ALBERT

Description: Soil aeration is a critical factor for oxygen‐limited subsoil processes, as transport by diffusion and advection is restricted by the long distance to the free atmosphere. Oxygen transport into the soil matrix is highly dependent on its connectivity to larger pore channels like earthworm and root colonised biopores. Here we hypothesize that the soil matrix around biopores represents different connectivity depending on biopore genesis and actual coloniser. We analysed the soil pore system of undisturbed soil core samples around biopores generated or colonised by roots and earthworms and compared them with the pore system of soil, not in the immediacy of a biopore. Oxygen partial pressure profiles and gas relative diffusion was measured in the rhizosphere and drilosphere from the biopore wall into the bulk soil with microelectrodes. The measurements were linked with structural features such as porosity and connectivity obtained from X‐ray tomography and image analysis. Aeration was enhanced in the soil matrix surrounding biopores in comparison to the bulk soil, shown by higher oxygen concentrations and higher relative diffusion coefficients. Biopores colonised by roots presented more connected lateral pores than earthworm colonised ones, which resulted in enhanced aeration of the rhizosphere compared to the drilosphere. This has influenced biotic processes (microbial turnover/mineralization or root respiration) at biopore interfaces and highlights the importance of microstructural features for soil processes and their dependency on the biopore's coloniser.

Description: Deutsche Forschungsgemeinschaft http://dx.doi.org/10.13039/501100001659

Description: Copper (Cu) is an essential element for plants and microorganisms and at larger concentrations a toxic pollutant. A number of factors controlling Cu dynamics have been reported, but information on quantitative relationships is scarce. We aimed to (i) quantitatively describe and predict soil Cu concentrations (CuAR) in aqua regia considering site‐specific effects and effects of pH, soil organic carbon (SOC) and cation exchange capacity (CEC), and (ii) study the suitability of mixed‐effects modelling and rule‐based models for the analysis of long‐term soil monitoring data. Thirteen uncontaminated long‐term monitoring soil profiles in southern Germany were analysed. Since there was no measurable trend of increasing CuAR concentrations with time in the respective depth ranges of the sites, data from different sampling dates were combined and horizon‐specific regression analyses including model simplifications were carried out for 10 horizons. Fixed‐ and mixed‐effects models with the site as a random effect were useful for the different horizons and significant contributions (either of main effects or interactions) of SOC, CEC and pH were present for 9, 8 and 7 horizons, respectively. Horizon‐specific rule‐based cubist models described the CuAR data similarly well. Validations of cubist models and mixed‐effects models for the CuAR concentrations in A horizons were successful for the given population after random splitting into calibration and validation samples, but not after independent validations with random splitting according to sites. Overall, site, CEC, SOC and pH provide important information for a description of CuAR concentrations using the different regression approaches. Highlights: Information on quantitative relationships for factors controlling Cu dynamics is scarce. Site, CEC, SOC and pH provide important information for a description of Cu concentrations. Validations of cubist models and mixed‐effects models for A horizons were successful for a closed population of sites.

Description: Bavarian State Ministry of the Environment and Consumer Protection http://dx.doi.org/10.13039/501100010219

Description: Ministry of Agriculture and Environment Mecklenburg‐Western Pomerania

Description: In recent years, German cities were heavily impacted by pluvial flooding and related damage is projected to increase due to climate change and urbanisation. It is important to ask how to improve urban pluvial flood risk management. To understand the current state of property level adaptation, a survey was conducted in four municipalities that had recently been impacted by pluvial flooding. A hybrid framework based on the Protection Motivation Theory (PMT) and the Protection Action Decision Model (PADM) was used to investigate drivers of adaptive behaviour through both descriptive and regression analyses. Descriptive statistics revealed that participants tended to instal more low‐ and medium‐cost measures than high‐cost measures. Regression analyses showed that coping appraisal increased protection motivation, but that the adaptive behaviour also depends on framing factors, particularly homeownership. We further found that, while threat appraisal solely affects protection motivation and responsibility appraisal affects solely maladaptive thinking, coping appraisal affects both. Our results indicate that PMT is a solid starting point to study adaptive behaviours in the context of pluvial flooding, but we need to go beyond that by, for instance, considering factors of the PADM, such as responsibility, ownership, or respondent age, to fully understand this complex decision‐making process.

Description: Bundesministerium für Bildung und Forschung http://dx.doi.org/10.13039/501100002347

Description: Little research attention has been given to validating clusters obtained from the groundwater geochemistry of the waterworks' capture zone with a prevailing lake‐groundwater exchange. To address this knowledge gap, we proposed a new scheme whereby Gaussian finite mixture modeling (GFMM) and Spike‐and‐Slab Bayesian (SSB) algorithms were utilized to cluster the groundwater geochemistry while quantifying the probability of the resulting cluster membership against each other. We applied GFMM and SSB to 13 geochemical parameters collected during different sampling periods at 13 observation points across the Barnim Highlands plateau located in the northeast of Berlin, Germany; this included 10 observation wells, two lakes, and a gallery of drinking production wells. The cluster analysis of GFMM yielded nine clusters, either with a probability ≥0.8, while the SSB produced three hierarchical clusters with a probability of cluster membership varying from 〈0.2 to 〉0.8. The findings demonstrated that the clustering results of GFMM were in good agreement with the classification as per the principal component analysis and Piper diagram. By superimposing the parameter clustering onto the observation clustering, we could identify discrepancies that exist among the parameters of a certain cluster. This enables the identification of different factors that may control the geochemistry of a certain cluster, although parameters of that cluster share a strong similarity. The GFMM results have shown that from 2002, there has been active groundwater inflow from the lakes towards the capture zone. This means that it is necessary to adopt appropriate measures to reverse the inflow towards the lakes.

Description: Article impact statement: The probability of cluster membership quantified using an algorithm should be validated against another probabilistic‐based classifier.

Description: Federal Ministry of Education and Research http://dx.doi.org/10.13039/501100002347

Description: © The Author(s), 2022. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Aymonnier, K., Ng, J., Fredenburgh, L. E., Zambrano-Vera, K., Muenzer, P., Gutch, S., Fukui, S., Desjardins, M., Subramaniam, M., Baron, R. M., Raby, B. A., Perrella, M. A., Lederer, J. A., & Wagner, D. D. Inflammasome activation in neutrophils of patients with severe COVID-19. Blood Advances, 6(7), (2022): 2001–2013, https://doi.org/10.1182/bloodadvances.2021005949.

Description: Infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) engages the inflammasome in monocytes and macrophages and leads to the cytokine storm in COVID-19. Neutrophils, the most abundant leukocytes, release neutrophil extracellular traps (NETs), which have been implicated in the pathogenesis of COVID-19. Our recent study shows that activation of the NLRP3 inflammasome is important for NET release in sterile inflammation. However, the role of neutrophil inflammasome formation in human disease is unknown. We hypothesized that SARS-CoV-2 infection may induce inflammasome activation in neutrophils. We also aimed to assess the localization of inflammasome formation (ie, apoptosis-associated speck-like protein containing a CARD [ASC] speck assembly) and timing relative to NETosis in stimulated neutrophils by real-time video microscopy. Neutrophils isolated from severe COVID-19 patients demonstrated that ∼2% of neutrophils in both the peripheral blood and tracheal aspirates presented ASC speck. ASC speck was observed in neutrophils with an intact poly-lobulated nucleus, suggesting early formation during neutrophil activation. Additionally, 40% of nuclei were positive for citrullinated histone H3, and there was a significant correlation between speck formation and nuclear histone citrullination. Time-lapse microscopy in lipopolysaccharide -stimulated neutrophils from fluorescent ASC reporter mice showed that ASC speck formed transiently and at the microtubule organizing center long before NET release. Our study shows that ASC speck is present in neutrophils from COVID-19 patients with respiratory failure and that it forms early in NETosis. Our findings suggest that inhibition of neutrophil inflammasomes may be beneficial in COVID-19.

Description: P.M. received an Individual Marie Skłodowska-Curie Actions fellowship by the European Commission (796365 - COAGULANT). This work was supported by the National Institutes of Health (NIH)/Research Program Award grant R35 HL135765 (D.W.), by the NIH/National Heart, Lung, and Blood Institute grant T32 HL007633-35 (J.N.), and by the NIH/National Institute of Allergy and Infectious Diseases grant U01AI138318 (J.L and M.P); by the Massachusetts Consortium on Pathogen Readiness (MassCPR) Evergrande COVID‐19 Response Fund Award to B.R.; and by a generous gift to D.W. from the Steven Berzin family.

Description: Application of farmyard manure (FYM) is common practice to improve physical and chemical properties of arable soil and crop yields. However, studies on effects of FYM application mainly focussed on topsoils, whereas subsoils have rarely been addressed so far. We, therefore, investigated the effects of 36‐year FYM application with different rates of annual organic carbon (OC) addition (0, 469, 938 and 1875 g C m−2 a−1) on OC contents of a Chernozem in 0–30 cm (topsoil) and 35–45 cm (subsoil) depth. We also investigated its effects on soil structure and hydraulic properties in subsoil. X‐ray computed tomography was used to analyse the response of the subsoil macropore system (≥19 μm) and the distribution of particulate organic matter (POM) to different FYM applications, which were related to contents in total OC (TOC) and water‐extractable OC (WEOC). We show that FYM‐C application of 469 g C m−2 a−1 caused increases in TOC and WEOC contents only in the topsoil, whereas rates of ≥938 g C m−2 a−1 were necessary for TOC enrichment also in the subsoil. At this depth, the subdivision of TOC into different OC sources shows that most of the increase was due to fresh POM, likely by the stimulation of root growth and bioturbation. The increase in subsoil TOC went along with increases in macroporosity and macropore connectivity. We neither observed increases in plant‐available water capacity nor in unsaturated hydraulic conductivity. In conclusion, only very high application of FYM over long periods can increase OC content of subsoil at our study site, but this increase is largely based on fresh, easily degradable POM and likely accompanied by high C losses when considering the discrepancy between OC addition rate by FYM and TOC response in soil. Highlights A new image processing procedure to distinguish fresh and decomposed POM. The increase of subsoil C stock based to a large extend on fresh, labile POM. Potential of arable subsoils for long‐term C storage by large FYM application rates is limited. The increase in TOC has no effect on hydraulic properties of the subsoil.

Description: Deutsche Forschungsgemeinschaft http://dx.doi.org/10.13039/501100001659

Description: We present a workflow to estimate geostatistical aquifer parameters from pumping test data using the Python package welltestpy. The procedure of pumping test analysis is exemplified for two data sets from the Horkheimer Insel site and from the Lauswiesen site, Germany. The analysis is based on a semi‐analytical drawdown solution from the upscaling approach Radial Coarse Graining, which enables to infer log‐transmissivity variance and horizontal correlation length, beside mean transmissivity, and storativity, from pumping test data. We estimate these parameters of aquifer heterogeneity from type‐curve analysis and determine their sensitivity. This procedure, implemented in welltestpy, is a template for analyzing any pumping test. It goes beyond the possibilities of standard methods, for example, based on Theis' equation, which are limited to mean transmissivity and storativity. A sensitivity study showed the impact of observation well positions on the parameter estimation quality. The insights of this study help to optimize future test setups for geostatistical aquifer analysis and provides guidance for investigating pumping tests with regard to aquifer statistics using the open‐source software package welltestpy.

Description: Article impact statement: We present a workflow to infer parameters of subsurface heterogeneity from pumping test data exemplified at two sites using welltestpy.

Description: German Federal Environmental Foundation (DBU) http://dx.doi.org/10.13039/100007636

Description: In designed experiments, different sources of variability and an adequate scale of measurement need to be considered, but not all approaches in common usage are equally valid. In order to elucidate the importance of sources of variability and choice of scale, we conducted an experiment where the effects of biochar and slurry applications on soil properties related to soil fertility were studied for different designs: (a) for a field‐scale sampling design with either a model soil (without natural variability) as an internal control or with composited soils, (b) for a design with a focus on amendment variabilities, and (c) for three individual field‐scale designs with true field replication and a combined analysis representative of the population of loess‐derived soils. Three silty loam sites in Germany were sampled and the soil macroaggregates were crushed. For each design, six treatments (0, 0.15 and 0.30 g slurry‐N kg−1 with and without 30 g biochar kg−1) were applied before incubating the units under constant soil moisture conditions for 78 days. CO2 fluxes were monitored and soils were analysed for macroaggregate yields and associated organic carbon (C). Mixed‐effects models were used to describe the effects. For all soil properties, results for the loess sites differed with respect to significant contributions of fixed effects for at least one site, suggesting the need for a general inclusion of different sites. Analysis using a multilevel model allowed generalizations for loess soils to be made and showed that site:slurry:biochar and site:slurry interactions were not negligible for macroaggregate yields. The use of a model soil as an internal control enabled observation of variabilities other than those related to soils or amendments. Experiments incorporating natural variability in soils or amendments resulted in partially different outcomes, indicating the need to include all important sources of variability. Highlights Effects of biochar and slurry applications were studied for different designs and mixed‐effects models were used to describe the effects. Including an internal control allowed observation of, e.g., methodological and analytical variabilities. The results suggested the need for a general inclusion of different sites. Analysis using a multilevel model allowed generalizations for loess soils. The results indicated the need to include all important sources of variability.

Description: Temperate forest soils are often considered as an important sink for atmospheric carbon (C), thereby buffering anthropogenic CO2 emissions. However, the effect of tree species composition on the magnitude of this sink is unclear. We resampled a tree species common garden experiment (six sites) a decade after initial sampling to evaluate whether forest floor (FF) and topsoil organic carbon (Corg) and total nitrogen (Nt) stocks changed in dependence of tree species (Norway spruce—Picea abies L., European beech—Fagus sylvatica L., pedunculate oak—Quercus robur L., sycamore maple—Acer pseudoplatanus L., European ash—Fraxinus excelsior L. and small‐leaved lime—Tilia cordata L.). Two groups of species were identified in terms of Corg and Nt distribution: (1) Spruce with high Corg and Nt stocks in the FF developed as a mor humus layer which tended to have smaller Corg and Nt stocks and a wider Corg:Nt ratio in the mineral topsoil, and (2) the broadleaved species, of which ash and maple distinguished most clearly from spruce by very low Corg and Nt stocks in the FF developed as mull humus layer, had greater Corg and Nt stocks, and narrow Corg:Nt ratios in the mineral topsoil. Over 11 years, FF Corg and Nt stocks increased most under spruce, while small decreases in bulk mineral soil (esp. in 0–15 cm and 0–30 cm depth) Corg and Nt stocks dominated irrespective of species. Observed decadal changes were associated with site‐related and tree species‐mediated soil properties in a way that hinted towards short‐term accumulation and mineralisation dynamics of easily available organic substances. We found no indication for Corg stabilisation. However, results indicated increasing Nt stabilisation with increasing biomass of burrowing earthworms, which were highest under ash, lime and maple and lowest under spruce. Highlights We studied if tree species differences in topsoil Corg and Nt stocks substantiate after a decade. The study is unique in its repeated soil sampling in a multisite common garden experiment. Forest floors increased under spruce, but topsoil stocks decreased irrespective of species. Changes were of short‐term nature. Nitrogen was most stable under arbuscular mycorrhizal species.

Description: Deutsche Forschungsgemeinschaff (DFG)

Description: An earthquake of Mw=6.3 struck L’Aquila town (central Italy) on April 6, 2009 rupturing an approximately 18 km long SW-dipping normal fault. The aftershock area extended for a length of more than 35 km and included major aftershocks on April 7 and 9, and thousands of minor events. Surface faulting occurred along the SW-dipping Paganica fault with a continuous extent of ~2.5 km. Ruptures consist of open cracks and vertical dislocations or warps (0.1 maximum throw) with an orientation of N130°-N140°. Small triggered slip and shaking effects also took place along nearby synthetic and antithetic normal faults. The observed limited extent, and small surface displacement, of the Paganica ruptures with respect to the height of the fault scarps and vertical throws of paleoearthquakes along faults in the area, puts the faulting associated with the L’Aquila earthquake in perspective with respect to the maximum expected magnitude, and the regional seismic hazard.

Description: In press

Description: 3.2. Tettonica attiva

Description: JCR Journal

Description: open

Description: An earthquake of Mw = 6.3 struck L Aquila town (central Italy) on 6 April 2009 rupturing an ~18-km-long SW-dipping normal fault. The aftershock area extended for a length of more than 35 km and included major aftershocks on 7 and 9 April and thousands of minor events. Surface faulting occurred along the SW-dipping Paganica fault with a continuous extent of ~2.5 km. Ruptures consist of open cracks and vertical dislocations or warps (0.1m maximum throw) with an orientation of N130°–140°. Small triggered slip and shaking effects also took place along nearby synthetic and antithetic normal faults. The observed limited extent and small surface displacement of the Paganica ruptures with respect to the height of the fault scarps and vertical throws of palaeo-earthquakes along faults in the area put the faulting associated with the L' Aquila earthquake in perspective with respect to the maximum expected magnitude and the regional seismic hazard.

Description: Published

Description: 43-51

Description: 3.2. Tettonica attiva

Description: JCR Journal

Description: reserved

Description: Despite the advance in our understanding of the carbon exchange between terrestrial ecosystems and the atmosphere, semiarid ecosystems have been poorly investigated and little is known about their role in the global carbon balance. We used eddy covariance measurements to determine the exchange of CO2 between a semiarid steppe and the atmosphere over 3 years. The vegetation is a perennial grassland of Stipa tenacissima L. located in the SE of Spain. We examined diurnal, seasonal and interannual variations in the net ecosystem carbon balance (NECB) in relation to biophysical variables. Cumulative NECB was a net source of 65.7, 143.6 and 92.1 g C mˉ2 yrˉ1 for the 3 years studied, respectively. We separated the year into two distinctive periods: dry period and growing season. The ecosystem was a net source of CO2 to the atmosphere, particularly during the dry period when large CO2 positive fluxes of up to 15 μmol mˉ2 sˉ1 were observed in concomitance with large wind speeds. Over the growing season, the ecosystem was a slight sink or neutral with maximum rates of -2.3 μmol mˉ2 sˉ1. Rainfall events caused large fluxes of CO2 to the atmosphere and determined the length of the growing season. In this season, photosynthetic photon flux density controlled day-time NECB just below 1000 μmol mˉ2 sˉ1. The analyses of the diurnal and seasonal data and preliminary geological and gas-geochemical evaluations, including C isotopic analyses, suggest that the CO2 released was not only biogenic but most likely included a component of geothermal origin, presumably related to deep fluids occurring in the area. These results highlight the importance of considering geological carbon sources, as well as the need to carefully interpret the results of eddy covariance partitioning techniques when applied in geologically active areas potentially affected by CO2-rich geofluid circulation.

Description: Published

Description: 539–554

Description: 4.5. Studi sul degassamento naturale e sui gas petroliferi

Description: JCR Journal

Description: reserved

Description: We present an improved evaluation of the current strain and stress fields in Southern Apennines (Italy) obtained through a careful analysis of geodetic, seismological and borehole data. In particular, our analysis provides an updated comparison between the accrued strain recorded by geodetic data, and the strain released by seismic activity in a region hit by destructive historical earthquakes. To this end, we have used 9 years of GPS observations (2001-2010) from a dense network of permanent stations, a dataset of 73 well constrained stress indicators (borehole breakouts and focal mechanisms of moderate to large earthquakes), and published estimations of the geological strain accommodated by active faults in the region. Although geodetic data are generally consistent with seismic and geologic information, previously unknown features of the current deformation in southern Italy emerge from this analysis. The newly obtained GPS velocity field supports the well-established notion of a dominant NE-SW-oriented extension concentrated in a ~50 km wide belt along the topographic relief of the Apennines, as outlined by the distribution of seismogenic normal faults. Geodetic deformation is, however, non uniform along the belt, with two patches of higher strain-rate and shear stress accumulation in the north (Matese Mountains) and in the south (Irpinia area). Low geodetic strain-rates are found in the Bradano basin and Apulia plateau to the east. Along the Ionian Sea margin of southern Italy, in southern Apulia and eastern Basilicata and Calabria, geodetic velocities indicate NW-SE extension which is consistent with active shallow-crustal gravitational motion documented by geological studies. In the west, along the Tyrrhenian margin of the Campania region, the tectonic geodetic field is disturbed by volcanic processes. Comparison between the magnitude of the geodetic and the seismic strain-rates (computed using a long historical seismicity catalogue) allow detecting areas of high correlation, particularly along the axis of the mountain chain, indicating that most of the geodetic strain is released by earthquakes. This relation does not hold for the instrumental seismic catalogue, as a consequence of the limited time span covered by instrumental data. In other areas (e.g. Murge plateau in central Apulia), where seismicity is very low or absent, the yet appreciable geodetic deformation might be accommodated in aseismic mode. Overall, the excellent match between the stress and the strain-rate directions in much of the Apennines indicates that both earthquakes and ground deformation patterns are driven by the same crustal forces.

Description: Published

Description: 1270-1282

Description: 3.2. Tettonica attiva

Description: JCR Journal

Description: restricted

Description: Several volcanoes worldwide have shown changes in their stress state as a consequence of the deformation produced by the pressurization of a magmatic body. This study investigates seismic swarms occurring on the western flank of Mt. Etna in January 1997 - January 1998. Integrating seismic observations and geodetic data, we constrained the seismogenic fault system, and on the basis of stress tensor inversion and SHMAX analyses, we infer an inflating pressure source located at 5.5 km b.s.l. beneath the west portion of summit area. Evaluation of Coulomb failure stress (CFS) related to the proposed model, showed how a large part of the seismogenic fault underwent a significant CFS increase (500 kPa). We infer the presence of a sub-vertical faulted region, potentially weak, N50°E oriented beneath the western sector of Mt. Etna. This structure could be brought closer to failure thereby generating seismic swarms as the effect of elastic stress transfer induced by movement and/or overpressure of magmatic masses within the upper crust under the volcano.

Description: This research was funded by the INGV–DPC 2007–2009 Agreement (Project V4_Flank).

Description: Published

Description: 339-348

Description: 1.4. TTC - Sorveglianza sismologica delle aree vulcaniche attive

Description: JCR Journal

Description: restricted

Description: Methane soil flux measurements have been made in 38 sites at the geothermal system of Sousaki (Greece) with the closed chamber method. Fluxes range from –47.6 to 29,150 mg m-2 d-1 and the diffuse CH4 output of the system has been estimated at 19 t a-1. Contemporaneous CO2 flux measurements showed a moderate positive correlation between CO2 and CH4 fluxes. Comparison of the CO2/CH4 soil flux ratios with the CO2/CH4 ratio of the gases of the main gas manifestations provided evidence for methanotrophic activity within the soil. Laboratory CH4 consumption experiments confirmed the presence of methanotrophic microorganisms in soil samples collected at Sousaki. Consumption was generally in the range from –4.9 to –38.9 pmolCH4 h-1 g-1 but could sometimes reach extremely high values (–33,000 pmolCH4 h-1 g-1.). These results are consistent with recent studies on other geothermal systems that revealed the existence of thermoacidophilic bacteria exerting methanotrophic activity in hot, acid soils, thereby reducing methane emissions to the atmosphere.

Description: Published

Description: 97–107

Description: 4.5. Studi sul degassamento naturale e sui gas petroliferi

Description: JCR Journal

Description: reserved

Description: We present a new crustal model for the European plate, derived from collection and critical integration of information selected from the literature. The model covers the whole European plate from North Africa to the North Pole (20N - 90N) and from the Mid-Atlantic Ridge to the Urals (40W - 70E). The chosen parameterization represents the crust in three layers (sediments, upper crust and lower crust), and describes the 3D geometry of the interfaces and seismologically-relevant parameters — isotropic P- and S-wave velocity, plus density — with a resolution of 0.5 × 0.5 degrees on a geographical latitude-longitude grid. We selected global and local models, derived from geological assumptions, active seismic experiments, surface-wave studies, noise correlation, receiver functions. Model EPcrust presents significant advantages with respect to previous models: it covers the whole European plate; it is a complete and internally-consistent model (with all the parameters provided, also for the sedimentary layer); it is reproducible; it is easy to update in the future by adding new contributions; and it is available in a convenient digital format. EPcrust could be used to account for crustal structure in seismic wave propagation modeling at continental scale or to compute linearized crustal corrections in continental-scale seismic tomography, gravity studies, dynamic topography and other applications that require a reliable crustal structure. Because of its resolution, our model is not suited for local-scale studies, such as the computation of earthquake scenarios, where more detailed knowledge of the structure is required. We plan to update the model as new data will become available, and possibly improve its resolution for selected areas in the future.

Description: Published

Description: 352-364

Description: 3.3. Geodinamica e struttura dell'interno della Terra

Description: JCR Journal

Description: reserved

Description: This thematic issue of Geofluids includes 11 papers representing the three main topics discussed in the 10th edition of the International Conference on Gas Geochemistry (ICGG-10): (i) gas in petroleum systems and seepage, (ii) gas in geothermal systems and volcanoes and (iii) gas, seismicity and geohazards. ICGG-10 was held in 2009 in Romania, a country extraordinarily rich in surface gas manifestations, that offers innumerable opportunities for innovative studies on gas geochemistry. We briefly describe the present knowledge on gases occurring both in petroliferous sedimentary basins and geothermal areas of Romania. The 11 contributions of this special issue, which include data from eight countries, are then summarised. Based on these papers and other works presented at the ICGG-10, we find that significant advances in analytical capabilities, data treating and interpretation have led to innovative insights into the origin, distribution and environmental impact of gases migrating to the Earth’s surface. It is increasingly clear, in particular, that gas geochemistry can be more effective for petroleum exploration, volcano-tectonic, geodynamic and environmental studies, if multiparametric studies are performed and the data are interpreted in the geological context.

Description: Published

Description: 457-462

Description: 4.5. Studi sul degassamento naturale e sui gas petroliferi

Description: JCR Journal

Description: restricted

Description: Gas seepage from petroleum basins is the second largest natural source of methane to the atmosphere, after wetlands. The uncertainty in global emission estimates should be reduced by extending the flux database which is fundamental for defining the emission factors and the actual area of seepage adopted for up-scaling. As a contribution to this goal, we report a new seepage data-set for the Transylvanian Basin, one of the largest natural gas producing regions of Europe, that is characterized by the widespread occurrence of natural leakages of gas at the surface, including at least 73 mud volcanoes and gas seeps. In this study, methane flux was measured using closed-chambers, from 12 seepage sites, in correspondence with focused gas vents (mud volcano craters, bubbling pools, and flammable gas leaks), in the soil surrounding the vents, and at 15 sites located far from macroseep zones but close to gas fields. Fluxes from individual vents (macro-seeps) were found to reach orders of kg CH4 m)2 day)1 (up to 12 kg m)2 day)1) and diffuse fluxes from soils (miniseepage) were found to be up to a few g CH4 m)2 day)1. Far from seep zones, positive CH4 fluxes (microseepage) may occur locally, typically on the order of tens to hundreds of mg m)2 day)1. The values, as well as the occurrence of seepage even far from vent zones and in mud volcanoes that are apparently extinct, are coherent with results obtained in other countries. Gas fluxes from macro-seeps and soils may change seasonally, but the interannual variation of the average emission factor was found to be minimal. The total CH4 output for Transylvania macro-seeps is estimated conservatively to be around 680 t year)1; the total geo-CH4 seepage emission from the Transylvania petroleum system could be approximately 40 · 103 t year)1, and at least 100 · 103 t year)1 for all Romanian petroleum systems, that is roughly 10% of the total anthropogenic CH4 emission in the country.

Description: Published

Description: 463-475

Description: 4.5. Studi sul degassamento naturale e sui gas petroliferi

Description: JCR Journal

Description: restricted

Description: Natural gas seeps in the Alpine region are poorly investigated. However, they can provide useful information regarding the hydrocarbon potential of sedimentary Alpine units and related geofluid migration, typically controlled by pressurized gas accumulations and tectonics. A gas seep located near Giswil, in the Swiss Northern Alps, was investigated, for the first time, for molecular and isotopic gas composition, methane flux to the atmosphere, and gas flux variations over time. The analyses indicated that the gas was thermogenic (CH4 〉 96%; d13C1: )35.5& to )40.2&) and showed evidence of subsurface petroleum biodegradation (13C-enriched CO2, and very low C3+ concentrations). The source rock in the region is marine Type II kerogen, which is likely the same as that providing thermogenic gas in the nearby Wilen shallow well, close to Lake Sarnen. However, the lack of d13CCO2 and d13C3 data for that well prevented us from determining whether the Wilen and Giswil seeps are fed by the same reservoir and seepage system. Gas fluxes from the Giswil seep, measured using a closedchamber system, were significant and mainly from two major vents. However, a substantial gas exhalation from the soil occurs diffusely in an area of at least 115 m2, leading to a total CH4 output conservatively estimated to be at least 16 tonnes per year. Gas flux variations, monitored over a 1-month period by a special tent and flowmeter, showed not only daily meteorological oscillations, but also an intrinsic ‘pulsation’ with periods of enhanced flux that lasted 2–6 h each, occurring every few days. The pulses are likely related to episodes of gas pressure build-up and discharge along the seepage system. However, to date, no relationship to seismicity in the active Sarnen strike-slip fault system has been established.

Description: Published

Description: 476-485

Description: 4.5. Studi sul degassamento naturale e sui gas petroliferi

Description: JCR Journal

Description: restricted

Description: Key Points Nonacog beta pegol, a recombinant glycoPEGylated FIX with extended half-life, was developed to improve care for patients with hemophilia B. Weekly prophylaxis with nonacog beta pegol was well tolerated and was associated with low bleeding rates and an improved quality of life.

Description: Key Points SCD increases release of HMGB1. HMGB1 plays a major role in increasing TLR4 activity in SCD.

Description: Key Points Major CALR-mutated clones may be observed in polycythemia vera negative for JAK2 mutations.

Description: Key Points MPL is essential for the development of JAK2V617F-positive myeloproliferative neoplasms in vivo. Ablation or reduction of Mpl significantly reduces the pool of neoplastic hematopoietic stem cells.

Description: Key Points Obesity is associated with increased risk for persistent minimal residual disease after induction therapy for pediatric BP-ALL.

Description: Key Points TAILS proteomics identified 2938 human platelet proteins, pervasive proteolytic processing, and precise proteolytic cleavage sites in stored platelets. During storage, metalloproteinases were predominantly involved in proteolytic processing, while other proteinases were mainly involved in degradation.

Description: Key Points N-Ras expression is essential for the proliferative advantage of acute myeloid leukemias with oncogenic NRAS/Nras mutations. Mitogen-activated protein kinase kinase inhibition prolongs survival in Nras-mutant AML by reducing proliferation, but fails to undergo apoptosis.

Description: Key Points The hyperactive phenotype of lymphoma-associated mutations is caused by increased oligomerization propensity of the MyD88 TIR domain. The TIR domain of mutants interacts with wild-type MyD88, explaining why heterozygous mutation could be sufficient as a driver mutation.

Description: Key Points Normal maturation of human NK cells requires the expression of TOX2. TOX2 directly regulates the expression of T-BET during human NK cell development.

Description: Key Points Both mature KIR+ and immature KIR− NK cells exert antileukemic activity toward pediatric BCP-ALL in vivo. In vivo treatment with low-dose 5-aza-cytidine enhances immature and mature NK-cell counts and promotes antitumor response.

Description: Key PointsCells transformed by activating JAK1 mutations become resistant to JAK inhibitor by acquiring activating mutations in JAK3 and vice versa. JAK1 and JAK3 mutants cooperatively activate STAT transcription factors.

Description: Key PointsFactor XIII-A is exposed in protruding caps on the activated platelet surface. Platelet FXIII-A exerts antifibrinolytic function by cross-linking α2AP to fibrin.

Description: Key Points MCL-1 is critical for thymic lymphoma development mediated by loss of p53. MCL-1 is essential for sustained growth of p53-deficient thymic lymphoma cells.

Description: Invasive fungal diseases (IFDs) represent an important cause of treatment failure in adults with acute leukemia. Because of leukemia’s heterogeneity, the risk for IFDs is highly variable. We therefore apply a risk-adapted antifungal strategy with strong emphasis on pretreatment and day-15 posttreatment to allow earlier and more individualized interventions. We determine pretreatment risks for IFDs based on 4 factors: (1) host fitness for standard therapy (ie, fit, unfit, or frail); (2) leukemia resistance (high vs low probability of achieving complete remission [CR]); (3) anticipated treatment-related toxicity such as neutropenia, mucositis, and steroid-induced immunosuppression; and (4) patient exposure to opportunistic fungi. Accordingly, we stratify patients as high, intermediate, or low risk for IFDs and apply risk-adapted antifungal strategies, including primary or secondary prophylaxis and diagnostic-based preemptive or empiric therapy. Prevention of IFDs also relies on optimizing organ function, decreasing exposure to opportunistic fungi, and improving net state of immunosuppression with use of better-tolerated and investigational agents for unfit patients and those with adverse leukemia biology. Novel targeted and safe therapies that can achieve higher rates of sustained CR among patients with adverse genetics offer the best promise for reducing the burden of IFDs in these patients.

Description: Key Points Ibrutinib affects collagen and VWF-mediated platelet activation. The bleeding diathesis correlates with defects in collagen-induced platelet aggregation and firm adhesion on VWF at arterial shear rate.

Description: Key Points The expression level of patient HLA-C allotypes affects GVHD and mortality after HCT from HLA-C-mismatched unrelated donors. Transplant outcome can be improved by avoiding high-risk HLA-C-mismatched donors when no matched stem cell source is available.

Description: Key Points Largest prospective trial for adult Burkitt lymphoma/leukemia patients. Substantial cure rates and high treatment-realization rates in all age groups.

Description: Key Points Caspase-9 is required for normal development of myeloid, lymphoid, and erythroid cells in mice. Loss of caspase-9 results in increased DNA damage and mutation burden after exposure to alkylating agents.

Description: Allogeneic hematopoietic stem cell transplantation (HSCT) has been considered as the treatment of choice for patients with high-risk chronic lymphocytic leukemia (HR-CLL; ie, refractory to purine analogs, short response [

Description: Key Points DIAPH1 (mDia1) is involved in both Rho-mediated actin polymerization and microtubule assembly and stability during proplatelet formation.

Description: Key Points Posttransplantation cyclophosphamide is effective as sole GVHD prophylaxis for myeloablative HLA-matched–related or –unrelated BMT. Despite low chronic GVHD with PTCy, relapse and survival are comparable with outcomes reported using other GVHD prophylactic approaches.

Description: Key Points PML/RARA loss or detachment from target promoters suffices to differentiate APL cells. PML/RARA degradation by arsenic thus explains arsenic-induced differentiation.

Description: Key Points Inhibiting LEDGF interaction with a novel fragment of MLL represents an attractive approach to develop new drugs for MLL leukemias. Structural studies reveal a new pocket on the LEDGF IBD suitable for targeting by small-molecule inhibitors.

Description: Antithrombotic treatment of splanchnic vein thrombosis (SVT) is a clinical challenge. Depending on the site of thrombosis, patients are at risk of developing liver insufficiency, portal hypertension, or bowel infarction and may experience recurrence in both the splanchnic veins and other vein segments. To prevent recurrence, anticoagulant therapy should be started as soon as possible after diagnosis and is often continued for an indefinite period of time. However, active bleeding is not infrequent at the time of SVT diagnosis, and major risk factors for bleeding, such as esophageal varices or a low platelet count, are frequently present in these patients. In real-world clinical practice, a proportion of SVT patients are left untreated because the risks associated with anticoagulant therapy are felt to exceed its benefits. However, the majority of patients receive anticoagulant drugs, with heterogeneous timing of initiation, drug choice, and dosages. Evidence to drive treatment decisions is limited because no randomized controlled trials have been carried out in these patients. This review provides practical guidance for the use of anticoagulant drugs in patients presenting with SVT, including symptomatic as well as incidentally detected events.

Description: Key Points Whole-transcriptome sequencing reveals NPM1-TYK2 gene fusion in cutaneous CD30-positve lymphoproliferative disorders. NPM1-TYK2 activates STAT signaling and is a therapeutic target in a subset of cutaneous CD30-positive lymphoproliferative disorders.

Description: Key Points GATA1 is downregulated in RPS19-deficient cells and zebrafish through upregulation of p53, TNF-α, and p38 MAPK. Treatment of rps19-deficient zebrafish with the TNF-α inhibitor etanercept rescues their erythroid and developmental defects.

Description: Key Points IFN-γ alone leads to aplastic anemia by disrupting the generation of common myeloid progenitors and lineage differentiation. The inhibitory effect of IFN-γ on hematopoiesis is intrinsic to hematopoietic stem/progenitor cells.

Description: Key Points Short-term preprocedure interruptions of either apixaban or warfarin are associated with a low rate of stroke or systemic embolism. Some patients taking apixaban or warfarin are able to undergo procedures safely without a preprocedure interruption of anticoagulation.

Description: Key PointsGPIbα–VWF-A1 bond kinetics regulates platelet–VWF interactions and can be altered to correct defects in hemostasis or prevent thrombosis. Targeting a distinct GPIbα−VWF-A1 binding interface may offer a unique therapeutic approach to reducing platelet-driven thrombosis.

Description: Key Points Telomerase RNA component hTR, but not the core enzymatic protein component hTERT, protects T cells from apoptosis. hTR prevents dexamethasone-induced apoptosis specifically when in a telomerase enzymatically inactive state.

Description: Key Points Severe thrombocytopenia is associated with a strongly impaired host defense during pneumonia-derived Klebsiella pneumoniae sepsis. Platelet counts between 5 and 13 × 109/L of normal prevent bleeding and confer protection against distant organ damage during gram-negative sepsis.

Description: Key Points Phagocytosis of IgG-opsonized blood cells by human macrophages is inhibited by intravenous immunoglobulins. This inhibition is independent of IgG-Fc sialylation but improves with IgG preparations that bind FcγRs more avidly.

Description: Key Points Transcriptomes and enhancers of human CD4+ Tfh and non-Tfh T effector cells reveal cell type–specific differences. These data are a significant resource for understanding mechanisms of normal and perturbed Tfh cell function.

Description: Key Points High levels of the anti-apoptotic factor BCL-2 can be therapeutically exploited by the BH3 mimetic ABT-199 in human T-ALL.

Description: Key Points We established hypoxia-resistant cells that can mimic in vivo conditions of hypoxic bone marrow. Exosomal miR-135b derived from these cell lines enhanced endothelial tube formation under hypoxia via the HIF-FIH signaling pathway.

Description: Key Points Comprehensive proteomics analysis in human monocytes exposed to APS-IgG has identified and characterized several novel proteins. These proteins have functional relevance to the APS.

Description: Key Points BCR activation enhances eIF4A m7GTP cap-binding. The 5′UTR of CARD11 suppresses protein translation.

Description: Hyperhaemolysis is a rare, poorly understood complication of red cell transfusion. We report the outcomes of SCT in 2 boys of Middle Eastern origin who developed life-threatening hyperhaemolysis each following their third transfusion for β-TM at the ages of 2.5 and 4 years. The diagnosis of hyperhaemolysis was based on laboratory evidence for haemolysis, post-transfusion haemoglobin (Hb) levels lower than pre-transfusion, positive Coomb’s tests and no allo-antibody able to be identified. Haemolysis was intravascular, extravascular and severe. Precipitous drops in Hb made transfusion unavoidable. The first boy responded to prednisolone, intravenous immunoglobulin (IVIG) and splenectomy. Haemosidderosis and fibrosis were present on liver biopsy, he was therefore regarded as a Class 3 thalassaemia patient and received hydroxyurea, azathioprine, erythropoietin, desferrioxamine, busulfan, cyclophosphamide and fludarabine conditioning prior to an HLA identical sibling SCT. He is alive and well 7 years post BMT with 30% stable donor chimerism and a normal Hb. The second child’s hyperhaemolysis failed to respond to prednisolone, IVIG, rituximab and splenectomy. Provision of the large number of suitably matched red cell units required was problematic. After receiving hydroxyurea, azathioprine, desferrioxamine, busulfan, cyclophosphamide, fludarabine, thiotepa and antithymocyte globulin (ATG) preparation for a CD3/CD19 depleted maternal haplotype peripheral blood SCT, the haemolysis finally stopped. Post-transplant severe veno-occlusive disease and multi-organ failure (MOF) required dialysis and ventilation. The maternal graft was rejected so 28 days after the first transplant he received a 4/6 mismatched unrelated cord blood transplant (UCBT) following further fludarabine and ATG, which fully engrafted. He recovered from MOF and was discharged from hospital 47 days after the UCBT, transfusion independent. On day +86 he contracted Respiratory Syncytial Virus chest infection with acute intravascular haemolysis necessitating transfusions. Fulminant liver failure developed, presumably due to iron toxicity, and death occurred on day +102, having received 112 transfusions in the 12 months since presentation. In conclusion, avoiding red cell transfusion is not always possible in hyperhaemolysis, especially in β-TM. Patients may quickly become classified as Class 3 in terms of predicting BMT outcome. Immune modulation therapy and SCT was effective in 1 case but only temporarily stopped haemolysis in the other, despite full engraftment ultimately being achieved with a mismatched UCBT. SCT should be considered early in cases of hyperhaemolysis in β-TM because it can potentially cure both and result in transfusion independence. Disclosures Off Label Use: Intravenous immunoglobulin and rituximab for treatment of haemolytic anaemia; hydroxyurea, azathioprine, fludarabine, erythropoeitin, busulphan, cyclophosphamide, thiotepa and antithymocyte globulin for use in stem cell transplantation in children with thalassaemia.

Description: Background: Post-remission treatment for AML is very aggressive and many times a SCT is needed. Comparisons between Allo-SCT and Auto-SCT ve always shown more Transplant Related Mortality (TRM) but less Cumulative Incidence of Relapse (CIR) in the first group. Our study describes, not only the long-term survival outcomes, but also the quality of life in long survivors. Methods: Retrospective study of 274 patients diagnosed with non-promyelocytic AML who underwent SCT between 1982 and 2011 in our center. Characteristics in the 162 Allo-SCT and the 112 Auto-SCT groups of patients were respectively: median age of 38 and 45 years old, secondary AML in 20% and 10%, refractory to Induction AML in 16% and 3%, pre-SCT status different from Complete Remission (CR) in 13% and 3% and year of SCT before 2005 in 53% and 86%. No significant differences between both groups were found in other risk factors as hyperleucocytosis at diagnosis or adverse cytogenetics. Results: With a median follow-up of 55 months [2-316], Overall Survival (OS) until 1997 in Allo-SCT and Auto-SCT were respectively, 40% and 61% at 1 year and 28% and 45% at 5 years (figure 1), but from 1997, 66% and 70% at 1 year and 47% and 48% at 5 years (figure 2). Disease Free Survival (DFS) until 1997 in Allo-SCT and Auto-SCT were respectively, 50% and 65% at 1 year and 38% and 46% at 5 years, but from 1997, 67% and 63% at 1 year and 52% and 47% at 5 years. In the last 15 years, no differences were found between both groups in OS nor DFS. CIR in Allo-SCT and Auto-SCT were respectively, 18% and 32% at 1 year and 24% and 50% at 5 years, without dependence on year of SCT. No relapse was observed later in any group. TRM until 1997 in Allo-SCT and Auto-SCT were respectively, 30% and 7% at 1 year and 35% and 9% at 5 years, but from 1997, 16% and 2% at 1 year and 25% and 4% at 5 years. Multivariable analysis showed that the only risk factor with a negative impact on OS was not having achieved CR at the time of SCT. Other variables as older age, hyperleucocytosis at diagnosis, adverse cytogenetics, secondary AML or sooner year of SCT lost their univariable analysis significance. Allo-SCT: From the 162 patients, 72(44%) are alive by this moment, 43(60%) with ECOG 0, 21(29%) with ECOG 1 and the other 8(11%) with ECOG 2, basically because of graft versus host disease (GVHD in 39 patients, 21 steroid-dependent and 3 refractory to any treatment). All of them have been in CR during the last 2 years of follow-up. In contrast, 90(56%) patients have died: 52(58%) because of SCT complications (20 infections, 16 GVHD, 8 toxicity and 8 mixed causes), 33(37%) because of disease and 5(5%) because of other causes. With a median follow-up of 43 months [2-316], there have been 4 secondary neoplasm, all of them solid ones, which appeared with a median of 242 months [179-311] from SCT. None of them had previously received radiotherapy. Auto-SCT: From the 112 patients, 43(38%) are alive by this moment, 32(74%) with ECOG 0 and the other 11(26%) with ECOG 1. All of them have been in CR during the last 2 years of follow-up. In contrast, 69(62%) patients have died: 45(65%) because of disease, 14(20%) because of SCT complications and 10(15%) because of other causes. With a median follow-up of 93 months [5-230], there have been 6 secondary neoplasm, 5 of them hematologic ones, which appeared with a median of 90 months [76-115] from SCT. None of them had received radiotherapy, but previously treated hematopoietic stem cells. Only one is alive at the time of last follow-up. Conclusions: In one hand, despite the high incidence of relapse in Auto-SCT in any period, OS is lower in Allo-SCT during the first years [1982-1996], although it has a tendency towards OS in Auto-SCT from 1997 because of the decrease in TRM, which is more significative in Allo-SCT. In the other hand, DFS is slightly higher in Allo-SCT during the last years [1997-2011], although the quality of life in long survivors is worse, basically because of GVHD. In summary, we have not really found differences between Allo-SCT and Auto-SCT in terms of OS and DFS in our series, so both procedures are efficient to treat AML (near 50% of the patients in both groups are alive at 5 years from SCT in recent years). The decrease in TRM until 4% at 5 years in Auto-SCT makes it a good choice, particularly for older patients without risk factors. However, the development of secondary hematologic neoplasms is a relevant fact, with an incidence of 11% and a high late mortality. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Description: By reducing treatment intensity allogeneic hematopoietic stem cell transplantation (allo-HSCT) has become feasible for elderly patients. Different reduced-intensity conditioning (RIC) regimens are available, but there is little consensus about the optimal preparative regimen to use, in particular with regard to the outcomes counterbalancing the aim of feasibility and tolerability with higher rates of relapse. Here, we retrospectively evaluate the outcome of sequential therapy employing RIC with fludarabine 30 mg/m2, cytarabine 2g/m2 and amsacrine 100 mg/m2 for 4 days (FLAMSA; Schmid C et al. JCO 2005) followed by busulfan 10 x 0.8 mg/kg (FLAMSA-Bu) compared to RIC utilizing fludarabine 5 x 30 mg/m2, carmustine (BCNU) 2 x 150 mg/m2 and melphalan 110 mg/m2 (FBM; Marks R et al. Blood 2008) in elderly patients treated consecutively at our institution between July 2005 and October 2012. We analyzed the course of 114 patients (pts) with acute myeloid leukemia (AML; n=99) or myelodysplasia (MDS; n=15) aged ≥ 59 years with 59 pts aged ≥ 66 years who were treated with either FLAMSA-Bu (n=66; n=24 ≥ 66 years) or FBM (n=48; n=35 ≥ 66 years). All patients received sero-therapy with anti-thymocyteglobuline (ATG). Median patient age was 66 years for the entire cohort (68 years FBM; 64 years FLAMSA-Bu). 36 patients (75%) of the FBM and 42 patients (63 %) of the FLAMSA-Bu group suffered from high risk disease defined as relapsed or refractory AML or refractory anemia with excess of blasts in transformation (RAEB-T). The hematopoietic cell transplantation comorbidity index (HCT-CI) was higher for the patients of the FBM group than for the FLAMSA-Bu group with 26 (54 %) versus (vs) 24 patients (36 %) scoring ≥ 2 (p 0.085). Graft source after conditioning with FBM/FLAMSA-Bu was bone marrow (1/2), G-CSF mobilized peripheral blood stem cells (40/62) and double-umbilical cord-blood (7/1). In 23 pts (20 %) HLA-matched related and in 91 pts (80 %) HLA-matched unrelated donor transplantation was performed. Engraftment failure was observed in 1 patient after FLAMSA-Bu, while engraftment was achieved in all evaluable patients of the FBM group in a median of 23 days vs 18 days after FLAMSA-Bu (p 0.003), while 7 pts with double-umbilical cord-blood transplantation where included in the FBM group vs 1 pt in the FLAMSA-Bu group. Non-hematological treatment-related acute toxicity ≥ CTC III (gastrointestinal, hepatic, cardiovascular, renal, centralnervous system) occurred in 12/48 pts (25 %) after FBM and in 18/66 pts (27 %) after FLAMSA-Bu. Incidence of severe acute (III-IV) and chronic GvHD was 22.9 %/16.6 % for FBM vs 18.2 %/19.7 % for FLAMSA-Bu, respectively. After conditioning with FBM 2/48 pts vs 9/66 pts after FLAMSA-Bu were diagnosed with a secondary malignancy (p 0.08). Non-relapse mortality (NRM) after 12 months was 26.8 % for FBM versus 25.2 % for the FLAMSA-Bu group. Incidence of relapse after FBM vs FLAMSA-Bu conditioning was 22.9 % vs 15.2 % after 1 year and 31.3 % vs 16.7 % after 2 years. Occurrence of relapse was significantly related to an incomplete or mixed chimerism (donor cells ≤ 95 % in peripheral blood and/or bone marrow) at day +30 (p 0.001). After a median follow up of 31.4 months (range 4.4-97.5) estimated overall survival (OS) and relapse-free survival (RFS) after 2 years was 55.4 % and 51.4 % for the FBM vs 58 % and 56.7 % for the FLAMSA-Bu group, respectively. Analyzing different subgroups, FBM conditioning might be favorable for pts aged ≥ 66 years when suffering from high risk AML (n=26): Within this group 1-year OS after FBM vs FLAMSA-Bu was 71.4 % vs 66.7 % (p 0.58) and 1-year RFS was 71.4 % vs 58.3 % (p 0.59), respectively. Notably, for pts at highest risk (aged ≥ 66 years and suffering from secondary or therapy-related AML; n=24) the benefit of FBM conditioning becomes more pronounced: 1-year OS after FBM vs FLAMSA-Bu was 62.5 % vs 37.5 % (p 0.26) and 1-year RFS 54.2 % vs 37.5 % (p 0.17). Both conditioning regimens are feasible, and provide similar rates of acute toxicity, NRM and GvHD. There might be evidence for a benefit of conditioning with FBM for the subgroup of “the oldest patients at highest risk”. Taking into account that there is an increasing group of ‘medically fit’ elderly patients in the field of allogeneic transplantation, prospective clinical trials are needed to investigate different conditioning regimens considering their special requirements. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Cerebral sinus venous thrombosis (CSVT) is potentially life-threatening thrombosis with mortality around 10%. Venous thromboembolism (VTE) is a common complication in children with cancer. These children have several thrombotic risk factors such as the malignancy itself, severe infections, prothrombotic medication and immobilization. The treatment of acute lymphoblastic leukemia (ALL) includes steroids and asparaginase (ASP), raising the VTE risk. In children with ALL the central nervous system (CNS) is a common localization for VTE. However, retrospective studies on small numbers of patients, larger studies and population-based data in children are scarce. The five Nordic countries, Estonia and Lithuania have a common treatment protocol for children with ALL between 1 and 18 years of age with prospective registration of toxicities, including CSVT offering a unique opportunity to study CSVT in this patient group. This is to our knowledge the largest report of children with ALL and CSVT describing the incidence, symptoms, treatment and the effect of CSVT on ALL treatment. Methods We assessed the symptoms, treatment, clinical risk factors and outcome of all children between ages 1 and 17 years at diagnosis of B-cell precursor or T-cell ALL between June 2008 and July 2013 and with CSVT. Data were collected from the patients’ medical records and the NOPHO leukemia registry. Results In total, 20 (1.9%) of the 1038 children with ALL treated according to the NOPHO ALL 2008 protocol developed CSVT. The cumulative incidence of CSVT was 2.0%. All the thromboses occurred within the first 5 months of treatment. The most common symptoms at the diagnosis of CSVT were headache, convulsions, weakness/fatigue and cerebral nerve palsy/hemiparesis/hemiplegia. The most frequent localizations for CSVT were sinus sagittalis (n=16) and sinus transversus (n=10). However, in most cases multiple cerebral veins were involved ( 70%). Median D-dimer at time of the CSVT diagnosis was 0.85 mg/L (range 0.19-4.7 mg/L) with 5 patients having normal D-dimer. We could not identify any clinical risk factors for CSVTs. CSVT was associated with steroids (treatment within 2 weeks before the diagnosis of CSVT) in 16/20 and with Pegylated asparaginase in 16/20. Fifteen patients were later screened for the inherited thrombophilic factors; one child had heterozygous prothrombin G20110A mutation and another heterozygous factor V (R506Q) Leiden mutation. Most patients (19/20) were treated with anticoagulants: mostly low molecular weight heparin (LMWH). The median treatment with LMWH was 26 weeks (range 14-119 weeks). No bleeding complications were observed in connection with LMWH. Two deaths were directly related to CSVT. Asparaginase was omitted from the treatment in 7 and delayed or reduced in 5 of the cases raising the risk for subsequent suboptimal leukaemia treatment. Of the surviving 18 patients, follow-up imaging revealed complete recanalization in 7 and partial recanalization in 7 cases. No imaging was available for the remaining 4 patients. Conclusions The incidence of CSVT in children with ALL was approximately 2%. No statistically significant clinical predictors for CSVT were identified. The mortality related to CSVT was 10%. Anticoagulation with LMWH was the treatment of choice in most cased and was well tolerated. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Busulfan (Bu) is used in the conditioning regimen for hematopoietic stem cell transplantation (HSCT). Therapeutic drug monitoring (TDM) of Bu with subsequents adjustments doses based on a “target” therapeutic concentration may reduce toxicity after HSCT. Objectives: To evaluatethe impact of TDM of Bu and clinical outcomes in patients with acute leukemia that underwent to allogeneic matched related donor (MRD) and allogeneic matched unrelated donor (MUD) HSCT. Patients and methods: From January 2009 to January 2014, we prospectively analyzed 42 patients with diagnosis of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) who underwent TDM of Bu (IV or oral) before transplantation (test dose) and TDM on 1st day of conditioning regimen. Samples were collected at 0, 30’, 60’ and subsequently every hour until 6 hours after administration of busulfan. The plasma was extracted by HPLC (High Performance Liquid Chromatography). All the patients that were submitted to TDM had a test dose 15 days to 48 hours before transplantion. The dose of Bu was adjusted during the first day of the conditioning regimen based on test dose. At the same time we analyzed 21 patients in the retrospective group who did not underwent TDM (from 2004 to 2010). Results: In the retrospective group (n=21), all of them underwent to MRD transplantation. Six (46.2%) were in first complete remission (CR1), 18(85.7%) patients received Bu and cyclophosphamide (BuCy) and the mean of age was 38 years (18-55 Yo). The median of CD34+ cells was 5.4 x 106/kg. The second group consisted by patients that received oral Bu (n= 21): 7 (33.3%) underwent to MUD transplantation, 14 (66.6%) to MRD transplantation, 8 (44.4%) patients were second complete remission (CR2), 4 (22.2%) had active disease status with a mean age of 32.7 years (14-58 Yo). Fifteen (71.4%) received BuCy and 16 (76.2%) received cells from peripheral blood. The median of CD34+ cells was 5.8 x106/kg. The median area under the curve (AUC) in 24 hours was 4950 μMol.min (3196.6- 8212 μMol.min). The third group was IV Bu (n= 21): 7 (33.3%) patients underwent MRD and 14 (66.6%) MUD transplantation, 7 (33.3%) patients were CR1, 7 (33.3%) had active disease or prior HSCT with a mean of age 52.7 years (20-74 Yo). The majority of patients received fludarabine and Bu (n=18; 85.7%) as conditioning and bone marrow was the main source. Immunosuppression was based on FK-506 and methotrexate (90.5% of patients). The median of AUC in 24 hours was 5690 μMol.min (3539.6- 8881.8 μMol.min). The cumulative incidence (CI) of sinusoidal obstructive syndrome (SOS) in the retrospective group and IV Bu were 9.5% for both, while in the group oral Bu it was at 19% (p = 0.566). The median AUC of Bu received during conditioning for those who died of SOS was lower in oral Bu than IV Bu (4872 uMol.min vs 5732 uMol.min respectively). The CI of acute graft-versus-host disease (aGVHD) at D+100 was 38.1% in the retrospective group, 40.6% in oral Bu and 42.9% in IV Bu. Chronic GVHD was 13.6% in oral Bu, 34% in IV Bu and 42.9% in the retrospective group (p = 0.142). The CI of relapse at D+100 was 19% in IV Bu, 4.8% in the retrospective group and oral Bu did not have this event. The IC of death at D+100 was 34.9% in group oral Bu, 9.5% in IV Bu and 14.3% in the retrospective (p = 0.102). The CI of relapse at 1.5 years was 35.8% in the IV Bu, 34.8% in oral Bu and 14.3% in the retrospective group. The CI of death at 1.5 years was 9.5% in group IV Bu, 53.5% in oral Bu and 34.3% in the retrospective (p = 0.015). Among patients who died until D+100, the median of AUC was 5732 μMol.min (5578.5-6818.5 μMol.min) during the conditioning for IV Bu and 4872 μMol.min (3448-8212 μMol.min) for oral Bu. The range between the AUC was large and there was no correlation with patients who died. Conclusion: In acute leukemia SOS had an impact on mortality at D+100 after HSCT (p

Description: Introduction: The application of nanoparticles in dendritic cell (DC)-based cancer immunotherapy represents a promising strategy to enhance antigen-specific T cell immune responses. This study was to investigate the effect of bPEI-SPIONs on antigen-specific T cell responses elicited by DCs loaded with apoptotic U266 myeloma tumor antigen in the presence or absence of bPEI-SPIONs. Materials and Methods: The myeloma tumor antigens were prepared as following: 1) apoptotic U266 cells by UBV-irradiation and overnight incubation (16 h irradiated cells) and 2) apoptotic U266 cells by UVB-irradiation and 2 h incubation in the absence (2 h irradiated cells) or 3) presence of bPEI-SPIONs (bPEI-SPION 2 h irradiated cells). Monocyte-derived immature DCs were activated with TNF-α, loaded with several kinds of myeloma tumor antigens 2 h after TNF-α stimulation, and cultured for 2 days. Results: Optimal concentration of bPEI-SPIONs was 16 µg/mL to uptake into tumor cells and the bPEI-SPIONs render U266 cells sensitive to UVB-irradiation through reactive oxygen species (ROS) induction pathway hence accelerated the apoptotic cell death. 2 h irradiated cells and bPEI-SPION 2 h irradiated cells released immunogenic proteins, including HSP70, HSP90, and HMGB1. bPEI-SPION 2 h irradiated cells were easily up-taken by DCs without alteration of surface phenotypes and migration capacities. DCs loaded with bPEI-SPION 2 h irradiated cells showed highest IL-12p70 production level and Th1 polarization compared to other DCs. Conclusion: These results suggest that bPEI-SPIONs are a promising tool to improve immunogenicity of myeloma tumor cells and to enhance Th1 polarization of DCs loaded with these tumor cells. Disclosures No relevant conflicts of interest to declare.

Description: ADAMTS-13 is a protease, member of the ADAMTS family (A Disintegrin and Metalloproteinase with ThromboSpondin type 1 repeats-13), which cleaves the cell bound large ultrapolymeric von Willebrand factor (vWF) strings. Circulating ADAMTS-13 is primarily synthesized and released from hepatic stellate and endothelial cells. Acquired functional deficiency of ADAMTS-13, usually due to inhibitory IgG autoantibodies, results in excessive platelet aggregation and disseminated vWF/platelet-rich thrombus formation. A possible association of low levels of ADAMTS-13 Ag with arterial thrombosis and endothelial dysfunction has also been reported. Hivert and colleagues (Blood 2012;120:3214-21) and our group have shown that increased levels of vWF, the only known substrate of ADAMTS-13, are associated with poorer prognosis in patients with symptomatic Waldenstrom’s Macroglobulinemia (WM). Thus, our aim was to investigate the possible association of ADAMTS-13 antigen (Ag) levels with features of WM and possible biologic implications of the ADAMTS-13 / vWF interaction in WM patients’ prognosis. Our study included 42 patients with symptomatic WM who were treated and followed in the Department of Clinical Therapeutics of the University of Athens (Greece), from 1999 to 2012, 22 patients with asymptomatic WM and 19 healthy controls of matched gender and age. For the purpose of this study we used stored serum, which had been collected before initiation of any therapy. ADAMTS-13 antigen levels were measured using a commercially available kit (R&D Systems, Minneapolis, MN, USA), which has a detection limit for ADAMTS-13 13 (ng/mL) with intra- and inter-Assay Precisions of

Description: Background: Extramedullary disease (EMD), strictly defined as an infiltrate of clonal plasma cells at an anatomic site distant from the bone marrow or adjacent soft tissue in a patient with underlying multiple myeloma, is an uncommon manifestation of multiple myeloma. Comparatively little is known about this disease entity, with no large case series published in the last decade. Patients and Methods: 663 consecutive patients with multiple myeloma who underwent autologous or allogeneic stem cell transplantation at a single, large, academic medical center in the United States from January 2005 to December 2011 were assessed for the presence or absence of EMD, as well as baseline demographic and biochemical characteristics, treatment regimens, and response to therapy. Results: A cohort of 55 patients with biopsy-proven EMD was identified, comprising 8.3% of the total study population. Among the patients with EMD, 13 (23.6%) were found to have EMD at the time of initial presentation, while the remainder developed EMD during the course of their illness. Patients with EMD received a median of 5 different treatment regimens during the course of their illness, most commonly with combinations of dexamethasone, thalidomide, lenalidomide, and bortezomib, as well as autologous hemotopoietic stem cell transplantation. Patients had received a median of 3 lines of therapy prior to experiencing an extramedullary relapse. Patients with EMD had markedly elevated maximum serum LDH levels (median 613.5 units/L) and low minimum hemoglobin levels (median 7.8 g/dL). Common cytogenetic abnormalities included deletion 13q, deletion 11q, t(11;14), and deletion 17p. Available immunohistochemical data suggest that EMD specimens had frequent expression of CXCR4, CD44, and CD56. The median overall survival data of these patients was 3.2 years (range, 0.9-9.5) and the median time from diagnosis of EMD to death was 0.5 years (range, 0.002-3.2). Conclusions: This report describes a large series of multiple myeloma patients with EMD who were treated in the era of stem cell transplant at a single academic medical center. Further studies to examine the molecular characteristics of extramedullary multiple myeloma are necessary to better define this entity and characterize therapeutic options that can prolong survival in this otherwise very vulnerable population. Disclosures Ghobrial: Millennium/Takeda: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Laubach:Novartis: Research Funding; Onyx Pharmaceuticals: Research Funding. Schlossman:Millennium: Consultancy. Mitsiades:Millennium Pharmaceuticals: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Amgen: Research Funding; Johnson & Johnson: Research Funding; DFCI: patent submission on stromal co-culture technologies Patents & Royalties.

Description: Autoimmune hemolytic anemia (AIHA) occurs in CLL at some time during the course of the disease in up to 7-10% of patients. The acute onset of AIHA may occur unrelated to therapy but has also been linked to treatment with chemo­therapeutic agents including chlor­ambucil, benda­mustine and particu­larly purine nucleosides such as fludarabine. Although the mechanism is still not well understood, chemo­therapy-induced changes in regulatory T-cells have been proposed as a trigger for autoimmunity and clinical hemolysis. In contrast to these cytotoxic therapies, ibrutinib, an inhibitor of Bruton’s tyrosine kinase recently approved for the treatment of CLL, appears to have a different mechanism of action and thus far has not been associated with AIHA in published reports. However, we report here a patient with CLL and a history of prior AIHA, who developed a recurrence of acute hemolysis after the initiation of ibrutinib. The patient is a 67-year-old man diagnosed with CLL in 2002 and treated for progressive disease with a single cycle of bendamustine in 2009. Although the lymphocytosis resolved rapidly, the hemoglobin also decreased from 14 g/dL to 5.2 g/dL by 3 weeks after the start of therapy. Due to the onset of Coombs-positive AIHA, chemotherapy was dis­con­tinued. Hemolysis resolved with prednisone therapy and did not recur after a slow taper. The CLL then remained asymptomatic until 2012 when night sweats developed at a white blood cell (wbc) count of 95,000/µL. Benda­mustine was re-started and despite a negative Coombs test prior to treatment, Coombs-positive AIHA developed again with the hemoglobin falling from normal to 7.0 g/dL within 4 weeks. After stabilization with transfusions and steroids, an additional cycle of bendamustine plus rituximab was administered without further complications and the patient’s symptoms and lympho­cytosis resolved. After the discontinuation of prednisone, hemolysis did not recur clinically although the Coombs test remained 1+ positive through early 2014. By May 2014 the wbc count had increased to 144,000/µL with the onset of a mild anemia (Hgb 12.3 g/dL) and symptomatic night sweats. Due to the history of repeated chem­otherapy-associated AIHA, alter­native therapy with ibrutinib, which had not been associated with AIHA, was instituted at 420 mg daily. However, within 2 weeks the hemoglobin decreased to 7.0 g/dL while the wbc count increased to 300,000/µL. A reticulocyte count was 16%, total bilirubin 3.2 mg/dL, haptoglobin

Description: Introduction Our previous work showed that in Multiple Myeloma (MM) the immune function is impaired, including immunosuppressive properties of granulocytes due to their increased amount of arginase-1 and reduced phagocytic activity (Parrinello, manuscript in preparation). It is currently unknown if granulocyte dysfunction occurs in progression from MGUS to MM. Aim Providing a gene expression profile of mature granulocytes isolated from peripheral blood at the steady-statein MGUS and MM. Methods Using oligonucleotide microarrays we first evaluated the gene expression profile of granulocytes at the steady state in 5 MM, 3 MGUS and 3 healthy subjects matched for sex and age. Then, we validated the first up-regulated gene PROK-2, obtained from preliminary findings in granulocytes from peripheral blood in 85 consecutive newly diagnosed MGUS (N=45), MM (N=40) and 15 healthy subjects, in RT-PCR (validation set). Results We found 708 genes differentially expressed (467 up- and 241 down regulated) in MGUS versus healthy granulocytes at the steady state. The set of annotated, differentially expressed genes could befunctionally organized by “gene ontology” (http://www.geneontology.org/) into the following major categories: i) receptors and signal transduction (including up-regulation of CD14, Toll-like receptor 5 (TLR-5), IL-7 Receptor (CD127), IL-11 receptor, TGF-beta receptor 2, hematopoietic cells kinase (HCK), IFNAR1); ii) negative regulation of adaptive immune response (including up regulation of CD127, STAT6, IFNAR1, OSCAR, PROK-2 and down regulation of p50, p65,NFKBIA, IL8, ELK-1, HIF-1 alpha, CEBP-beta, CEBP-zeta). In MM samples we confirmed a statistically significant up-regulation of PROK-2 (a key molecule of VEGF-independent angiogenesis), CD14 (mediator hypersensitive innate immune response to lipopolysaccharide) and HCK (the hematopoietic cell kinase, involved in neutrophil migration and degranulation). In the validation set, PROK-2 expression was two times higher in MGUS than healthy subjects (p=.02) and up to ten times higher in MM (p=.001). In MM patients, increased levels of PROK-2 were positively associated with advanced bone disease and unfavourable cytogenetics. Conclusion Granulocytic impairment is present in MGUS and worsened in MM patients due to increased expression of genes that negatively regulate adaptive immune response. PROK-2 is a key molecule involved in the granulocyte dysfunction and could be involved in the progression from MGUS to MM. Disclosures Musto: Celgene: Honoraria; Janssen: Honoraria.

Description: Background Ixazomib is the first oral proteasome inhibitor to be investigated clinically for the treatment of MM. Phase 1 studies have shown single-agent activity and manageable toxicities in RRMM (Kumar et al. Blood 2014) and phase 1/2 studies have suggested the feasibility and activity of weekly oral ixazomib plus Rd in previously untreated MM (Kumar et al. ASH 2012; Richardson et al. ASH 2013). These findings have led to ongoing phase 3 trials of weekly ixazomib 4 mg + Rd in RRMM and previously untreated MM. However, the early-phase studies were conducted in Western pts. This phase 1, open-label multicenter study aimed to determine the safety, tolerability, and pharmacokinetics (PK) of weekly ixazomib alone or with Rd in Japanese pts with RRMM (Japic Clinical Trials Information no. 121822). Methods Primary objectives were to evaluate the safety and tolerability, including dose-limiting toxicities (DLTs) and adverse events (AEs), and the PK of ixazomib alone or with Rd. A secondary objective was evaluation of antitumor activity. Japanese pts aged ≥20 years with RRMM who had received at least 2 prior regimens, which must have included bortezomib, thalidomide or lenalidomide, and corticosteroids, were eligible. All had measurable disease and ECOG performance status of 0–2. Pts with grade ≥2 peripheral neuropathy or grade ≥2 diarrhea at study entry were excluded. Pts received ixazomib 4 mg on days 1, 8, and 15 of 28-day cycles, alone or with Rd (lenalidomide 25 mg on days 1–21, dexamethasone 40 mg on days 1, 8, 15, and 22), per the regimen used in the ongoing phase 3 trials. AEs were graded per NCI-CTCAE v4.03. Blood samples for PK analysis were taken at multiple time points prior to and after dosing on days 1 and 15 of cycle 1. Responses were assessed per IMWG uniform response criteria. Results Fourteen pts were enrolled; 8 (57%) were male, median age was 62.5 yrs (range 53–71), 4 pts were aged ≥65 yrs, median number of prior therapies was 7. Seven pts received single-agent ixazomib and 7 received ixazomib + Rd. One pt in each cohort was excluded from the DLT-evaluable population. Two patients experienced DLTs in cycle 1: 1 pt receiving single-agent ixazomib had grade 4 thrombocytopenia and grade 3 diarrhea, hypertension, hypokalemia, hyponatremia, and nausea; 1 pt in the ixazomib + Rd cohort had grade 4 thrombocytopenia and neutropenia. All events were considered treatment-related. At data cut-off (Jan 6 2014), 6 pts remained on treatment and 8 had discontinued due to: progressive disease (PD; n=3), AEs (n=3), symptomatic deterioration, and protocol violation (each n=1). At data cut-off, pts (n=14) had received a median of 6 cycles of ixazomib (range 1–21); the 7 pts in the ixazomib + Rd cohort had received a median of 4 cycles (range 1–12) of ixazomib + Rd. Thirteen (93%) pts experienced treatment-related AEs; the most common were neutropenia (71%), thrombocytopenia (71%), leukopenia (64%), lymphopenia (57%), and diarrhea (50%). There were no cases of peripheral neuropathy. Nine (64%) pts had grade ≥3 AEs; the most common were lymphopenia (50%), neutropenia (43%), and thrombocytopenia (36%). Two (14%) pts (single-agent cohort) had serious AEs (grade 2 bronchitis in 1 pt, and grade 4 thrombocytopenia and grade 3 hypokalemia in 1 pt). Three pts discontinued due to AEs; 1 due to diarrhea in the single-agent cohort, and 1 due to neutropenia and 1 due to thrombocytopenia in the ixazomib + Rd cohort. There were no deaths. PK data showed ixazomib was rapidly absorbed with a Tmax at 1.08–1.83 hrs. Terminal half-life (geometric mean) was 5.7 days for single-agent ixazomib and 5.2 days for ixazomib + Rd. There were no substantial differences in the ixazomib PK profile between the two cohorts. Thirteen pts were response-evaluable. One pt (ixazomib + Rd cohort) had a partial response; at data cut-off, this pt remained in response with a 100% M-protein reduction (unconfirmed VGPR) and duration of response of ~10.8 months. Seven pts had stable disease (including 3 with M-protein reductions of 25–50%), 2 had PD, and 3 were not assessable. Conclusions These data suggest that ixazomib 4 mg alone or with Rd is feasible and tolerable in Japanese pts with RRMM. The AEs were manageable, reflecting the AE profile seen in Western populations, supporting the use of this dose and schedule in Japanese pts. Disclosures Handa: Celgene: Research Funding; Yakult: Research Funding; Kirin: Research Funding; Chugai: Research Funding. Off Label Use: Investigational agent ixazomib for the treatment of Japanese patients with relapsed and/or refractory multiple myeloma.. Matsushima:Takeda Pharmaceutical Company Limited : Employment.

Description: Myeloma (MM) cells grow and expand almost exclusively in the bone marrow while creating a cellular microenvironment suitable for MM cell growth and survival (MM niche). In pursuing the molecular mechanisms whereby MM cells gain drug resistance in the “MM niche”, we have found that the serine/threonine kinase Pim-2 is constitutively over-expressed in MM cells, and further up-regulated by co-cultures with bone marrow stromal cells (BMSCs) as well as osteoclasts (Leukemia, 2011), and that Pim-2 is an important therapeutic target in MM for the progression of MM tumor and bone disease (Leukemia, 2014). The ABC transporter BCRP is preferentially expressed in drug resistant MM cells as well as in MM progenitors or stem cells. BCRP has been demonstrated to be phosphorylated by Pim kinases to trigger its dimerization and function; Pim inhibition may suppress the BCRP function to sensitize BCRP-expressing MM cells to chemotherapeutic agents. In the present study we therefore explored whether Pim inhibition is able to target and impair BCRP-expressing drug-resistant MM cells and MM progenitors. We analyzed an ABC transporter activity in BCRP-expressing RPMI8226 and KMS11 cells by intracellular accumulation and retention of BCRP substrates with auto-fluorescence emission, mitoxantrone and doxorubicin, in flow cytometry. Treatment with Pim inhibitors, SMI-16a or SMI-4a, increased the incorporation of these drugs into the MM cells and enhanced their subsequent intracellular retention after 6-hour incubation without these drugs, although BCRP expression on their surface was only marginally affected by the Pim inhibition. Interestingly, acidic conditions up-regulated Pim-2 expression while reducing the accumulation and retention of these drugs in BCRP-expressing RPMI8226 and KMS11 cells. However, the Pim inhibitors efficaciously restored the drug accumulation and retention reduced by extracellular acidification, and enhanced the cytotoxic activity of the BCRP substrate doxorubicin against RPMI8226 cells rather preferentially in acidic conditions. Furthermore, the Pim inhibition minimized the sizes of “side populations”, highly drug-resistant fractions with enhanced BCRP activity, and the ability of colony formation in RPMI8226 and KMS11 cells, which was more marked in acidic conditions. We previously demonstrated the in vivo effects of the Pim inhibitors in human INA-6 cell-bearing SCID-rab MM models and syngeneic mouse MM models with an intra-tibial inoculation of 5TGM1 MM cells (Leukemia, 2014). To further examine the acid-tropism of anti-tumorigenic activity of Pim inhibition, we pretreated murine 5TGM1 MM cells in vitro with or without SMI16a at pH6.8 for 24 hours, and transplanted to the tibiae in mice the same numbers of viable MM cells remaining in each treatment group. Treatment with SMI16a at pH6.8 almost completely abrogated in vivo tumorigenic capacity of 5TGM1 cells, while MM cells without the treatment rapidly grew and expanded in and outside of the tibiae, suggesting targeting clonogenic MM cells by Pim inhibition preferentially in acidic conditions. Taken together, Pim-2 may become an important therapeutic target of drug-resistant BCRP-expressing MM cells and their progenitors which appear to gain more drug resistance in acidic bone lesions. Combinatory treatment with Pim inhibitors warrants further study to overcome drug resistance in MM cells, including their tumorigenic cancer stem cells. Disclosures No relevant conflicts of interest to declare.

Description: Multiple myeloma (MM) is a devastating bone marrow (BM) cancer characterized by clonal proliferation of plasma cells. Despite the emergence of novel therapeutics MM remains a fatal disease. The tumor microenvironment plays a critical role in promoting MM growth. We have recently demonstrated that a population of BM myeloid-derived suppressor cells is involved in regulation of MM progression. These cells abundantly produce the pro-inflammatory protein S100A9. Tasquinimod (ABR-215050, Active Biotech/IPSEN) is a quinoline-3-carboxamide derivative that binds to S100A9 and blocks its interaction with receptors TLR4, RAGE, and CD147. Here we investigated whether pharmacological inhibition of S100A9 with tasquinimod inhibits MM progression. A panel of MM murine (DP42) and human (RPMI-8226, H929, and U266) cell lines was cultured in the presence of tasquinimod or vehicle control and cell viability was determined using MTT assay. Treatment with tasquinimod did not affect MM cell viability. We then evaluated the anti-tumor effect of tasquinimod in vivo in a MM syngeneic model. In this model, murine MM DP42 cells are injected i.v., home to the BM, and grow as MM that closely resembles the human disease. On day 2 after tumor cell injection mice were randomly assigned to the treatment or control groups. Treatment group received tasquinimod at a dose of 30 mg/kg/day in drinking water for 28 days. We found that tasquinimod significantly improved survival of MM-bearing mice (p

Description: Introduction: Capillary zone electrophoresis (CZE) and measurement of total immunoglobulins (total Ig) are standard techniques for the identification and quantification of monoclonal immunoglobulins (M-Ig). Heavy/light chain (HLC) pair analysis allows discrimination between Igκ and Igλ and as a result allows measurement of both the monoclonal involved and polyclonal uninvolved HLC pair. We compared measurement of M-Ig by CZE and total Ig to HLC levels. Methods: 93 samples from 8 patients with IgA intact immunoglobulin multiple myeloma (MM) were analysed. M-Ig was measured using CZE Sebia Capillarys 2 system, total IgA (tIgA) and IgAκ and IgAλ HLC concentrations on a SPAPLUS analyser. IgA HLC levels were measured with Hevylite® and compared to published normal ranges (IgAκ (g/L): 0.57-2.08, IgAλ (g/L): 0.44-2.04, IgAκ/IgAλ: 0.78-1.94). Passing and Bablok fit analysis was used to determine correlation between the assays. Results: Measurement of the involved HLC pair (iHLC) (median: 12.45g/L; range: 0.64-44.71g/L) compared well with CZE (n=34; median: 11.04g/L; range: 1.24-37.71g/L.; y= 1.2x + -2.65, R2= 0.94). Measurement of iHLC (median: 0.88. range: 0.05-21.55g/L) also compared well with tIgA measurement (n=65 median=1.44g/L; range: 0.227-21.11g/L, y=0.85x + -0.26, R2=0.98). Percent changes in iHLC concentrations from baseline through treatment compared well with CZE (n=28; y=1.59x + 0.15 R2=0.93) and tIgA (n=57; y=1.06x + 0.01, R2=0.96). Of the 34 samples with quantifiable M-protein by CZE, 32(94%) had an abnormal HLC ratio. The two discrepant samples were follow up samples from the same patient, where HLC normalised alongside total IgA entering the normal reference range. In addition, all 15 samples (15/65; 23%) where tIgA concentration was above the normal reference range all had abnormal HLC ratios. M-Ig was not detected by CZE in 48/82 (57%) samples, 46/48 of the samples (96%) had a normalised HLC ratio. In 38/65 (58%) samples, tIgA concentrations were within the normal reference range, and 34 (90%) had a normalised HLC ratio. HLC ratio for all patient samples normalised in subsequent samples following treatment. Conclusion: The measurement of M-Ig is comparable between Hevylite® and both CZE and tIgA. The presented data indicate that Hevylite® is a more sensitive test for detecting residual disease and warrants prospective studies on larger cohorts of patients. Acknowledgments:This work was partially supported by the National Science Fund (D02-35/2009). Disclosures Guenova: Novartis Pharma Sevices Bulgaria: Consultancy, Research Funding, Speakers Bureau; Roche Bulgaria: Consultancy, Research Funding, Speakers Bureau; Amgen Bulgaria: Consultancy, Research Funding, Speakers Bureau; Sanofi-Aventis Bulgaria: Consultancy, Research Funding, Speakers Bureau.

Description: Background We have a poor understanding of the vaccination immune response and outcomes in multiple myeloma (MM). As MM patients (Pts) are living longer and therapies are immunomodulatory there is an unmet medical need to further characterize the role of the immune system. A common reason for hospitalization or death in MM Pts is infection. As an initial step in MM Cancer Care Delivery Research (CCDR), we evaluated the current vaccination practice patterns in MM Pts at Aurora Health Care using the EMR and data analytics. Methods An IRB approved study reviewed MM Pts from 5/15/2012 to 5/15/2014. Data collected included demographics, influenza (FV) and pneumonia vaccination (PV) history, hospitalization episodes, cost associated with hospitalization, and admission and discharge diagnoses. Pts were considered PV positive if vaccinated within 5 years prior to study with any PV type. FV was none (no FV in 2012-2014), optimal [FV in 2012 and (2013 or 2014)], or suboptimal [FV in 2012 or (2013 or 2014)]. Data was analyzed using SAS and STATA 12. Results A total of 1131 MM Pts were identified. Race included 70% white, 13% black, and 17% mixed, other or information not available. MM median age at diagnosis was 71 and only 4% (47) had prior autologous stem cell transplantation. PV rate was 30%. FV was 55% none, 24% suboptimal and 20% optimal. There was no statistically significant difference in the rate of PV and FV when stratified vs age, gender, and race. Over two years there were a total of 662 hospitalization events involving 317 MM Pts. The total cost of hospitalization was approx $35M. The average charge per hospitalized patient was $110K (range: $2K -1.3M) with an average $52K per hospitalization encounter (range: 2K – 648K). The rate of PV and FV vaccination among Pts with index hospitalization is significantly higher than non-hospitalized patients. There was no difference in hospitalization cost based on vaccination status. (See Table 1) Discussion Vaccination rates were low and did not correlate with hospital outcomes. This may be explained as a limitation for a retrospective EMR analysis without accounting for temporal relationship of vaccines – i.e. possible vaccination after admission. Alternatively, this may indicate that our current methods of vaccination in MM are not effective. Other limitations include need for a more granular review of treatment regimens and infectious complications. Additional surrogate markers are needed to understand the effect of vaccines and the immune system on health care outcomes such as hospitalizations, cost, and survival. This will be addressed in prospective registries and immunologic studies at our center and may be queried at other health systems. Table 1 – Vaccination Status and Hospitalizations Vaccination Status % Hospitalization Events, % Hospitalization Charge, $ PV – No 70% 20% $16M PV – Yes 30% 52% $18M FV – None 55% 16% $9M FV – Suboptimal 24% 42% $13M FV - Optimal 20% 43% $12M Disclosures No relevant conflicts of interest to declare.

Description: The combination of fludarabine, cyclophosphamide and rituximab (FCR) is still currently regarded as the standard regimen for treatment of physically fit patients with chronic lymphocytic leukemia (CLL). This therapy can be associated with significant toxicity, and patient adherence to the protocol may often be difficult outside of clinical trials. This retrospective study aimed to evaluate the efficacy and safety of FCR therapy in the real life setting, with particular focus on the influence of dose reduction on treatment outcome. A total of 132 CLL patients (≤70 years of age) treated with FCR as frontline therapy from 10 medical centers, were reviewed. The majority of patients were males (73.5%, n=97) and younger than 60 years (78%, n=103). Eleven patients had Binet stage A (8.3%), 72 (54.5%) were stage B and 49 (37.1%) had Binet stage C. Results of FISH analysis were available for 99 patients, with high risk cytogenetics of del(11q) in 21 patients (21.2%) and del(17p) in 9 cases (9.1%). The majority (56.5%, n=74) received rituximab at a dose of 500mg/m2 and the rest 375mg/m2. Almost half of the patients (49.2%, n=65) were given a reduced dose of chemotherapy (

Description: Introduction: Chronic lymphocytic leukemia (CLL) is a heterogeneous disease with variable clinical course. Several studies have been conducted to predict outcome in patients with CLL and also have been going on. A proliferation inducing ligand (APRIL) has been shown to involve in survival and resistance to apoptosis in CLL, and APRIL molecule has been investigated as a prognostic marker in CLL patients. However, there are limited and controversial data regarding APRIL and its impact on prognosis in CLL. We aimed to compare serum APRIL levels in CLL patients with those of age and gender matched healthy subjects, and to investigate the relationship between APRIL and the other common prognostic factors, and to determine whether serum APRIL levels predict time to first treatment in CLL. Methods: After ethical approval and informed consent were obtained, between May and December 2012, venous blood samples were driven from 96 CLL patients’ and 25 healthy controls’, and serum APRIL levels were measured by ELISA. Demographic data and the prognostic markers were obtained from the patients’ files, and patients have been followed for a minimum of 12 months. We tested the correlation between APRIL with the, clinical and biological parameters, and used the log rank test to compare their Kaplan Meier curves. Results: Patients were divided into three groups: Treatment naive (group A, n=49), chemotherapy receiving (group B, n=25) and who had previously received chemotherapy (group C, n=22). Median APRIL level was higher in group A (2.78 vs 1.29; p=0.034) and group C (3.54 vs 1.29; p=0.001) when compared to healthy controls, but was not different in group B (1.56 vs 1.29; p=0.3) (Figure 1). Serum APRIL level in group A was negatively correlated with hemoglobin levels (r=-0.298; p=0.037) and platelet counts (r=-0.321; p=0.025) whereas no correlation with age, Rai and Binet stages, lymphocyte counts, β2-microglobulin and CD38 levels were detected. Group A patients were also divided into 2 subgroups (APRIL levels low, n=20 and APRIL levels high, n=29) using median natural logarithm of serum APRIL level as cut off. April low and high subgroups were similar with respect to demographic data and prognostic factors. Median time to first treatment was not reached in the APRIL low group, but was 104 months in the APRIL high group (p=0.13, log-rank test). Conclusions: Among the treatment naive patients, serum APRIL levels only negatively correlate with hemoglobin levels and platelet counts. These correlations seem to be associated with tumor burden rather than the prognosis, because APRIL levels were not different in chemotherapy receiving patients compared to healthy controls. Since a median survival time could not be reached in the APRIL low group, short follow up time might be an explanation why the APRIL levels did not predict the time to first treatment. In conclusion, our findings let us to think APRIL levels are not a useful marker to predict prognosis in patients with CLL. Figure 1. Median APRIL levels of CLL patients and healthy controls (ng/mL) Figure 1. Median APRIL levels of CLL patients and healthy controls (ng/mL) Disclosures No relevant conflicts of interest to declare.

Description: Bendamustine has been demonstrated to be effective for the treatment of CLL, either alone compared with chlorambucil (Knauf et al, JCO 2009 and BJH 2012) or in combination with monoclonal antibodies such as rituximab both in second or more lines (Fischer et al, JCO 2011) and in first line treatment (Fischer et al, JCO 2012). However, the relationship between its activity with clinical and biological prognosticators has been addressed only in few studies. For this purpose, we evaluated the efficacy and safety of bendamustine, in a real-life contest, on 56 patients, median age 66 years (41-80), median number of previous regimens 1 (0-3, 32% previously untreated). Bendamustine was given for a median number of 6 cycles (70-90 mg/m2), in 82% of cases with rituximab at conventional doses. Overall (ORR) and complete response (CRR) rates were 73% and 44.6%, respectively. Obviously, CRR was higher (83.3%) for 18 patients treated in first line. A significant correlation was found between lower ORR and lymphocyte doubling time 30%) of alpha-4 integrin CD49d (OR 13.0; P=0.018), an important marker of bad prognosis in CLL (Bulian et al, JCO 2014). On the other hand, no significant correlations were found between ORR and CD38, ZAP-70 or IGHV mutational status. Similarly, no significant correlations were noted between ORR and FISH cytogenetics, excluding del(17)p, or NOTCH1 mutations, thus confirming the independence of response to bendamustine from some well-known important biologic prognostic factors. In fact, multivariate analysis confirmed a significant relationship only between ORR and TP53/del(17)p (OR 0.020; P=0.0015) and concomitant rituximab (OR 0.019; P=0.0074). The estimated 1-year OS and PFS were 57% and 86%, respectively. Side effects included grade 3-4 neutropenia, infections, thrombocytopenia and anemia which occurred in 21%, 12%, 12% and 5% of patients, respectively. Grade 3-4 non-hematologic toxicity, including infusion-related reactions, heart or kidney or liver failure were found almost exclusively in elderly patients treated with bendamustine after two or more lines of therapy (12.5%). In multivariate analisys of OS, calculated from the end of treatment with bendamustine, only response to bendamustine (P=0.008) was confirmed to be an independent prognostic factor, while both the number of previous therapies and the concomitant use of rituximab demonstrated no statistical significance. These our results confirm both the activity and safety of bendamustine, particularly in combination with rituximab, also in the setting of elderly patients, often affected by two or three comorbidities. Noteworthy, this effectiveness appears to be present also in patients with unfavorable clinical and biological features, excluding del(17)p or TP53 mutations, in which the employment either of modern oral BCR inhibitors or of BH3 mimetics anti-Bcl-2 will be definitely active, also in combination with the same bendamustine. Disclosures No relevant conflicts of interest to declare.

Description: BACKGROUND: Chemoimmunotherapy for chronic lymphocytic leukemia (CLL) has been the standard of care for initial treatment. A randomized demonstrated both a progression free survival (PFS) and overall survival (OS) advantage when rituximab was added to fludarabine (F) and cyclophosphamide (C). Alemtuzumab (Campath) (CAM), an anti-CD52 monoclonal antibody, is an effective therapy for patients with both previously untreated and relapsed CLL. Its role in combination with chemotherapy is less certain. METHODS: We conducted a multicenter phase II clinical trial of FC followed by subcutaneous CAM in previously untreated CLL. Patients were eligible if they met standard criteria for initiating therapy, or if they were asymptomatic with the prognostically adverse immunoglobulin heavy chain variable (IGHV) gene unmutated status. Patients received fludarabine (25 mg/m2/day, days 1-3) and cyclophosphamide (250 mg/m2/day, days 1-3) every 28 days for six treatment cycles, followed by a 3-8 week rest period. Disease response was assessed, including minimal residual disease (MRD) status by sensitive flow cytometry in those in complete remission (CR). Patients who achieved less than a CR were eligible to receive standard dose CAM (30 mg thrice weekly for 12 weeks); those who were in CR but MRD positive could receive reduced dose CAM (30 mg weekly for 12 weeks). The primary outcome was duration of response (DOR). Secondary outcomes included the response rates after FC and after the addition of CAM, as well as the safety profile of the regimen. RESULTS: We enrolled 25 patients from November 2004 to June 2007 at 3 centers. The median age of the participants was 62 years (range 42-75). Detailed information was available for 17 patients pre-treatment: high risk Rai stage in 9, IGHV unmutated in 9 including 4 patients who were IGHV unmutated as their indication for treatment. Five patients had trisomy 12, 4 had 13q deletion, 1 each had 17p deletion and 11q deletion, and 6 had no abnormality. One patient was excluded from the analysis due to a diagnosis of mantle cell lymphoma after eligibility review. Four patients had no response evaluation and were considered treatment failures. Seventeen (71%) patients had a CR after 6 cycles of FC, including 11 who were MRD negative, one had a partial response (PR), and two had progressive disease (PD). Four of the 6 patients who were MRD positive received CAM after FC. Two required only a single dose to become MRD negative, and 2 received 12 weekly doses. One of these patients became MRD negative. The median DOR for those achieving CR was 38 months (range 12-105 months). There were no treatment related deaths. Five patients experienced a SAE including one with febrile neutropenia, two with pneumonia, and two with autoimmune hemolytic anemia. There were ten additional treatment emergent adverse events including two that were grade 3 (mucositis and fever) and one CMV reactivation while receiving CAM. Two patients developed treatment related myelodysplasia, one died and the other underwent allogeneic stem cell transplant. There were two deaths due to Richter’s transformation. During long term follow up, there have been five additional deaths. CONCLUSIONS: The CR rate after FC was higher than that reported in prior trials of previously untreated patients and the incidence of MRD negative CR was surprisingly high. The DOR was consistent with prior experience with FC. Too few patients received CAM to draw any conclusions about its role as a consolidative therapy given subcutaneously on a weekly schedule. Both the FC and CAM therapies were well tolerated, with few adverse events associated with their use. Disclosures No relevant conflicts of interest to declare.

Description: Background Many studies have examined the disparities in cancer diagnosis, treatment and survival among different subgroups classified based on race, socioeconomic status and age. Not as many studies have examined disease characteristics in rural populations, perhaps because of the lack of a consensus in the United States regarding the definition of "rural". In these few studies, many disparities were reported in association with rural residence such as lower levels of utilization of cancer screening tests, lower likelihood of receiving guideline-appropriate therapy and shorter survival. Objectives To examine multiple myeloma disease characteristics and survival in rural patients of southern New Mexico in comparison to their urban counterparts. Methods Patients presented to Memorial Medical Center (MMC) and its associated cancer center from January 2003 to December 2013 with multiple myeloma were enrolled in the study. Demographic and clinical data were collected. The charts were also examined for evidence of offering or discussing Autologous Stem Cell Transplant (ASCT) as a treatment option with patients, and whether it was actually performed. Patient staging at diagnosis according to International Staging System (ISS) for myeloma was determined, if possible. Urban vs. rural classification was based on the Rural-Urban Commuting Area codes (RUCA) version 2.0; a census tract-based classification scheme that utilizes the standard bureau of census urban definition in combination with work commuting information. Categorization D of RUCA was chosen. It defines urban as all places that have 30% or more of their workers going to a Census Bureau-defined Urbanized Area. Results A total of 87 patients were initially enrolled in the study. Four patients were excluded because sufficient evidence to establish the diagnosis could not be verified. Two additional patients who had solitary plasmacytoma with no evidence of systemic involvement were excluded as well. Patients were classified based on their residence at the time of diagnosis as rural (29 patients) and non-rural (52 patients). There was no difference between the mean age at diagnosis between the two groups with mean being 66.20 for non-rural group and 67.77 for the rural group. The type of heavy chain protein was generally similar for both groups with 55.74 % of patients diagnosed with Immunoglobulin G heavy chain disease. The average duration of initial presenting symptom prior to diagnosis was 7.36 weeks for the whole sample, 13.6 week for the rural group, and 4.56 weeks for the non rural group, which suggests that non-rural patients were more likely to seek medical attention sooner than their rural counterparts (p=0.0037). Tobacco consumption was higher among patients in the rural group compared to non-rural group. Nearly half (47.37%) of rural patients were diagnosed at stage 3 according to ISS staging system, while only 31.03% of non-rural patients were diagnosed at the same stage. Rural patients were more likely to be diagnosed at a more advanced disease stage (p=0.063). The nature of the chief presenting problem was generally similar in both groups with the exception of the higher likelihood of patients in non-rural group to be diagnosed at an asymptomatic stage. In the whole sample, 33.33% of patients had evidence of being offered or educated about ASCT as a treatment option, and 18.52% of patients actually receiving it. There was no different between the two groups in this regard. Median survival time for the whole sample was 57 months. Patients in the rural group had a median survival of 39.03 months (95% CI 18.96- 57.99), while non-rural patients had a median survival of 68.99 months (95% CI 25.95-90.02) ( p