ALBERT

Beschreibung: Background: New experimental methods must be developed to study interaction networks in systems biology. To reduce biological noise, individual subjects, such as single cells, should be analyzed using high throughput approaches. The measurement of several correlative physical properties would further improve data consistency. Accordingly, a considerable quantity of data must be acquired, correlated, catalogued and stored in a database for subsequent analysis. Results: We have developed openBEB (open Biological Experiment Browser), a software framework for data acquisition, coordination, annotation and synchronization with database solutions such as openBIS. OpenBEB consists of two main parts: A core program and a plug-in manager. Whereas the data-type independent core of openBEB maintains a local container of raw-data and metadata and provides annotation and data management tools, all data-specific tasks are performed by plug-ins. The open architecture of openBEB enables the fast integration of plug-ins, e.g., for data acquisition or visualization. A macro-interpreter allows the automation and coordination of the different modules. An update and deployment mechanism keeps the core program, the plug-ins and the metadata definition files in sync with a central repository. Conclusions: The versatility, the simple deployment and update mechanism, and the scalability in terms of module integration offered by openBEB make this software interesting for a large scientific community. OpenBEB targets three types of researcher, ideally working closely together: (i) Engineers and scientists developing new methods and instruments, e.g., for systems-biology, (ii) scientists performing biological experiments, (iii) theoreticians and mathematicians analyzing data. The design of openBEB enables the rapid development of plug-ins, which will inherently benefit from the "house keeping" abilities of the core program. We report the use of openBEB to combine live cell microscopy, microfluidic control and visual proteomics. In this example, measurements from diverse, complementary techniques are combined and correlated.

Beschreibung: Probabilistic Boolean network (PBN) modelling is a semi-quantitative approach widely used for thestudy of the topology and dynamic aspects of biological systems. The combined use of rule-basedrepresentation and probability makes PBN appealing for large-scale modelling of biological networkswhere degrees of uncertainty need to be considered.A considerable expansion of our knowledge in the field of theoretical research on PBN can be observedover the past few years, with a focus on network inference, network intervention and control. Withrespect to areas of applications, PBN is mainly used for the study of gene regulatory networks thoughwith an increasing emergence in signal transduction, metabolic, and also physiological networks. Atthe same time, a number of computational tools, facilitating the modelling and analysis of PBNs, arecontinuously developed.A concise yet comprehensive review of the state-of-the-art on PBN modelling is offered in this ar-ticle, including a comparative discussion on PBN versus similar models with respect to conceptsand biomedical applications. Due to their many advantages, we consider PBN to stand as a suitablemodelling framework for the description and analysis of complex biological systems, ranging frommolecular to physiological levels.

Beschreibung: Background: A close match of the HLA alleles between donor and recipient is an important prerequisite for successful unrelated hematopoietic stem cell transplantation. To increase the chances of finding an unrelated donor, registries recruit many hundred thousands of volunteers each year. Many registries with limited resources have had to find a trade-off between cost and resolution and extent of typing for newly recruited donors in the past. Therefore, we have taken advantage of recent improvements in NGS to develop a workflow for low-cost, high-resolution HLA typing. Results: We have established a straightforward three-step workflow for high-throughput HLA typing: Exons 2 and 3 of HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 are amplified by PCR on Fluidigm Access Array microfluidic chips. Illumina sequencing adapters and sample specific tags are directly incorporated during PCR. Upon pooling and cleanup, 384 samples are sequenced in a single Illumina MiSeq run. We developed "neXtype" for streamlined data analysis and HLA allele assignment. The workflow was validated with 1140 samples typed at 6 loci. All neXtype results were concordant with the Sanger sequences, demonstrating error-free typing of more than 6000 HLA loci. Current capacity in routine operation is 12,000 samples per week. Conclusions: The workflow presented proved to be a cost-efficient alternative to Sanger sequencing for high-throughput HLA typing. Despite the focus on cost efficiency, resolution exceeds the current standards of Sanger typing for donor registration.

Beschreibung: Abstract 3080 As compared to single-unit CBT, DCBT may improve engraftment and protect against relapse. Therefore, we have adopted DCBT for both children and adults with acute leukemia, myelodysplasia (MDS), and myeloproliferative diseases (MPD). However, determinants of disease-free survival (DFS) have yet to be fully established. Furthermore, whether DFS after DCBT is comparable in patients of European and non-European ancestry is of special interest. Therefore, we analyzed the DFS of 75 DCBT recipients with acute leukemia in morphologic remission or aplasia (n = 69), and MDS/MPD with ≤ 5% blasts (n = 6) transplanted from 10/2005-4/2011. Nearly all patients had high-risk disease. Children 0–15 years (n = 23) had the following characteristics: median age 9 years (range 0.9–15); median weight 37 kg (range 7–72); 30% European; and 26% CMV sero-positive. Diagnoses were 43% AML (or biphenotypic), 52% ALL, and 4% MDS/MPD, and all received high-dose conditioning. The children received grafts with a median infused TNC × 107/kg of 3.3 (larger unit) and 2.6 (smaller unit), and 2% of units were 6/6 HLA-A, -B antigen, -DRB1 allele matched, 63% 5/6, and 35% 4/6. Adults ≥ 16 years (n = 52) had the following characteristics: median age 41 years (range 16–69); median weight 69 kg (range 47–105); 48% European; 69% CMV sero-positive; and diagnoses were 63% AML (or biphenotypic), 27% ALL, and 10% MDS/MPD. Fifty percent received high-dose and 50% reduced intensity conditioning. Their units had a median infused TNC/kg of 2.7 and 1.9, and 3% were 6/6 HLA-matched, 47% 5/6, and 50% 4/6. All patients (pediatric and adult) received calcineurin-inhibitor/ mycophenolate mofetil immunosuppression, and none received anti-thymocyte globulin. Sustained donor neutrophil engraftment was seen in 91% of children and 94% of adults at medians of 20 and 26 days, respectively. The incidence of grade II-IV acute GVHD by day 180 was 44% in children and 58% in adults. Day 100 transplant-related mortality (TRM) was 9% in children and 19% in adults. The 2-year relapse incidence was 9% in children and 6% in adults. With a median follow-up of survivors of 26 months (range 4–70), 2-year Kaplan-Meier estimate of DFS was 78% in children and 64% in adults (Figure). Differences in survival by age did not reach significance. Univariate analysis of variables potentially influencing 2-year DFS (with log rank estimates of significance) in all patients is shown in the Table. There were no differences in 2-year DFS according to ancestry, remission status, and conditioning intensity. There was also no difference in 2-year DFS according to engrafting unit-recipient HLA-match (4-6/6 or 10 allele), or engrafting unit infused TNC dose/kg. However, patients who were CMV seronegative had a higher 2-year DFS (85% vs 55%, p = 0.018). Multivariate analysis revealed recipient CMV serostatus was a predictor of DFS independent of patient age, and its effect was mediated by an influence on TRM. We have previously shown that DCBT extends transplant access to minority patients. We now demonstrate that DCBT can achieve high and comparable DFS in both European and non-European pediatric and adult patients with acute leukemia and MDS/MPD. While these are very encouraging results further investigation in racial/ethnic sub-groups is needed. Nonetheless, our findings support DCBT as an immediate alternative therapy for high-risk acute leukemia in patients without suitable unrelated volunteer donors, especially given the very low incidence of relapse. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Abstract 3136 Background: Follicular lymphoma (FL) is the most common indolent non-Hodgkin lymphoma (NHL) in the Western hemisphere and is likely incurable with chemotherapy alone. Despite a median overall survival exceeding 12 years in the modern era, a fraction of patients experience an aggressive clinical course characterized by short remission duration(s) and/or chemorefractory disease. While high-dose therapy and autologous stem cell rescue (HDT-ASCR) is an accepted treatment modality for prolonging progression-free survival (PFS) it is unlikely to provide cure. Allogeneic stem cell transplantation (alloSCT) is a definitive therapy that has been shown to produce long-term disease free remissions. Unfortunately, alloSCT is limited by the risk of transplant-related mortality (TRM) despite the advent of non-myeloablative (NMA) conditioning regimens. Given that many FL patients whom fail HDT-ASCR may not proceed to alloSCT, and the inherent difficulty in deciding the appropriate consolidative transplant modality, we conducted a retrospective exploratory analysis of clinical features of early and multiply relapsed FL patients undergoing first HDT-ASR or alloSCT at a single center in the modern, post-rituximab era. Methods: We retrospectively reviewed all patients with early relapsed and/or refractory FL that proceeded to HDT-ASCR or NMA alloSCT as first transplant at MSKCC between 2006 and 2010. Chemosensitive disease was defined as a partial response (PR) or complete response (CR) to the last treatment regimen prior to transplant by computed tomography scans per IWG Criteria (Cheson et al JCO 1999). Events were defined as progression of FL post-transplant or death from any cause. Event-free survival (EFS) and overall-survival (OS) were estimated using Kaplan-Meier method. Results: We identified 40 patients with relapsed or refractory FL who had undergone either first HDT-ASCR (N=20) or alloSCT (N=20). All patients had FL grade 1–3a without pathologic evidence of transformation at the time of re-induction prior to transplant consolidation. Patient characteristics are outlined in the table below: All HDT-ASCR patients received BEAM (carmustine, etoposide, cytarabine, melphalan) conditioning. All alloSCT patients received a uniform non-myeloablative conditioning regimen (cyclophosphamide, fludarabine, total body irradiation 200 cGy). The median follow-up for survivors is 34 months. The estimated 3 year-EFS and OS was 60% and 62% with HDT-ASCR and 79% and 85% with alloSCT respectively (p=ns). FL patients with remission duration ≤ 12 months prior to re-induction therapy proceeding to consolidative HDT-ASCR had significantly shorter EFS compared to those patients with previous remission duration 〉 12 months (p

Beschreibung: Abstract 3147 Introduction: Poor graft function (PGF) without immune rejection, defined as persistent cytopenias with hypocellular marrow and full donor myeloid chimerism, can be a life-threatening complication after allogeneic HSCT. It is commonly caused by viral infectious, myelosuppressive drugs like antivirals, and graft-vs-host disease (GvHD). Treatment options include supportive therapy with transfusions and growth factors and in severe cases administration of additional hematopoietic stem cells (HSCs) from the same donor without conditioning (stem cell boost). The incidence, natural history, and the indications for stem cell boost therapy are not well defined. Aims: To assess the incidence, etiologies, and indications for stem cell boost for PGF in a homogeneous group of patients with advanced MDS and AML who underwent TCD HSCT from matched or mismatched related or unrelated donors after conditioning with the same myeloablative regimen. Patients and methods: Poor graft function was defined as persistent neutropenia (ANC

Beschreibung: Abstract 3144 Based on encouraging results with the use of clofarabine (CLO) for reinduction treatment of acute leukemias, we have developed two allograft protocols for patients with hematologic malignancies with a cytoreductive regimen, using CLO in combination with melphalan (Mel) and thiotepa (Thio). Patients on protocol #1 received unmodified bone marrow (BMT), peripheral blood stem cells (PBSCT), or unmodified double unit cord blood (dCBT). Patients on protocol #2 received CD34+ T-cell depleted stem cells (TCD-SCT). Cytoreduction consisted of CLO 20 mg/m2/day × 5, Thio 10 mg/Kg/day × 1 and Mel 70 mg/m2/day × 2. Graft-versus-host disease (GvHD) prophylaxis consisted of tacrolimus (Tacro) and methotrexate (MTX) with unmodified BMT or PBSCT, tacro and mycophenolate mofetil (MMF) with unmodified dCBT, and none with TCD-SCT. Rabbit ATG at 2.5 mg/Kg × 2 or 3 doses was used for the prevention of rejection with the TCD-SCT. To date, 64 pts were treated with this regimen including: unmodified BMT/PBSCT 27 patients, dCBT 15 patients, and TCD-SCT 22 patients. The median age for patients was 10.2 years (range 0.9–58.7) for unmodified SCT and 41.5 (range 0.6–67.2) for TCD-SCT. This was the second SCT for 13 of 27 pts in the BMT-PBSCT group, 2 of 15 pts in the CBT group, and 4 of 22 pts in the TCD group. Patient diagnoses included acute lymphoblastic leukemia (ALL) (N=36), acute myelogenous leukemia (AML) (N=23), and myelodysplastic syndrome (MDS) (N=5). Patients with ALL or AML in first remission (CR1) or CR2 and MDS in CR1 or refractory anemia (RA) were categorized as having good risk disease (GRD), while all other pts were considered to have poor risk disease (PRD), irrespective of all other factors. There were 15 of 27 pts with PRD in the BMT/PBSCT group, 10 of 15 pts in the CBT group, and 9 of 22 pts in the TCD-SCT group. For the unmodified BMT/PBSCT group, donors were HLA-matched related (N=11), mismatched related (N=1), matched unrelated (N=12), or mismatched unrelated (N=3). All CBT recipients received double-unit grafts from 2 mismatched unrelated donors. For the TCD-SCT group, donors were HLA-matched related (N=8), mismatched related (N=1), matched unrelated (N=4), or mismatched unrelated (N=9). Engraftment occurred in 59 of 61 evaluable pts; three pts died before engraftment. One pt recipient of unmodified BMT/PBSCT suffered a late graft failure, and one pt recipient of CBT suffered an early graft failure in the context of sepsis. Grade 2–4 acute GvHD occurred in 8/26 (31%) evaluable pts in the BMT/PBSCT group, 5/13 (38%) evaluable pts in the CBT group, and 4/20 (20%) evaluable pts in the TCD-SCT group. With a median follow-up of 20.5 months for the unmodified SCT groups and 15.4 months for the TCD group, the overall survival (OS) and disease-free survival (DFS) rates were: 53.7% and 41.0% for the BMT/PBSCT group, 51.3% and 41.5% for the CBT group, and 64.1% and 60.7% for the TCD-SCT group. This cytoreductive regimen represents a promising approach for the transplantation of patients with acute leukemias without the use of total body irradiation. This regimen is also sufficiently immunosuppressive to insure consistent engraftment of T-cell depleted transplants. Lastly, it appears to be relatively well tolerated for younger pts requiring a second SCT. Disclosures: Off Label Use: Clofarabine.

Beschreibung: Abstract 3130 Background: Allogeneic stem cell transplantation (alloSCT) remains the only curative therapy for patients with indolent B cell non-Hodgkin's Lymphoma (B-NHL) as well as patients with aggressive B-NHL that have failed prior autologous stem cell transplants (ASCT). Non-myeloablative (NMA) alloSCT has been universally implemented for B-NHL given the extensive prior therapy and advanced age of the typical patient undergoing alloSCT for these diseases and resultant 40–50% incidence of transplant-related mortality (TRM) with myeloablative conditioning per registry series'. NMA alloSCT relies on graft-versus-lymphoma (GVL) effect for disease control. While the literature has suggested improved event-free survival (EFS) for B-NHL patients with a negative fluorine-18-deoxyglucose positron emission tomography (FDG-PET) scan prior to ASCT in chemosensitive patients, this has not been elucidated in a conventional NMA alloSCT treatment platform. Our hypothesis is that FDG avidity pre-NMA alloSCT is not prognostic to progression-free survival (PFS) in chemosensitive patients with B-NHL. Methods: This is a retrospective study of adult B-NHL patients with disease assessed by CT and FDG-PET prior to undergoing a T-cell replete NMA alloSCT from a 9/10 or 10/10 HLA matched related or unrelated donor conditioned with cyclophosphamide 50 mg/kg x1, fludarabine 25 mg/m2 daily over 5 days and 2 Gy total body irradiation, with or without peri-allo-SCT rituximab 375 mg/m2 administered approximately day (d) -8, d+21, d+28, d+25, d+42 as per an internal protocol. Patients receiving an unrelated donor graft received equine anti-thymocyte globulin 30 mg/kg administered daily d-3 and d-2. Graft-versus-host disease (GVHD) prophylaxis consisted of tacrolimus, sirolimus and methotrexate (n=51) or cyclosporin-A and mycophenolate mofetil (n=15). Event-free survival (EFS) was calculated using Kaplan-Meier method. An event is defined as progression of disease (PD) or death from any cause. Results: This analysis includes 66 patients with a median age of 54 (range 33–69) years at the time of allo-SCT. Diagnoses included: 16 CLL/SLL, 30 FL, 11 DLBCL/transformed B-NHL, 6 MCL and 3 other. Patients were previously treated with a median of 3 prior chemotherapy regimens (range 1–6) and 12 patients had undergone a previous ASCT. Fifty-one patients (77%) were chemosensitive at the time of alloSCT by CT criteria, as defined by either a complete or partial remission (Cheson et al, JCO 1999; Hallek et al, Blood 2005 for CLL/SLL). Of these 51 patients, 18 had FDG-avidity on FDG-PET at time of allo-SCT above liver background per Deauville criteria (Meignan et al, Leukemia and Lymphoma 2009). The median hematopoietic comorbidity index (HCT-CI) was 1 (range 0–8). Twenty-six patients received a graft from a 10/10 HLA matched related donor, 34 from a 10/10 HLA matched unrelated donor and 6 from a 9/10 HLA mismatched unrelated donor. Eighty-six percent of the patients received peri-alloSCT rituximab. The median follow-up for survivors is 29 (range 3–67) months. Of the 15 events, 10 were TRM (7 GVHD-related deaths) and 5 PD (3 chemosensitive patients and 2 chemorefractory patients pre-alloSCT). Only 1 event has occurred beyond 13 months. The estimated 2 year EFS is 77%. There was no difference in EFS according to: CR vs PR, graft source, number of previous lines of therapy, GVHD prophylaxis, peri-alloSCT rituximab, previous ASCT, or HCT-CI. The estimated EFS at 2 years for chemosensitive patients per CT criteria is 86% compared to 50% for patients without chemosensitivity pre-allo-HCT (figure 1, p