ALBERT

Description: Background: Tardigrades are multicellular organisms, resistant to extreme environmental changes suchas heat, drought, radiation and freezing. They outlast these conditions in an inactive form(tun) to escape damage to cellular structures and cell death. Tardigrades are apparentlyable to prevent or repair such damage and are therefore a crucial model organism for stresstolerance. Cultures of the tardigrade Milnesium tardigradum were dehydrated byremoving the surrounding water to induce tun formation. During this process and thesubsequent rehydration, metabolites were measured in a time series by GC-MS.Additionally expressed sequence tags are available, especially libraries generated from theactive and inactive state. The aim of this integrated analysis is to trace changes intardigrade metabolism and identify pathways responsible for their extreme resistanceagainst physical stress. Results: In this study we propose a novel integrative approach for the analysis of metabolicnetworks to identify modules of joint shifts on the transcriptomic and metabolic levels. Wederive a tardigrade-specific metabolic network represented as an undirected graph with3,658 nodes (metabolites) and 4,378 edges (reactions). Time course metabolite profilesare used to score the network nodes showing a significant change over time. The edges arescored according to information on enzymes from the EST data. Using this combinedinformation, we identify a key subnetwork (functional module) of concerted changes inmetabolic pathways, specific for de- and rehydration. The module is enriched in reactionsshowing significant changes in metabolite levels and enzyme abundance during thetransition. It resembles the cessation of a measurable metabolism (e.g. glycolysis andamino acid anabolism) during the tun formation, the production of storage metabolites andbioprotectants, such as DNA stabilizers, and the generation of amino acids and cellularcomponents from monosaccharides as carbon and energy source during rehydration. Conclusions: The functional module identifies relationships among changed metabolites (e.g.spermidine) and reactions and provides first insights in important altered metabolicpathways. With sparse and diverse data available, the presented integrated metabolitenetwork approach is suitable to integrate all existing data and analyse it in a combinedmanner.

Description: Introduction The current standard to investigate centrosome aberrations on a single cell level is labeling of known centrosomal as well as core centriolar proteins by immunofluorescence (IF) staining and subsequent manual quantification of centrosomes and centrioles. This approach is, however, very time-consuming and prone to inter-observer variations. In order to systematically evaluate centrosomal aberrations as potential predictors of malignancy, reliable high throughput analyses of IF images are required. To address this unmet need, we developed a semi-automated workflow using the highly versatile data analysis platform Konstanz Information Miner (KNIME) (Berthold et al., 2008). Input data U2OS-PLK4 cells (Konotop et al., Cancer Res. 2016) were induced for centrosomal amplification and immediately processed including IF staining against pericentrin and centrin and image acquisition using a Zeiss Cell Observer and 40× 1.3 NA Plan Apochromat objective. Per condition, 300 cells were observed and allocated manually to the phenotype classes - cells with normal centrosomes, cells with clustered amplified centrosomes and cells with declustered amplified centrosomes. The workflow settings were trained with 20% of the entire data. KNIME workflow and neural network for centrosome analysis Our KNIME workflow for centrosome analysis is composed of three main functional parts: (1) Input node groups, where image data is loaded and user-specific settings are pre-defined, (2) the Image Analysis metanode which carries out the central workflow functions and is outlined in the Figure and (3) the output node groups where all data is organized into results data tables and verification views are generated. Briefly, the Image Analysis metanode identifies nuclei, cells and centrosome areas (single or clustered centrosomes) based on thresholding and extracts various features of all objects. The centrosome detection is supposed to be highly sensitive to ensure a low number of false-negative detections. False-positive detections are filtered out or tagged as "uncertain" by a pre-defined set of rules, however, the thresholds used in these rules are adapted automatically based on features of "reference spots". These are identified centrosome candidates with a high likelihood to be true centrosomes. The workflow structure allows easy adjustment to changing parameters for a broad spectrum of user applications with a similar readout. To discriminate between cells with normal and amplified centrosomes we used a feed-forward neural network classifier that assigns the cells into these classes by evaluating relevant extracted feature parameters. The network is a multilayer perceptron (layer widths 70, 10, 10, 10, 2) with nonlinear sigmoid activation functions; the output layer carries a softmax activation and yields a probability distribution over the two classes "normal" and "clustered". The training was performed by maximizing the cross-entropy loss on a dataset of 554 manually-labelled samples, 154 of which were retained for validation. Comparison of manual and automated quantification As expected, centrosomal amplification increased upon TET-induction according to manual quantification (clustered 71% vs. 21%, declustered 7.4% vs. 0.3%). The KNIME workflow was used for feature extraction and to assign the cells into the phenotype class declustered in case of 〉2 spots per cell. It tagged 43/615 cells (7%) as uncertain and almost all of the remaining cells were labeled in agreement with the manual count (one false-positive and one false-negative). Subsequently, using the features exported from the KNIME workflow, a neural network was trained to discriminate between normal and clustered amplified centrosomes. The training loss converged to 95%, i.e. 381 of 400 training samples were correctly classified, and on the hitherto unseen validation samples the network correctly classified 88%, i.e. 135 of 154 were labeled in agreement with the manual method. The combined detection of centrosome amplification by the KNIME workflow and neural network was (TET-induced vs. control): clustered 79% vs. 21%, declustered 5.6% vs. 0.3%. Conclusions We present a reliable semi-automated workflow for high throughput analysis of IF images. This tool will be particularly useful for screens of centrosomal aberrations, but can also be easily adjusted for different experimental and infrastructural setups. Figure Disclosures Goldschmidt: Mundipharma: Research Funding; MSD: Research Funding; Amgen: Consultancy, Research Funding; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Dietmar-Hopp-Stiftung: Research Funding; John-Hopkins University: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; John-Hopkins University: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Chugai: Honoraria, Research Funding; Molecular Partners: Research Funding; Janssen: Consultancy, Research Funding. Schönland:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Prothena: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Medac: Other: Travel Grant. Krämer:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Research Funding; BMS: Research Funding.

Description: BACKGROUND: Rituximab (R) in combination with DHAP is a widely accepted salvage regimen in patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL). DHAP combines cisplatin (100 mg/m2) typically administered intravenously (i. v.) by continuous infusion over 24 hours, followed on day 2 by cytarabine (2 g/m2) in a 3-hour infusion repeated after 12 hours, and oral administration of dexamethasone (40 mg/d) for 4 consecutive days. A common adverse effect of this protocol consists of renal toxicity which may result in dose reduction or treatment discontinuation. Therefore novel approaches to overcome renal toxicity of R-DHAP are urgently warranted. Assuming that a lower single dose of cisplatin over several days would reduce renal toxicity, our institution has chosen to administer cisplatin at a dosage of 25 mg/m2 per day as a 3-hour infusion over 4 consecutive days. In this study we systematically examine efficacy and renal toxicity of this modified R-DHAP regimen. METHODS: We retrospectively analyzed data of 122 patients with relapsed/refractory DLBCL who were treated at our institution from July 2002 to July 2013. Patients were grouped according to the number of R-DHAP courses applied and renal function was evaluated in each subgroup. Creatinine serum levels before each R-DHAP cycle and two to three weeks after the last R-DHAP were assessed and GFR was calculated according to the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation. RESULTS: Overall, 256 R-DHAP cycles were administered. 31 (25%), 61 (50%), 14 (12%) and 16 (13%) patients received one, two, three or four R-DHAP courses, respectively. Dose adjustments needed to be applied in only 5 (4%) patients who in total received 11 R-DHAP courses. A step-by-step evaluation of renal function after each R-DHAP course revealed that a GFR decrease can be observed after each chemotherapy cycle. However, in none of the subgroups GFR was lower than 60 ml/min/1.73 m2. In most patients, only renal impairment stage I and II was observed. Renal impairment stage III was observed in 12 patients (10%) and stage IV only in one patient (1%). The overall response rate of the modified R-DHAP protocol was 54% (CR 17%, CRu 5%, PR 32%, SD 15% and PD 29%, not known 4%). CONCLUSION: A modified R-DHAP regimen with administration of cisplatin 25 mg/m2 over 4 consecutive days is effective and safe in relapsed/refractory DLBCL and leads only to minimal renal toxicity. Disclosures Witzens-Harig: Pfizer: Honoraria, Research Funding; Roche: Honoraria.

Description: Background: Systemic light chain amyloidosis (AL) is a rare and life-threatening protein-deposition disorder. The diagnosis and especially quantification of the underlying, usually small clonal B cell disorder in patients with very low levels of free kappa or lambda light chains in serum (FLC) can be challenging. DFLC (difference of involved minus uninvolved FLC) response to therapy is hardly assessable for initial values below 50 mg/l. Consequently, these patients are frequently excluded from prospective and retrospective studies. Objective: Characterization of AL amyloidosis patients with dFLC

Description: Introduction Genomic instability is the basic prerequisite for a Darwinian-type evolution of neoplasia and as such represents a fundamental hallmark of cancer. Centrosomal aberrations have been identified as potent drivers of genomic instability (Cosenza et al., Cell Reports 2017; Krämer et al., Leukemia 2003). The current standard to investigate centrosomal aberrations in cancer patients is immunofluorescence (IF) staining. Although this method is fast and easily scalable, its diagnostic significance is controversially discussed. Moreover, ultrastructural analysis of centrosomes in cancer patients is required to gain a mechanistical understanding of the relationship between genomic instability and centrosomal aberrations. To address this, we combined semi-automated analysis of immunofluorescence (IF) images with high-throughput electron tomography (ET) of different cell lines and subentities of primary plasma cell neoplasia, which serve as surrogate for clonal evolution. Methods CD138+ plasma cells were isolated from bone marrow aspirates of consenting patients with plasma cell neoplasia. Each sample was split to be subsequently processed for IF and ET. The IF workflow included (1) chemical fixation, (2) staining for nuclei, cells, centrin and pericentrin, (3) semi-automated acquisition of 〉1000 cells, (4) semi-automated analysis of IF data using the software Konstanz Information Miner (KNIME) (Berthold et al., GfKL 2007). The ET workflow included (1) chemical fixation (2) agarose embedding, (3) dehydration and epoxy resin embedding, (4) serial sectioning at 200 nm, (5) semi-automated screening for centrioles with transmission electron microscopy (TEM) (Schorb et al., Nature Methods 2019), (6) semi-automated acquisition of previously identified centriole regions with serial section ET. Results So far, four patients with relapsed refractory myeloma as well as two cell lines (U2OS-PLK4, RPMI.8226) have been screened with TEM. No centrosomal amplification was apparent by IF in any of these patients. Within 5598 cells, 205 centrosomes have been detected. A total of 659 electron tomograms were performed on 141 regions of interest that were distributed on average over five sections. One patient with highly refractory multiple myeloma (resistance to eight prior therapies) showed over-elongated and partially fragmented centrioles (Figure), similar to recently reported findings in tumor cell lines (Marteil et al., Nature Communications 2018). Six out of 10 mother centrioles in this patient were longer than 500 nm, which is supposed to be the physiological length. The dimensions (mean [range]) of mother (decorated with appendages) and daughter centrioles in this patient were: length 919 nm [406 nm - 2620 nm] and 422 nm [367 nm - 476 nm]; diameter 221 nm [99 nm - 470 nm] and 236 nm [178 nm - 450 nm]. Moreover, the mother centrioles showed multiple sets of appendages (mean [range]: 5.9 [2 - 13]), while one set of appendages would be physiological. This is an ongoing study and additional results are expected by the date of presentation. Conclusions We present a semi-automated methodological setup that combines high-throughput IF and cutting-edge ET to study centrosomal aberrations. To our knowledge, this is the first study that systematically analyzes the centrosomal phenotype of cancer patients at the ultrastructural level. Our preliminary IF results suggest that supernumerary centrosomes in plasma cell neoplasia might be less common than previously reported. Moreover, we for the first time describe and characterize over-elongated centrioles in myeloma patients, reminiscent of previous findings in tumor cell lines. With increasing numbers of patients, we will be also able to correlate results from IF and ET to address the current uncertainty with respect to IF screens for centrosomal aberrations. Better insight into centrosomal aberrations will likely increase our understanding on karyotype evolution in plasma cell neoplasia and possibly facilitate the development of novel targeted therapies. Figure Disclosures Goldschmidt: John-Hopkins University: Research Funding; John-Hopkins University: Research Funding; MSD: Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Dietmar-Hopp-Stiftung: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Research Funding; Molecular Partners: Research Funding; Janssen: Consultancy, Research Funding; Mundipharma: Research Funding; Chugai: Honoraria, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Müller-Tidow:MSD: Membership on an entity's Board of Directors or advisory committees. Schönland:Medac: Other: Travel Grant; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Prothena: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Krämer:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Daiichi-Sankyo: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Research Funding.

Description: Daratumumab has shown promising first results in systemic amyloid light-chain (AL) amyloidosis. We analyzed a consecutive series of 168 patients with advanced AL receiving either daratumumab/dexamethasone (DD, n = 106) or daratumumab/bortezomib/dexamethasone (DVD, n = 62). DD achieved a remission rate (RR) of 64% and a very good hematologic remission (VGHR) rate of 48% after 3 months. Median hematologic event-free survival (hemEFS) was 11.8 months and median overall survival (OS) was 25.6 months. DVD achieved a 66% RR and a 55% VGHR rate. Median hemEFS was 19.1 months and median OS had not been reached. Cardiac organ responses were noted in 22% with DD and 26% with DVD after 6 months. Infectious complications were common (Common Terminology Criteria [CTC] grade 3/4: DD 16%, DVD 18%) and likely related to a high rate of lymphocytopenia (CTC grade 3/4: DD 20%, DVD 17%). On univariable analysis, hyperdiploidy and gain 1q21 conferred an adverse factor for OS and hemEFS with DD, whereas translocation t(11;14) was associated with a better hemEFS. N-terminal prohormone of brain natriuretic peptide 〉8500 ng/L could not be overcome for survival with each regimen. Multivariable Cox regression analysis revealed plasma cell dyscrasia (difference between serum free light chains [dFLC]) 〉180 mg/L as an overall strong negative prognostic factor. Additionally, nephrotic-range albuminuria with an albumin-to-creatinine-ratio (ACR) 〉220 mg/mmol was a significantly adverse factor for hemEFS (hazard ratio, 2.1 and 3.1) with DD and DVD. Daratumumab salvage therapy produced good results and remission rates challenging any therapy in advanced AL. Outcome is adversely influenced by the activity of the underlying plasma cell dyscrasia (dFLC) and nephrotic-range albuminuria (ACR).

Description: Key PointsAL patients with an initial dFLC