR 2.3, 95% CI 1.1-4.8, p=0.032). Conclusion: Our results confirm data from adults and reveal that the frequency of LSC at diagnosis in pediatric AML patients can distinguish patients more likely to fail current treatment regimens as these patients develop significantly more relapses). Identifying this patient group creates opportunities for more personalized medicine and the development of therapies directed against LSC, sparing normal hematopoietic stem cells. Disclosures Kaspers: Janssen-Cilag: Research Funding. Cloos:Takeda: Honoraria.

Description: Introduction: Second-Harmonic Generation microscopy (SHG) has provided progresses in extracellular matrix research, mainly regarding automated analysis of collagen fibers. Nodular sclerosis-classical Hodgkin lymphoma (NS) often has a rich collagen deposition, which remains poorly explored in its biological significance and potential prognostic role. The aim of this study was to characterize the collagen component of NS using SHG, and to investigate its clinical value. Methods: Hematoxylin and eosin stained slides from 53 consecutive samples of paraffin embedded NS tissue were analyzed by SHG imaging in an Inverted Zeiss LSM 780-NLO. HIV-positive individuals were excluded. For comparative purposes, 11 reactive lymph nodes (RL) were also randomly selected. In each slide, 3 capsular areas and 3 sclerotic regions (perinodular septa - PS - in NS and fibrotic foci around germinal centers in RL) were chosen. Collagen near blood vessels was not considered. We evaluated quantity, uniformity and organization of the fibers with ImageJ and OrientationJ plug-in in 4 hotspots from each image. Shapiro-Wilk test was used to assess data normality, while t-tests and Pearson correlations were performed to analyze collagen parameters between two groups. Overall survival (OS) was defined as time from diagnosis until death from the disease or last follow-up. Event-free survival (EFS) was set as time from diagnosis until progressive disease, death from the disease or last follow-up. Collagen data were used in survival analyses as continuous variables (in Cox-hazards model) or categorical ones (by choosing the mean value as a threshold for Kaplan-Meier curves and log-rank test). Significance was set at p 3). The first-line treatment was ABVD in 40 cases (75.5%) and BEACOPP in 13 patients (24.5%). Radiotherapy was performed in 27 (50.9%) patients. Tumor PS presented more dense (p

Description: Key Points Chronic graft-versus-host disease is associated with a global Breg defect. This defect is particularly accentuated in the CD24hiCD27+ Breg compartment.

Description: Key Points The outcome of HSCT in this large SCN cohort is acceptable. Given the TRM, a careful selection of HSCT candidates should be undertaken.

Description: Background: Although T cell immunotherapy is considered a promising therapeutic approach in B cell malignancies, autologous T cell based therapy proved to be far less effective in CLL than in more aggressive B cell malignancies. This has been attributed to an acquired state of T cell dysfunction. Disturbances in conventional (αβ-)T cells include expansion of CD4+ and CD8+ T cells, increased expression of exhaustion markers and impaired cytotoxicity and cytokine production. Vγ9Vδ2-T cells are a conserved subset of cytotoxic T lymphocytes with potent antitumor activity, due to recognition of phosphoantigen-induced changes in CD277 in tumor cells. Aminobisphosphonate (ABP) treatment leads to intracellular accumulation of phosphoantigens and increased Vγ9Vδ2 antitumor responses. Vγ9Vδ2-T cells have been shown to effectively kill malignant B cell lines in vitro. Moreover, in clinical trials Vγ9Vδ2-T cells have been shown to recognize and kill B cell lymphomas. Whether Vγ9Vδ2-T cells could be exploited for CLL immunotherapy has not yet been explored. The aim of this study is to investigate the phenotype and function of Vγ9Vδ2-T cells in CLL patients, in order to determine whether Vγ9Vδ2-T cells can effectively kill CLL cells. Results: Frequencies of Vγ9Vδ2-T cells do not differ between untreated CLL patients (n=46) and age-matched healthy controls (HC) (n=20) as assessed by flow cytometry. Vγ9Vδ2-T cell subpopulations are skewed towards effector type (CD27- CD45RA-) in CLL patients, while numbers of naïve (CD27+ CD45RA+) Vγ9Vδ2-T cells are decreased. Expression of exhaustion markers PD-1 and BTLA is comparable between CLL and HC, as is expression of CD16, mediating antibody-dependent cellular cytotoxicity. Next, we compared the functionality of Vγ9Vδ2-T cells from CLL patients and HC. We first examined cytokine production and CD107a expression, a marker of degranulation. Production of TNFα and IFNγ upon PMA/ionomycin stimulation was significantly diminished in CLL Vγ9Vδ2-T cells as compared to HC Vγ9Vδ2-T cells. Similarly, CD107a expression was significantly reduced. Overnight coculture with primary CLL cells or the Vγ9Vδ2-T cell sensitive Daudi lymphoma cell line also induced expression of TNFα, IFNγ and CD107a. However, upon co-culture, HC Vγ9Vδ2-T cells expressed significantly more TNFα, IFNγ and CD107 than CLL Vγ9Vδ2-T cells. Subsequently, we compared cytotoxicity of Vγ9Vδ2-T cells towards Daudi cells. HC-derived Vγ9Vδ2-T cells killed Daudi cells 3-4 times more effectively at 1:5 and 1:2.5 effector:target ratios. Although ABP pretreatment of Daudi cells increased both CLL-derived and HC-derived Vγ9Vδ2-mediated killing, differences between CLL and HC could not be overcome. We then looked at Vγ9Vδ2-T cell cytotoxicity towards CLL cells. Vγ9Vδ2-T cells from HCs effectively recognized and killed primary CLL cells, irrespective of ABP pretreatment. CLL-derived Vγ9Vδ2-T cells killed allogeneic CLL cells significantly less efficiently. Finally, we investigated whether the Vγ9Vδ2-T cell dysfunction in CLL patients was reversible upon ex vivo activation without the presence of leukemic B cells. Purified Vγ9Vδ2-T cells were cocultured with mature monocytic-derived dendritic cells in the presence of ABP for 8 days. Following these culture conditions, no difference was observed in production of TNFα, IFNγ and IL-4 upon PMA/ionomycin stimulation between HC- and CLL-derived activated Vγ9Vδ2-T cells. Likewise, there was no difference in CD107a expression. The activated Vγ9Vδ2-T cells of HCs and CLL patients were equally effective at killing Daudi cells. Conclusion: Vγ9Vδ2-T cells are capable of recognizing and killing CLL cells. Yet, CLL-derived Vγ9Vδ2-T cells are functionally impaired in terms of cytokine production and cytotoxic capacity in comparison to age-matched HCs. Functional impairments of Vγ9Vδ2-T cells are reversible upon ex vivo activation. If dysfunction can be overcome effectively, the antileukemic properties of autologous Vγ9Vδ2-T cells can be efficiently employed. Disclosures No relevant conflicts of interest to declare.

Description: Recessive mutations in SEC23B gene cause congenital dyserythropoietic anemia type II (CDAII), a rare hereditary disorder hallmarked by ineffective erythropoiesis, iron overload, and reduced expression of hepatic hormone hepcidin (Iolascon, 2013). The most recently described hepcidin regulator is the erythroblast-derived hormone erythroferrone (ERFE), a member of TNF-α superfamily that specifically inhibits hepcidin production in experimental models (Kautz, 2014). However, the function of ERFE in humans remains to be investigated. To determine whether dysregulation of ERFE expression is associated with ineffective erythropoiesis and iron-loading in CDAII, we studied the ERFE-encoding FAM132B gene expression in 48 SEC23B-related CDAII patients and 29 age and gender matched healthy controls (HCs). Twelve new cases and four novel SEC23B mutations were described. Samples were obtained after informed consent, according to the Declaration of Helsinki. Genomic DNA, mutational screening, RNA isolation, cDNA preparation, and qRT-PCR were performed as previously described (Russo, 2013). All patients were young adults (17.0±2.5 years at diagnosis), with increased serum ferritin (395.4±67.6 ng/mL) and transferrin saturation (71.9±5.4 %). We observed a statistically significant overexpression of FAM132B gene in peripheral blood mononuclear cells from CDAII patients (9.09±0.08) compared to HCs (8.32±0.12, p

Description: Introduction: The differential diagnosis of hereditary and acquired thrombocytopenias can be challenging, especially when between immune thrombocytopenia (ITP) and less well characterized hereditary thrombocytopenias (HT) such as MYH9-related disorders. Fundamental differences in the management of these two conditions add clinical relevance to the search for novel parameters that differentiate these conditions. The immature platelet count (IPF) represents the fraction of platelets with higher RNA content, and in analogy to the reticulocyte count for erythropoiesis is a biomarker of thrombopoietic activity. In a recent report (Miyazaki et al, 2015), IPF values that were more than 5-fold higher than those observed in ITP patients were reported in a population of 15 patients with HT. However, whether this increased values represented a real increase in thrombopoietic activity, or reflected a technical limitation of IPF determination in large platelets could not be clarified. Here, we aimed to evaluate the role of IPF determination in the differential diagnosis between HT and several forms of acquired thrombocytopenia in a larger and more diverse population of patients. We also evaluated thrombopoietin (TPO) levels in HT compared to ITP, to further investigate the mechanisms by which extremely large IPF values are observed in HT. Methods: IPF and mean platelet volume (MPV) were prospectively determined using a Sysmex XE5000 hematologic analyzer (as part of the complete blood count) in a cohort of patients with post-chemotherapy thrombocytopenia (n=56), bone marrow failure (myelodysplastic syndromes and aplastic anemia; n=22), ITP (ITP; n=105) and inherited thrombocytopenias (n=27). The latter population consists of a well-defined cohort of individuals with HT thrombocytopenia characterized by clinical, familial, laboratory and molecular data. TPO levels were determined by ELISA (R&D Systems) in 21 HT patients and 22 ITP patients matched for platelet count and age. A group of 178 healthy volunteers were used to determine normal IPF and MPV values. Results: Median platelet counts were similar in post-chemotherapy patients (CTx) (32.0*109/L), bone marrow failure (BMF) (33.5*109/L), ITP (52.0*109/L) and HT (52.0*109/L) (P=0.15). Similar IPF levels were observed in CTx and BMF patients (5.6%; IQR 3.4-8.8% and 6.5%; IQR 3.5-13.7%. Compared to these two groups, higher IPF values were observed in both ITP (12.3%; 7.0-21.0%) and HT patients (29.8%; 17.5-56.4%) (both P values 〈 0.05). In addition, IPF were significantly higher in HT compared to ITP (Kruskall-Wallis test and Dunn's post test,P=0.001). MPV values were different between HT and CTx/BMF groups, but could not differentiate ITP from HT. TPO levels. The accuracy of IPF to discriminate HT from all other causes of thrombocytopenia estimated by ROC analysis was 0.88 (CI95%0.8-0.96, p

Description: Introduction Despite current ABO/RhD matching and strict antibody screening policies, still every year transfused patients experience life-threatening hemolytic reactions due to boostering of previously immunization to red blood cell (RBC) antigens. Prevention can be optimized by administering RBC units at least matched on the most immunogenic antigens to high risk patients. In this respect, we set out to assess the immunogenicity of RBC antigens. Methods We performed an incident new user cohort study among previously non-transfused, non-alloimmunized patients who received RBC transfusions between 2006 and 2013 in six Dutch hospitals. Patients developing alloantibodies were followed up until their first RBC alloantibody identification and all non-alloimmunized patients until the last negative screen. To compute dose-specific alloimmunization risks and thereby evaluate the immunogenicity of various RBC antigens, only antigen-positive units transfused to all patients lacking this antigen should be considered. Per definition, alloimmunized patients met this condition. RBC phenotypes of non-alloimmunized patients were however unknown since phenotyping is routinely limited to ABO and RhD antigens. For each RBC antigen we therefore randomly extracted a subgroup of non-immunized patients whose size was based on the known proportion of antigen-negative individuals in the Caucasian population. The given antigen-positive units transfused to these 'antigen-negative cohorts' functioned to estimate the number of antigen-positive units transfused to the true antigen-negative, non-alloimmunized individuals in the source population. Multiple imputation was used to complete the dataset regarding some missing donor antigen phenotypes. We then calculated cumulative immunization incidences for each RBC antigen according to the total number of mismatched (i.e. antigen-positive) units using Kaplan-Meier survival tables. Women under 45 years of age were analyzed separately as in the Netherlands they receive c, E and K matched blood. Results In 474 of 21,512 patients (2.0%), 537 first formed antibodies were detected, the majority against E and K antigens. Cumulative immunization incidences after 40 RBC units transfused increased to 7.6% (CI 4.8-11.2). Due to lower frequencies of Rh and K immunizations, women under 45 years, who received blood matched on these antigens, demonstrated significantly lower cumulative immunization incidences compared to the remainder of the study population (4.4% (CI 0.2-20.5) versus 7.6% (CI 4.8-11.2) after 40 units received, log-rank p 0.013). Anti-c was only formed by RhD-positive patients while the lack of RhD expression led to significantly less E immunizations (cumulative immunization incidences 1.7% (CI 0.0-32.0) and 3.7% (CI 1.4-7.9) after 40 RBC units received for RhD-negative and RhD-positive patients respectively (log-rank p

Description: Background: The International Prognostic Scoring System (IPSS) for MDS has recently been revised (IPSS-R). However both scoring systems were developed after exclusion of therapy-related cases and data on its usefulness in treatment-related MDS (tMDS) is limited. Aims and Methods: We analyzed 1837 pts from Spanish, German, Swiss, Austrian, US, Italian, and Dutch centers diagnosed 1975-2015. Complete data to calculate the IPSS/-R was available in 1511 pts. The impact of prognostic features was analyzed by uni- and multivariable models and estimated by a measure of concordance for censored data (Dxy). Results: Median age was 68 years. 1% of pts had 5q-syndrome, 13% RCUD, 4% RARS, 27% RCMD/-RS, 18% RAEB 1, 18% RAEB 2, 4% CMML 1, 2% CMML 2, 3% MDS-U, and 7% AML (RAEB-T) according to WHO-classification. Regarding cytogenetics 38% exhibited good, 14% intermediate, and 48% poor-risk according to IPSS, and 2% very good, 36% good, 17% intermediate, 15% poor, and 31% very poor according to IPSS-R. Prognostic risk groups were 12% IPSS low, 34% int 1, 36% int 2, and 18% high, while the IPSS-R was very low in 8%, low in 20%, intermediate in 17%, high in 23%, and very high in 32%. The most frequent primary diseases were NHL 28%, breast cancer 16%, myeloma 6%, prostate cancer 6%, Hodgkins disease 5%, and 4% gastrointestinal tumors. Patients received chemotherapy in 75% and radiotherapy in 47%. Regarding chemotherapeutic drugs, most pts received combination regimens containing alkylating agents in 65%, topoisomerase inhibitors in 44%, antitubulin agents in 26%, and antimetabolites in 26%. Median follow-up from MDS diagnosis was 59 months, median survival 16 months. Since a disease altering treatment is, at least in higher risk disease, which is overrepresented in tMDS, standard of care, we decided to analyze treated as well as untreated pts to avoid a selection bias. This included stem cell transplantation in 16% with a median survival of 24 months. Features with influence on survival and time to AML in univariable analysis included FAB, WHO, IPSS, IPSS-R, cytogenetics, hb, platelets, marrow and peripheral blasts, ferritin, LDH, fibrosis, ß2-microglobulin, and use of alkylating agents for the treatment of primary disease. For hemoglobin, platelets, LDH, fibrosis, and ß2-microglobulin the influence was stronger on survival. Year of diagnosis, age, gender, neutrophil count, WBC, use of chemo or radiotherapy as well as other chemotherapeutic agents had no marked influence on both outcomes. According to our results, both the IPSS (Dxy 0.29 for survival, 0.32 for AML) and IPSS-R (Dxy 0.34, 0.32 for AML) perform moderately in tMDS, but not as well as in primary MDS (pMDS). Therefore, existing prognostic models need to be adjusted to tMDS. However, this appears to be not without difficulties. The scores tested, as well as most prognostic variables themselves perform inferior compared to pMDS. It becomes even more complicated since tMDS in itself is even more heterogeneous than pMDS. Scores and variables perform differently depending on the primary disease or therapy. The IPSS/-R and its variables perform for example better in pts with solid tumors compared to hematologic diseases or in pts who have received radio- instead of chemotherapy, but also in pts after prostate compared to breast cancer. In addition to the integration of further variables, new cutoffs, or the weighting of existing variables, we are currently testing the possibility of separate score versions for different tMDS subgroups. Separate score versions for survival and time to AML would also give differing weights to most features. Hemoglobin, platelets and cytogenetics would get more weight for survival, while marrow blasts would be more important regarding AML. Conclusion: In contrast to early descriptions of tMDS, with poor risk cytogenetics in the vast majority of pts and a uniformly poor prognosis, surprisingly we find good risk karyotypes in a relatively large number of pts. Although, poor risk cytogenetics are still overrepresented, this indicates, different types of tMDS exist. Our analysis shows that many variables exhibit prognostic influence in tMDS and the IPSS or preferably IPSS-R can be applied in these pts. However, the prognostic power of both scores is inferior compared to pMDS, making an optimized tMDS score reasonable. Currently data from further IWG centers is integrated in our database and further analyses are performed to propose a tMDS specific score. Figure 1. Figure 1. Disclosures Komrokji: Novartis: Research Funding, Speakers Bureau; Pharmacylics: Speakers Bureau; Incyte: Consultancy; Celgene: Consultancy, Research Funding. Sekeres:TetraLogic: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Steensma:Celgene: Consultancy; Incyte: Consultancy; Amgen: Consultancy; Onconova: Consultancy. Valent:Novartis: Consultancy, Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Celgene: Honoraria. Platzbecker:Boehringer: Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Esteve:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria.

Description: BACKGROUND High dose therapy followed by autologous stem cell transplantation (ASCT) remains the standard of care, especially in Europe, for young and eligible multiple myeloma patients (usually younger than 65 years old). Immunoparesis is defined as a reduction (below the lower normal limit) in the levels of 1 or 2 uninvolved immunoglobulins (Ig) and it is related to a reversible suppression of B lymphocytes that correlates inversely with disease stage. B Lymphocyte reconstitution begins at 3 months after ASCT, with maximum B lymphocyte levels at 1 year after ASCT. AIMS The goal of the present study was to investigate the role of the immunoparesis recovery after ASCT as predictor of relapse or progression in multiple myeloma (MM). METHODS We reviewed medical records of MM patients who underwent to ASCT at University Hospital of Salamanca between 1992 and 2013. The primary endpoint was time to relapse or progression from ASCT. Ig (Ig G, Ig A e Ig M) were collected at the time of diagnosis, before ASCT, every 3 months during the first year after ASCT, and every year up to 5 years after ASCT among eligible patients until the relapse or disease progression. RESULTS 106 multiple myeloma patients who underwent ASCT were included in the analysis. Conventional chemotherapy was administered as induction regimen in 69 patients (65%), whereas novel agents were used in 37 patients (35%). Most patients had immunoparesis at diagnosis (91%) and at the moment of ASCT as well (94%). After a median follow-up of 62 months, median time to progression or relapse (TTP) from ASCT was 31 months (95 % CI: 24.1 - 37.1 months). MM patients with immunoparesis 1 year after ASCT had a significantly shorter median TTP as compared with patients without immunoparesis (33.5 months vs 94.2 months; HR: 2.14, 95% CI: 1.13-4.05; p=0.019). In the group of patients with reduction of both Igs, median TTP was slightly inferior than in the group with reduction of only one of them(33.5 vs 36.4 months, p=0.03). Presence of ISS 3, high-risk cytogenetics at diagnosis, less than partial response achieved before and three months after ASCT were also identified as predictors of progression. Multivariate analysis selected immunoparesis 1 year after ASCT as an independent variable for relapse or progression (HR: 5.97, 95% CI: 1.63-21.88; P=0.007). CONCLUSIONS The lack of immunoparesis recovery at 1 year after ASCT in MM patients is associated with significantly higher risk of relapse or progression and this group of patients could potentially benefit of continuous treatment after ASCT to enhance the immune recovery. Disclosures Ocio: Array BioPharma: Consultancy, Research Funding; Celgene: Consultancy, Honoraria; Amgen/Onyx: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy; Mundipharma: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; MSD: Research Funding; Pharmamar: Consultancy, Research Funding; Janssen: Honoraria. Puig:The Binding Site: Consultancy; Janssen: Consultancy. Mateos:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx: Consultancy; BMS: Consultancy; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees.