ALBERT

Description: A simulation and two re-analyses from 1985 to 2007 have been produced for the Mediterranean Sea using different assimilation schemes: a Reduced Order Optimal Interpolation (SOFA) and a three-dimensional variational scheme (OceanVar). The observational data set consists of vertical temperature and salinity in-situ profiles and along-track satellite sea-level anomalies; daily mean fields of satellite sea surface temperature are used for correcting the air-sea fluxes. This paper assesses the quality of the re-analyses with respect to observations and the simulation. Both the SOFA and OceanVar schemes give very similar root mean square errors and biases for temperature and salinity fields compared with the assimilated observations. The largest errors are at the thermocline level and in regions of large eddy field variability. However, OceanVar gives 20% better results for sea-level anomaly root mean square error.

Description: This work was supported by the European Commision MyOcean Project (SPA.2007.1.1.01-development of upgrade capabilities for existing GMES fast-track services and related operational services; Grant Agreement: 218812-1-FP7-SPACE 2007- 1) and by the CIRCE project, founded by the European Commission’s 6th Framework Programme through contract no. 036961. We would also thank the Istituto Nazionale di Geofisica e Vulcanologia (INGV) and the Centro Euro-Mediterraneo per i Cambiamenti Climatici (CMCC) for facilities support.

Description: In press

Description: 4.6. Oceanografia operativa per la valutazione dei rischi in aree marine

Description: JCR Journal

Description: reserved

Description: Author Posting. © American Meteorological Society, 2010. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Physical Oceanography 40 (2010): 2743–2756, doi:10.1175/2010JPO4339.1.

Description: Analysis of modern and historical observations demonstrates that the temperature of the intermediate-depth (150–900 m) Atlantic water (AW) of the Arctic Ocean has increased in recent decades. The AW warming has been uneven in time; a local 1°C maximum was observed in the mid-1990s, followed by an intervening minimum and an additional warming that culminated in 2007 with temperatures higher than in the 1990s by 0.24°C. Relative to climatology from all data prior to 1999, the most extreme 2007 temperature anomalies of up to 1°C and higher were observed in the Eurasian and Makarov Basins. The AW warming was associated with a substantial (up to 75–90 m) shoaling of the upper AW boundary in the central Arctic Ocean and weakening of the Eurasian Basin upper-ocean stratification. Taken together, these observations suggest that the changes in the Eurasian Basin facilitated greater upward transfer of AW heat to the ocean surface layer. Available limited observations and results from a 1D ocean column model support this surmised upward spread of AW heat through the Eurasian Basin halocline. Experiments with a 3D coupled ice–ocean model in turn suggest a loss of 28–35 cm of ice thickness after 50 yr in response to the 0.5 W m−2 increase in AW ocean heat flux suggested by the 1D model. This amount of thinning is comparable to the 29 cm of ice thickness loss due to local atmospheric thermodynamic forcing estimated from observations of fast-ice thickness decline. The implication is that AW warming helped precondition the polar ice cap for the extreme ice loss observed in recent years.

Description: This study was supported by JAMSTEC (IP and VI), NOAA (IP, VI, and ID), NSF (IP,VA,VI, ID, JT, andMS),NASA(IP andVI), BMBF (ID), and UK NERC (SB) grants.

Description: Author Posting. © American Meteorological Society, 2010. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Physical Oceanography 40 (2010): 2605–2623, doi:10.1175/2010JPO4132.1.

Description: Steady scale-invariant solutions of a kinetic equation describing the statistics of oceanic internal gravity waves based on wave turbulence theory are investigated. It is shown in the nonrotating scale-invariant limit that the collision integral in the kinetic equation diverges for almost all spectral power-law exponents. These divergences come from resonant interactions with the smallest horizontal wavenumbers and/or the largest horizontal wavenumbers with extreme scale separations. A small domain is identified in which the scale-invariant collision integral converges and numerically find a convergent power-law solution. This numerical solution is close to the Garrett–Munk spectrum. Power-law exponents that potentially permit a balance between the infrared and ultraviolet divergences are investigated. The balanced exponents are generalizations of an exact solution of the scale-invariant kinetic equation, the Pelinovsky–Raevsky spectrum. A small but finite Coriolis parameter representing the effects of rotation is introduced into the kinetic equation to determine solutions over the divergent part of the domain using rigorous asymptotic arguments. This gives rise to the induced diffusion regime. The derivation of the kinetic equation is based on an assumption of weak nonlinearity. Dominance of the nonlocal interactions puts the self-consistency of the kinetic equation at risk. However, these weakly nonlinear stationary states are consistent with much of the observational evidence.

Description: This research is supported by NSF CMG Grants 0417724, 0417732 and 0417466. YL is also supported by NSF DMS Grant 0807871 and ONR Award N00014-09-1-0515.

Description: Author Posting. © American Meteorological Society, 2011. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Physical Oceanography 41 (2011): 889–910, doi:10.1175/2010JPO4496.1.

Description: This paper examines interaction between a barotropic point vortex and a steplike topography with a bay-shaped shelf. The interaction is governed by two mechanisms: propagation of topographic Rossby waves and advection by the forcing vortex. Topographic waves are supported by the potential vorticity (PV) jump across the topography and propagate along the step only in one direction, having higher PV on the right. Near one side boundary of the bay, which is in the wave propagation direction and has a narrow shelf, waves are blocked by the boundary, inducing strong out-of-bay transport in the form of detached crests. The wave–boundary interaction as well as out-of-bay transport is strengthened as the minimum shelf width is decreased. The two control mechanisms are related differently in anticyclone- and cyclone-induced interactions. In anticyclone-induced interactions, the PV front deformations are moved in opposite directions by the point vortex and topographic waves; a topographic cyclone forms out of the balance between the two opposing mechanisms and is advected by the forcing vortex into the deep ocean. In cyclone-induced interactions, the PV front deformations are moved in the same direction by the two mechanisms; a topographic cyclone forms out of the wave–boundary interaction but is confined to the coast. Therefore, anticyclonic vortices are more capable of driving water off the topography. The anticyclone-induced transport is enhanced for smaller vortex–step distance or smaller topography when the vortex advection is relatively strong compared to the wave propagation mechanism.

Description: Y. Zhang acknowledges the support of theMIT-WHOI Joint Programin Physical Oceanography, NSF OCE-9901654 and OCE-0451086. J. Pedlosky acknowledges the support of NSF OCE- 9901654 and OCE-0451086.

Description: Author Posting. © American Meteorological Society, 2011. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Climate 24 (2011): 2648–2665, doi:10.1175/2010JCLI3435.1.

Description: North Pacific Subtropical Mode Water (NPSTMW) is an essential feature of the North Pacific subtropical gyre imparting significant influence on regional SST evolution on seasonal and longer time scales and, as such, is an important component of basin-scale North Pacific climate variability. This study examines the seasonal-to-interannual variability of NPSTMW, the physical processes responsible for this variability, and the connections between NPSTMW and basin-scale climate signals using an eddy-permitting 1979–2006 ocean simulation made available by the Estimating the Circulation and Climate of the Ocean, Phase II (ECCO2). The monthly mean seasonal cycle of NPSTMW in the simulation exhibits three distinct phases: (i) formation during November–March, (ii) isolation during March–June, and (iii) dissipation during June–November—each corresponding to significant changes in upper-ocean structure. An interannual signal is also evident in NPSTMW volume and other characteristic properties with volume minima occurring in 1979, 1988, and 1999. This volume variability is correlated with the Pacific decadal oscillation (PDO) with zero time lag. Further analyses demonstrate the connection of NPSTMW to the basin-scale ocean circulation. With this, modulations of upper-ocean structure driven by the varying strength and position of the westerlies as well as the regional air–sea heat flux pattern are seen to contribute to the variability of NPSTMW volume on interannual time scales.

Description: Support for this research was provided by the Partnership for Advancing Interdisciplinary Modeling (PARADIGM), a National Ocean Partnership Program and by a NASA Modeling, Analysis, and the Prediction (MAP) project called Estimating the Circulation and Climate of the Ocean, Phase II (ECCO2).

Description: Author Posting. © American Meteorological Society, 2011. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Climate 24 (2011): 4844–4858, doi:10.1175/2011JCLI4130.1.

Description: The factors that determine the heat transport and overturning circulation in marginal seas subject to wind forcing and heat loss to the atmosphere are explored using a combination of a high-resolution ocean circulation model and a simple conceptual model. The study is motivated by the exchange between the subpolar North Atlantic Ocean and the Nordic Seas, a region that is of central importance to the oceanic thermohaline circulation. It is shown that mesoscale eddies formed in the marginal sea play a major role in determining the mean meridional heat transport and meridional overturning circulation across the sill. The balance between the oceanic eddy heat flux and atmospheric cooling, as characterized by a nondimensional number, is shown to be the primary factor in determining the properties of the exchange. Results from a series of eddy-resolving primitive equation model calculations for the meridional heat transport, overturning circulation, density of convective waters, and density of exported waters compare well with predictions from the conceptual model over a wide range of parameter space. Scaling and model results indicate that wind effects are small and the mean exchange is primarily buoyancy forced. These results imply that one must accurately resolve or parameterize eddy fluxes in order to properly represent the mean exchange between the North Atlantic and the Nordic Seas, and thus between the Nordic Seas and the atmosphere, in climate models.

Description: This study was supported by the National Science Foundation under Grants OCE-0726339 and OCE-0850416.

Description: Author Posting. © American Meteorological Society, 2011. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Physical Oceanography 41 (2011): 1741–1755, doi:10.1175/2011JPO4437.1.

Description: An in-depth data analysis was conducted to understand the occurrence of a strong sea surface temperature (SST) front in the central Bay of Bengal before the formation of Cyclone Nargis in April 2008. Nargis changed its course after encountering the front and tracked along the front until making landfall. One unique feature of this SST front was its coupling with high sea surface height anomalies (SSHAs), which is unusual for a basin where SST is normally uncorrelated with SSHA. The high SSHAs were associated with downwelling Rossby waves, and the interaction between downwelling and surface fresh waters was a key mechanism to account for the observed SST–SSHA coupling. The near-surface salinity field in the bay is characterized by strong stratification and a pronounced horizontal gradient, with low salinity in the northeast. During the passage of downwelling Rossby waves, freshening of the surface layer was observed when surface velocities were southwestward. Horizontal convergence of freshwater associated with downwelling Rossby waves increased the buoyancy of the upper layer and caused the mixed layer to shoal to within a few meters of the surface. Surface heating trapped in the thin mixed layer caused the fresh layer to warm, whereas the increase in buoyancy from low-salinity waters enhanced the high SSHA associated with Rossby waves. Thus, high SST coincided with high SSHA. The dominant role of salinity in controlling high SSHA suggests that caution should be exercised when computing hurricane heat potential in the bay from SSHA. This situation is different from most tropical oceans, where temperature has the dominant effect on SSHA.

Description: This work was supported by the NOAA/Office of Climate Observation (OCO) program.

Description: Author Posting. © American Meteorological Society, 2011. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Physical Oceanography 41 (2011): 1160–1181, doi:10.1175/2011JPO4547.1.

Description: Tropical instability waves are triggered by instabilities of the equatorial current systems, and their sea level signal, with peak amplitude near 5°N, is one of the most prominent features of the dynamic topography of the tropics. Cross-spectral analysis of satellite altimetry observations shows that there is sea level variability in the Pacific Ocean as far north as Hawaii (i.e., 20°N) that is coherent with the sea level variability near 5°N associated with tropical instability waves. Within the uncertainty of the analysis, this off-equatorial variability obeys the dispersion relation for nondivergent, barotropic Rossby waves over a fairly broad range of periods (26–38 days) and zonal wavelengths (9°–23° of longitude) that are associated with tropical instability waves. The dispersion relation and observed wave properties further suggest that the waves are carrying energy away from the instabilities toward the North Pacific subtropical gyre, which, together with the observed coherence of the sea level signal of the barotropic waves with that of the tropical instability waves, suggests that the barotropic Rossby waves are being radiated from the tropical instability waves. The poleward transport of kinetic energy and westward momentum by these barotropic Rossby waves may influence the circulation in the subtropics.

Description: Funding for this research came from WHOI’s TropicalResearch Initiative, the Charles D. Hollister Fund for Assistant Scientist Support, the John E. and Anne W. Sawyer Endowed Fund in Special Support of Scientific Staff, and Grant OCE-0845150 from the National Science Foundation.

Description: Author Posting. © American Meteorological Society, 2011. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Physical Oceanography 41 (2011): 1874–1893, doi:10.1175/2011JPO4604.1.

Description: A two-dimensional cross-shelf model of the New England continental shelf and slope is used to investigate the mean cross-shelf and vertical circulation at the shelf break and their seasonal variation. The model temperature and salinity fields are nudged toward climatology. Annual and seasonal mean wind stresses are applied on the surface in separate equilibrium simulations. The along-shelf pressure gradient force associated with the along-shelf sea level tilt is tuned to match the modeled and observed depth-averaged along-shelf velocity. Steady-state model solutions show strong seasonal variation in along-shelf and cross-shelf velocity, with the strongest along-shelf jet and interior onshore flow in winter, consistent with observations. Along-shelf sea level tilt associated with the tuned along-shelf pressure gradient increases shoreward because of decreasing water depth. The along-shelf sea level tilt varies seasonally with the wind and is the strongest in winter and weakest in summer. A persistent upwelling is generated at the shelf break with a maximum strength of 2 m day−1 at 50-m depth in winter. The modeled shelfbreak upwelling differs from the traditional view in that most of the upwelled water is from the upper continental slope instead of from the shelf in the form of a detached bottom boundary layer.

Description: WGZ was supported by the Woods Hole Oceanographic Institution postdoctoral scholarship program. GGGandDJMwere supported byONRGrant N-00014- 06-1-0739.

Description: Author Posting. © American Meteorological Society, 2011. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Physical Oceanography 41 (2011): 166-185, doi:10.1175/2010JPO4470.1.

Description: Field observations of turbulent kinetic energy (TKE), dissipation rate ε, and turbulent length scale demonstrate the impact of both density stratification and nonlocal turbulent production on turbulent momentum flux. The data were collected in a highly stratified salt wedge estuary using the Mobile Array for Sensing Turbulence (MAST). Estimates of the dominant length scale of turbulent motions obtained from the vertical velocity spectra provide field confirmation of the theoretical limitation imposed by either the distance to the boundary or the Ozmidov scale, whichever is smaller. Under boundary-limited conditions, anisotropy generally increases with increasing shear and decreased distance to the boundary. Under Ozmidov-limited conditions, anisotropy increases rapidly when the gradient Richardson number exceeds 0.25. Both boundary-limited and Ozmidov-limited conditions demonstrate significant deviations from a local production–dissipation balance that are largely consistent with simple scaling relationships for the vertical divergence in TKE flux. Both the impact of stratification and deviation from equilibrium turbulence observed in the data are largely consistent with commonly used turbulence closure models that employ “nonequilibrium” stability functions. The data compare most favorably with the nonequilibrium version of the L. H. Kantha and C. A. Clayson stability functions. Not only is this approach more consistent with the observed critical gradient Richardson number of 0.25, but it also accounts for the large deviations from equilibrium turbulence in a manner consistent with the observations.

Description: The funding for this research was obtained from ONR Grant N00014-06-1-0292 and NSF Grants and OCE-08-25226 and OCE-08-24871.

Description: Author Posting. © American Meteorological Society, 2010. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Physical Oceanography 40 (2010): 2768–2777, doi:10.1175/2010JPO4461.1.

Description: Although sustained observations yield a description of the mean equatorial current system from the western Pacific to the eastern terminus of the Tropical Atmosphere Ocean (TAO) array, a comprehensive observational dataset suitable for describing the structure and pathways of the Equatorial Undercurrent (EUC) east of 95°W does not exist and therefore climate models are unconstrained in a region that plays a critical role in ocean–atmosphere coupling. Furthermore, ocean models suggest that the interaction between the EUC and the Galápagos Islands (92°W) has a striking effect on the basic state and coupled variability of the tropical Pacific. To this end, the authors interpret historical measurements beginning with those made in conjunction with the discovery of the Pacific EUC in the 1950s, analyze velocity measurements from an equatorial TAO mooring at 85°W, and analyze a new dataset from archived shipboard ADCP measurements. Together, the observations yield a possible composite description of the EUC structure and pathways in the eastern equatorial Pacific that may be useful for model validation and guiding future observation.

Description: Karnauskas acknowledges the WHOI Penzance Endowed Fund in Support of Assistant Scientists.

Description: Author Posting. © American Meteorological Society, 2010. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Physical Oceanography 40 (2010): 2679–2695, doi:10.1175/2010JPO4395.1.

Description: Observations of stratification and currents between June 2007 and March 2009 reveal a strong overflow between 400- and 570-m depth from the Panay Strait into the Sulu Sea. The overflow water is derived from approximately 400 m deep in the South China Sea. Temporal mean velocity is greater than 0.75 m s−1 at 50 m above the 570-m Panay Sill. Empirical orthogonal function analysis of a mooring time series shows that the flow is dominated by the bottom overflow current with little seasonal variance. The overflow does not descend below 1250 m in the Sulu Sea but rather settles above high-salinity deep water derived from the Sulawesi Sea. The mean observed overflow transport at the sill is 0.32 × 106 m3 s−1. The observed transport was used to calculate a bulk diapycnal diffusivity of 4.4 × 10−4 m2 s−1 within the Sulu Sea slab (575–1250 m) ventilated from Panay Strait. Analysis of Froude number variation across the sill shows that the flow is hydraulically controlled. A suitable hydraulic control model shows overflow transport equivalent to the observed overflow. Thorpe-scale estimates show turbulent dissipation rates up to 5 × 10−7 W kg−1 just downstream of the supercritical to subcritical flow transition, suggesting a hydraulic jump downstream of the sill.

Description: This work was supported by the Office of Naval Research Grant N00014-09-1-0582 to Lamont-Doherty Earth Observatory of Columbia University; Grants ONR-13759000 and N00014-09-1-0582 to the Woods Hole Oceanographic Institution; Grant ONR-N00014-06-1-0690 to Scripps Institute of Oceanography; and a National Defense Science and Engineering Graduate Fellowship.

Description: Author Posting. © American Meteorological Society, 2010. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Physical Oceanography 40 (2010): 2713–2727, doi:10.1175/2010JPO4225.1.

Description: The authors explore the theoretical and empirical relationship between the nonlocal quantities of the entrainment ratio E, the appropriately depth- and time-averaged flux coefficient Γ, and the bulk Froude number Fro in density currents. The main theoretical result is that E = 0.125 Γ Fro2(CU3/CL)/cosθ, where θ is the angle of the slope over which the density current flows, CL is the ratio the turbulent length scale to the depth of the density current, and CU is the ratio of the turbulent velocity scale to the mean velocity of the density current. In the case of high bulk Froude numbers Γ Fro−2 and (CU3/CL) = Cϵ 1, so E 0.1, consistent with observations of a constant entrainment ratio in unstratified jets and weakly stratified plumes. For bulk Froude numbers close to one, Γ is constant and has a value in the range of 0.1–0.3, which means that E Fro2, again in agreement with observations and previous experiments. For bulk Froude numbers less than one, Γ decreases rapidly with bulk Froude number, explaining the sudden decrease in entrainment ratios that has been observed in all field and experimental observations.

Description: Support for MGW was provided by NSERC, the Canadian Foundation for Innovation, the Ontario Research Fund, and the Connaught Committee of the University of Toronto. CPC gratefully acknowledges the hospitality and support of the 2008 Summer Study Program in Geophysical Fluid Dynamics at Woods Hole Oceanographic Institution, where this project was initiated.

Description: Author Posting. © American Meteorological Society, 2011. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Physical Oceanography 41 (2011): 911–925, doi:10.1175/2011JPO4498.1.

Description: Motivated by discrepancies between Eulerian transport estimates and the behavior of Lagrangian surface drifters, near-surface transport pathways and processes in the North Atlantic are studied using a combination of data, altimetric surface heights, statistical analysis of trajectories, and dynamical systems techniques. Particular attention is paid to the issue of the subtropical-to-subpolar intergyre fluid exchange. The velocity field used in this study is composed of a steady drifter-derived background flow, upon which a time-dependent altimeter-based perturbation is superimposed. This analysis suggests that most of the fluid entering the subpolar gyre from the subtropical gyre within two years comes from a narrow region lying inshore of the Gulf Stream core, whereas fluid on the offshore side of the Gulf Stream is largely prevented from doing so by the Gulf Stream core, which acts as a strong transport barrier, in agreement with past studies. The transport barrier near the Gulf Stream core is robust and persistent from 1992 until 2008. The qualitative behavior is found to be largely independent of the Ekman drift.

Description: This work was supported by the National Science Foundation Grants CMG-82469600 and CMG-82579600 and by the Office of Naval Research Grant ONR-13108700.

Description: Author Posting. © American Meteorological Society, 2011. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Physical Oceanography 41 (2011): 1041–1056, doi:10.1175/2010JPO4313.1.

Description: Three autonomous profiling Electromagnetic Autonomous Profiling Explorer (EM-APEX) floats were air deployed one day in advance of the passage of Hurricane Frances (2004) as part of the Coupled Boundary Layer Air–Sea Transfer (CBLAST)-High field experiment. The floats were deliberately deployed at locations on the hurricane track, 55 km to the right of the track, and 110 km to the right of the track. These floats provided profile measurements between 30 and 200 m of in situ temperature, salinity, and horizontal velocity every half hour during the hurricane passage and for several weeks afterward. Some aspects of the observed response were similar at the three locations—the dominance of near-inertial horizontal currents and the phase of these currents—whereas other aspects were different. The largest-amplitude inertial currents were observed at the 55-km site, where SST cooled the most, by about 2.2°C, as the surface mixed layer deepened by about 80 m. Based on the time–depth evolution of the Richardson number and comparisons with a numerical ocean model, it is concluded that SST cooled primarily because of shear-induced vertical mixing that served to bring deeper, cooler water into the surface layer. Surface gravity waves, estimated from the observed high-frequency velocity, reached an estimated 12-m significant wave height at the 55-km site. Along the track, there was lesser amplitude inertial motion and SST cooling, only about 1.2°C, though there was greater upwelling, about 25-m amplitude, and inertial pumping, also about 25-m amplitude. Previously reported numerical simulations of the upper-ocean response are in reasonable agreement with these EM-APEX observations provided that a high wind speed–saturated drag coefficient is used to estimate the wind stress. A direct inference of the drag coefficient CD is drawn from the momentum budget. For wind speeds of 32–47 m s−1, CD ~ 1.4 × 10−3.

Description: The Office of Naval Research supported the development of the EM-APEX float system through SBIR Contract N00014-03-C-0242 to Webb Research Corporation and with a subcontract to APL-UW. Sanford and J. Girton were supported by the Office of Naval Research through GrantsN00014-04-1-0691 and N00014- 07-1-024, and J. Price was supported through Grant N00014-04-1-0109.

Description: Author Posting. © American Meteorological Society, 2011. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Physical Oceanography 41 (2011): 1182–1208, doi:10.1175/2010JPO4564.1.

Description: The authors use data collected by a line of tall current meter moorings deployed across the axis of the Kuroshio Extension (KE) jet at the location of maximum time-mean eddy kinetic energy to characterize the mean jet structure, the eddy variability, and the nature of eddy–mean flow interactions observed during the Kuroshio Extension System Study (KESS). A picture of the 2-yr record mean jet structure is presented in both geographical and stream coordinates, revealing important contrasts in jet strength, width, vertical structure, and flanking recirculation structure. Eddy variability observed is discussed in the context of some of its various sources: jet meandering, rings, waves, and jet instability. Finally, various scenarios for eddy–mean flow interaction consistent with the observations are explored. It is shown that the observed cross-jet distributions of Reynolds stresses at the KESS location are consistent with wave radiation away from the jet, with the sense of the eddy feedback effect on the mean consistent with eddy driving of the observed recirculations. The authors consider these results in the context of a broader description of eddy–mean flow interactions in the larger KE region using KESS data in combination with in situ measurements from past programs in the region and satellite altimetry. This demonstrates important consistencies in the along-stream development of time-mean and eddy properties in the KE with features of an idealized model of a western boundary current (WBC) jet used to understand the nature and importance of eddy–mean flow interactions in WBC jet systems.

Description: This work was supported by National Science Foundation funding for the KESS program under Grants OCE-0220161 (SW, NGH, and SRJ), OCE- 0825550 (SW), OCE-0850744 (NGH), and OCE-0849808 (SRJ). SW was also supported by the MIT Presidential Fellowship. The financial assistance of the Houghton Fund, the MIT Student Assistance Fund, and WHOI Academic Programs is also gratefully acknowledged.

Description: Author Posting. © American Meteorological Society, 2011. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Atmospheric and Oceanic Technology 28 (2011): 1065–1071, doi:10.1175/JTECH-D-10-05030.1.

Description: In this work a new methodology is proposed to correct the thermal lag error in data from unpumped CTD sensors installed on Slocum gliders. The advantage of the new approach is twofold: first, it takes into account the variable speed of the glider; and second, it can be applied to CTD profiles from an autonomous platform either with or without a reference cast. The proposed methodology finds values for four correction parameters that minimize the area between two temperature–salinity curves given by two CTD profiles. A field experiment with a Slocum glider and a standard CTD was conducted to test the method. Thermal lag–induced salinity error of about 0.3 psu was found and successfully corrected.

Description: This work is part of the SINOCOP and GliderBal projects funded by CSIC and Govern Balear, respectively.

Description: Author Posting. © American Meteorological Society, 2011. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Climate 24 (2011): 4973–4991, doi:10.1175/2011JCLI4083.1.

Description: The fourth version of the Community Climate System Model (CCSM4) was recently completed and released to the climate community. This paper describes developments to all CCSM components, and documents fully coupled preindustrial control runs compared to the previous version, CCSM3. Using the standard atmosphere and land resolution of 1° results in the sea surface temperature biases in the major upwelling regions being comparable to the 1.4°-resolution CCSM3. Two changes to the deep convection scheme in the atmosphere component result in CCSM4 producing El Niño–Southern Oscillation variability with a much more realistic frequency distribution than in CCSM3, although the amplitude is too large compared to observations. These changes also improve the Madden–Julian oscillation and the frequency distribution of tropical precipitation. A new overflow parameterization in the ocean component leads to an improved simulation of the Gulf Stream path and the North Atlantic Ocean meridional overturning circulation. Changes to the CCSM4 land component lead to a much improved annual cycle of water storage, especially in the tropics. The CCSM4 sea ice component uses much more realistic albedos than CCSM3, and for several reasons the Arctic sea ice concentration is improved in CCSM4. An ensemble of twentieth-century simulations produces a good match to the observed September Arctic sea ice extent from 1979 to 2005. The CCSM4 ensemble mean increase in globally averaged surface temperature between 1850 and 2005 is larger than the observed increase by about 0.4°C. This is consistent with the fact that CCSM4 does not include a representation of the indirect effects of aerosols, although other factors may come into play. The CCSM4 still has significant biases, such as the mean precipitation distribution in the tropical Pacific Ocean, too much low cloud in the Arctic, and the latitudinal distributions of shortwave and longwave cloud forcings.

Description: National Science Foundation, which sponsors NCAR and the CCSM Project. The project is also sponsored by the U.S. Department of Energy (DOE). Thanks are also due to the many other software engineers and scientists who worked on developing CCSM4, and to the Computational and Information Systems Laboratory at NCAR, which provided the computing resources through the Climate Simulation Laboratory. Hunke was supported within theClimate, Ocean and Sea Ice Modeling project at Los Alamos National Laboratory, which is funded by the Biological and Environmental Research division of the DOE Office of Science. The Los Alamos National Laboratory is operated by theDOENationalNuclear Security Administration under Contract DE-AC52-06NA25396. Raschwas supported by theDOEOffice of Science, Earth System Modeling Program, which is part of the DOE Climate Change Research Program. The Pacific Northwest National Laboratory is operated forDOEbyBattelle Memorial Institute under Contract DE-AC06-76RLO 1830. Worley was supported by the Climate Change Research Division of the Office of Biological and Environmental Research and by the Office ofAdvanced Scientific Computing Research, both in the DOE Office of Science, under Contract DE-AC05-00OR22725 with UT-Batelle, LLC.

Description: Author Posting. © American Meteorological Society, 2011. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Atmospheric and Oceanic Technology 28 (2011): 1351–1360, doi:10.1175/JTECH-D-10-05033.1.

Description: The Southern Ocean Flux Station was deployed near 47°S, 140°E. The extreme wind and wave conditions at this location require appropriate mooring design, which includes dynamic fatigue analysis and static analysis. An accurate estimate of the wave conditions was essential. A motion reference unit was deployed in a nearby test mooring for 6 months. The motion data provided estimates of significant wave height that agreed well with the Australian Bureau of Meteorology wave model, increasing confidence in the model performance in the Southern Ocean. The results of the dynamic fatigue analysis using three input wave datasets and implications for the mooring design are described. The design analysis predicts the fatigue life for critical mooring components and guided the final selection of links and chain shackles. The three input wave climatologies do not differ greatly, and this is reflected in minimal changes to mooring components for each of the fatigue analyses.

Description: Many years of logistic support for these deployments have been provided by the Australian Marine National Facility and the Australian Antarctic Sciences program (Award 1156). IMOS is funded through the Federal Government’s National Collaborative Research Infrastructure Strategy and the Super Science Initiative.

Description: Author Posting. © American Meteorological Society, 2010. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Climate 23 (2010): 6221–6233, doi:10.1175/2010JCLI3402.1.

Description: Enhanced decadal variability in sea surface temperature (SST) centered on the Kuroshio Extension (KE) has been found in the Community Climate System Model version 3 (CCSM3) as well as in other coupled climate models. This decadal peak has higher energy than is found in nature, almost twice as large in some cases. While previous analyses have concentrated on the mechanisms for such decadal variability in coupled models, an analysis of the causes of excessive SST response to changes in wind stress has been missing. Here, a detailed comparison of the relationships between interannual changes in SST and sea surface height (SSH) as a proxy for geostrophic surface currents in the region in both CCSM3 and observations, and how these relationships depend on the mean ocean circulation, temperature, and salinity, is made. We use observationally based climatological temperature and salinity fields as well as satellite-based SSH and SST fields for comparison. The primary cause for the excessive SST variability is the coincidence of the mean KE with the region of largest SST gradients in the model. In observations, these two regions are separated by almost 500 km. In addition, the too shallow surface oceanic mixed layer in March north of the KE in the subarctic Pacific contributes to the biases. These biases are not unique to CCSM3 and suggest that mean biases in current, temperature, and salinity structures in separated western boundary current regions can exert a large influence on the size of modeled decadal SST variability.

Description: Support for L.T. was provided by the NASA sponsored Ocean Surface Topography Science Team, under Contract 1267196 with the University of Washington, administered by the Jet Propulsion Laboratory. Support for Y.-O. K. comes from the NOAA Office of Global Programs (grant to C. Deser and Y.-O. Kwon) and the WHOI Heyman fellowship.

Description: Author Posting. © American Meteorological Society, 2011. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Climate 24 (2011): 762-777, doi:10.1175/2010JCLI3731.1.

Description: The meridional shifts of the Oyashio Extension (OE) and of the Kuroshio Extension (KE), as derived from high-resolution monthly sea surface temperature (SST) anomalies in 1982–2008 and historical temperature profiles in 1979–2007, respectively, are shown based on lagged regression analysis to significantly influence the large-scale atmospheric circulation. The signals are independent from the ENSO teleconnections, which were removed by seasonally varying, asymmetric regression onto the first three principal components of the tropical Pacific SST anomalies. The response to the meridional shifts of the OE front is equivalent barotropic and broadly resembles the North Pacific Oscillation/western Pacific pattern in a positive phase for a northward frontal displacement. The response may reach 35 m at 250 hPa for a typical OE shift, a strong sensitivity since the associated SST anomaly is 0.5 K. However, the amplitude, but not the pattern or statistical significance, strongly depends on the lag and an assumed 2-month atmospheric response time. The response is stronger during fall and winter and when the front is displaced southward. The response to the northward KE shifts primarily consists of a high centered in the northwestern North Pacific and hemispheric teleconnections. The response is also equivalent barotropic, except near Kamchatka, where it tilts slightly westward with height. The typical amplitude is half as large as that associated with OE shifts.

Description: This work was supported in part by the L’Institut universitaire de France (CF), the WHOI Heyman fellowship, and the NASAGrant withAwardNNX09AF35G(Y.-O. K), and grants through NOAA’s Climate Variability and Predictability Program (MAA).

Description: Author Posting. © American Meteorological Society, 2011. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Physical Oceanography 41 (2011): 241-246, doi:10.1175/2010JPO4557.1.

Description: The vertical dispersion of a tracer released on a density surface near 1500-m depth in the Antarctic Circumpolar Current west of Drake Passage indicates that the diapycnal diffusivity, averaged over 1 yr and over tens of thousands of square kilometers, is (1.3 ± 0.2) × 10−5 m2 s−1. Diapycnal diffusivity estimated from turbulent kinetic energy dissipation measurements about the area occupied by the tracer in austral summer 2010 was somewhat less, but still within a factor of 2, at (0.75 ± 0.07) × 10−5 m2 s−1. Turbulent diapycnal mixing of this intensity is characteristic of the midlatitude ocean interior, where the energy for mixing is believed to derive from internal wave breaking. Indeed, despite the frequent and intense atmospheric forcing experienced by the Southern Ocean, the amplitude of finescale velocity shear sampled about the tracer was similar to background amplitudes in the midlatitude ocean, with levels elevated to only 20%–50% above the Garrett–Munk reference spectrum. These results add to a long line of evidence that diapycnal mixing in the interior middepth ocean is weak and is likely too small to dictate the middepth meridional overturning circulation of the ocean.

Description: This material is based upon work supported by the National Science Foundation Grants OCE-0622825,OCE-0622670, OCE-0622630, and OCE-0623177.

Description: Author Posting. © American Meteorological Society, 2011. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Climate 24 (2011): 2429–2449, doi:10.1175/2010JCLI3997.1.

Description: Continuous estimates of the oceanic meridional heat transport in the Atlantic are derived from the Rapid Climate Change–Meridional Overturning Circulation (MOC) and Heatflux Array (RAPID–MOCHA) observing system deployed along 26.5°N, for the period from April 2004 to October 2007. The basinwide meridional heat transport (MHT) is derived by combining temperature transports (relative to a common reference) from 1) the Gulf Stream in the Straits of Florida; 2) the western boundary region offshore of Abaco, Bahamas; 3) the Ekman layer [derived from Quick Scatterometer (QuikSCAT) wind stresses]; and 4) the interior ocean monitored by “endpoint” dynamic height moorings. The interior eddy heat transport arising from spatial covariance of the velocity and temperature fields is estimated independently from repeat hydrographic and expendable bathythermograph (XBT) sections and can also be approximated by the array. The results for the 3.5 yr of data thus far available show a mean MHT of 1.33 ± 0.40 PW for 10-day-averaged estimates, on which time scale a basinwide mass balance can be reasonably assumed. The associated MOC strength and variability is 18.5 ± 4.9 Sv (1 Sv ≡ 106 m3 s−1). The continuous heat transport estimates range from a minimum of 0.2 to a maximum of 2.5 PW, with approximately half of the variance caused by Ekman transport changes and half caused by changes in the geostrophic circulation. The data suggest a seasonal cycle of the MHT with a maximum in summer (July–September) and minimum in late winter (March–April), with an annual range of 0.6 PW. A breakdown of the MHT into “overturning” and “gyre” components shows that the overturning component carries 88% of the total heat transport. The overall uncertainty of the annual mean MHT for the 3.5-yr record is 0.14 PW or about 10% of the mean value.

Description: This research was supported by the U.S. National Science Foundation under Awards OCE0241438 and OCE0728108, by the U.K. RAPID Programme (RAPID Grant NER/T/S/2002/00481), and by the U.S. National Oceanic and Atmospheric Administration, as part of its Western Boundary Time Series Program.

Description: Author Posting. © American Meteorological Society, 2010. This article is posted here by permission of American Meteorological Society for personal use, not for redistribution. The definitive version was published in Journal of Climate 23 (2010): 6115-6123, doi:10.1175/2010JCLI3607.1.

Description: Based on the Simple Ocean Data Assimilation (SODA) dataset and three types of Sverdrup streamfunction, an interdecadal variability of the eastward current in the middle South China Sea (SCS) during summer is identified. Both the pattern and strength of the summer Asian monsoon wind stress curl over the SCS contribute to the interdecadal variability of this current. From 1960 to 1979, the monsoon intensified and the zero wind stress curl line shifted southward. Both the core of positive wind stress curl in the northern SCS and the negative curl in the southern SCS moved southward and thus induced a southward shift of both the southern anticyclonic and northern cyclonic gyres, resulting in a southward displacement of the eastward current associated with these two gyres. In the meantime, the southern (northern) SCS anticyclonic (cyclonic) ocean gyre weakened (strengthened) and therefore also induced the southward shift of the eastward current near the intergyre boundary. In contrast, the eastward current shifted northward from 1980 to 1998 because the monsoon relaxed and the zero wind stress curl line shifted northward. After 1998, the eastward jet moved southward again as the zero wind stress curl line shifted southward and the SCS monsoon strengthened. The eastward current identified from the baroclinic streamfunction moved about 1.7° more southward than that from the barotropic streamfunction, indicating that the meridional position of the eastward current is depth dependent.

Description: This study was supported by the National BasicResearch Program (Grant 2007CB816003) and the National Natural Science Foundation of China (Grants 40976017, 40730843, and 40876004).

Description: Vitamin D insufficiency is common globally and low levels are linked to higher cancer incidence. Although vitamin D insufficiency is related to inferior prognosis in some cancers, no data exist for chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). We evaluated the relationship of 25(OH)D serum levels with time-to-treatment (TTT) and overall survival (OS) in newly diagnosed CLL patients participating in a prospective cohort study (discovery cohort) and a separate cohort of previously untreated patients participating in an observational study (confirmation cohort). Of 390 CLL patients in the discovery cohort, 119 (30.5%) were 25(OH)D insufficient. After a median follow-up of 3 years, TTT (hazard ratio[HR] = 1.66; P = .005) and OS (HR = 2.39; P = .01) were shorter for 25(OH)D-insufficient patients. In the validation cohort, 61 of 153 patients (39.9%) were 25(OH)D insufficient. After a median follow-up of 9.9 years, TTT (HR = 1.59; P = .05) and OS (HR 1.63; P = .06) were again shorter for 25(OH)D-insufficient patients. On pooled multivariable analysis of patients in both cohorts adjusting for age, sex, Rai stage, CD38 status, ZAP-70 status, immunoglobulin heavy chain variable (IGHV) gene mutation status, CD49d status, and cytogenetic abnormalities assessed by interphase fluorescent in situ hybridization testing, 25(OH)D insufficiency remained an independent predictor of TTT (HR = 1.47; P = .008), although the association with OS was not significant (HR = 1.47; P = .07). Vitamin D insufficiency is associated with inferior TTT and OS in CLL patients. Whether normalizing vitamin D levels in deficient CLL patients would improve outcome merits clinical testing.

Description: This is the first study to investigate the efficacy of intravenous iron in treating fatigue in nonanemic patients with low serum ferritin concentration. In a randomized, double-blinded, placebo-controlled study, 90 premenopausal women presenting with fatigue, serum ferritin ≤ 50 ng/mL, and hemoglobin ≥ 120 g/L were randomized to receive either 800 mg of intravenous iron (III)–hydroxide sucrose or intravenous placebo. Fatigue and serum iron status were assessed at baseline and after 6 and 12 weeks. Median fatigue at baseline was 4.5 (on a 0-10 scale). Fatigue decreased during the initial 6 weeks by 1.1 in the iron group compared with 0.7 in the placebo group (P = .07). Efficacy of iron was bound to depleted iron stores: In patients with baseline serum ferritin ≤ 15 ng/mL, fatigue decreased by 1.8 in the iron group compared with 0.4 in the placebo group (P = .005), and 82% of iron-treated compared with 47% of placebo-treated patients reported improved fatigue (P = .03). Drug-associated adverse events were observed in 21% of iron-treated patients and in 7% of placebo-treated patients (P = .05); none of these events was serious. Intravenous administration of iron improved fatigue in iron-deficient, nonanemic women with a good safety and tolerability profile. The efficacy of intravenous iron was bound to a serum ferritin concentration ≤ 15 ng/mL. This study was registered at the International Standard Randomized Controlled Trial Number Register (www.isrctn.org) as ISRCTN78430425.

Description: Recruitment of polymorphonuclear neutrophils (PMNs) remains a paramount prerequisite in innate immune defense and a critical cofounder in inflammatory vascular disease. Neutrophil recruitment comprises a cascade of concerted events allowing for capture, adhesion and extravasation of the leukocyte. Whereas PMN rolling, binding, and diapedesis are well characterized, receptor-mediated processes, mechanisms attenuating the electrostatic repulsion between the negatively charged glycocalyx of leukocyte and endothelium remain poorly understood. We provide evidence for myeloperoxidase (MPO), an abundant PMN-derived heme protein, facilitating PMN recruitment by its positive surface charge. In vitro, MPO evoked highly directed PMN motility, which was solely dependent on electrostatic interactions with the leukocyte's surface. In vivo, PMN recruitment was shown to be MPO-dependent in a model of hepatic ischemia and reperfusion, upon intraportal delivery of MPO and in the cremaster muscle exposed to local inflammation or to intraarterial MPO application. Given MPO's affinity to both the endothelial and the leukocyte's surface, MPO evolves as a mediator of PMN recruitment because of its positive surface charge. This electrostatic MPO effect not only displays a so far unrecognized, catalysis-independent function of the enzyme, but also highlights a principal mechanism of PMN attraction driven by physical forces.

Description: Abstract 195 ADAMTS13 contains multiple free thiols on its surface, which may form disulfide bonds with surface-exposed free thiols on plasma-derived von Willebrand factor (VWF). This interaction may prevent lateral association of apposed VWF under arterial shear stress. However, the functional consequence of ADAMTS13-VWF interaction without proteolysis is not known. We hypothesize that the interaction between the C-terminus of ADAMTS13 and the C-terminus of VWF inhibits thrombus formation under shear stress. Using a BioFlux microfluidic system, we showed that under arterial shear stress, 10 dyn/cm2, fluorescein-labeled platelets from PPACK (thrombin inhibitor) anti-coagulated human whole blood adhered to collagen (type I)-coated surface in a time-dependent manner. Addition of human recombinant full-length ADAMTS13 (10 nM) into whole blood dramatically reduced the surface coverage of fluorescein-labeled platelets. Conversely, addition of an inhibitory polyclonal anti-ADAMTS13 IgGs (150 ug/ml) to whole blood dramatically accelerated the accumulation of fluorescein-labeled platelets. These results suggest that this microfluidic system is highly sensitive for the assessment of anti-thrombotic function of ADAMTS13. Under the same conditions, we were able to further show that addition of recombinant C-terminal fragment of ADAMTS13 comprising of the 5th to 8th thrombospondin type 1 (TSP1) repeats and two CUB domains (T5C) or the 2nd to 8th TSP1 repeats and two CUB domains (T2C) into whole blood also inhibited the surface coverage of fluorescein-labeled platelets on collagen-coated surface in a concentration-dependent manner. In the presence of 0.1 μM and 0.5 μM of recombinant T2C or T5C, the surface coverage of fluorescein-labeled platelets was reduced by ∼40% and ∼60%, respectively. The inhibitory activity of these recombinant C-terminal fragments was nearly abolished if pre-treated with 40 mM of N-ethylmaleimide which blocked surface-exposed free thiols. Moreover, recombinant CUB domains at the highest concentration tested (1.0 μM) did not appear to alter the surface coverage of fluorecein-labeled platelets under the same conditions. These results suggest that the C-terminal TSP1 repeats of ADAMTS13 inhibit platelet adhesion and aggretion or thrombus formation through thiol-thiol interactions between ADAMTS13 and VWF (or other proteins). We conclude that the C-terminal TSP1 repeats may modulate thrombus formation independent of proteolytic activity. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 22 For many years it has been evident that it is challenging to express recombinant factor VIII (FVIII) in vitro and in vivo. In vitro studies suggest that high levels of FVIII expression can lead to cellular stress. We have demonstrated that adeno-associated viral (AAV) vector delivery results in long-term expression of therapeutic levels of FVIII in hemophilia A (HA) dogs up 10% of normal. In this model transgene expression is restricted to a portion of liver cells resulting in sustained FVIII levels. Notably, in dogs FVIII expression is sustained for periods longer than 7 years. Conceivably, the increased load per cell for cellular processing of FVIII may impact the cell's ability to synthesize and secrete functional FVIII, activation of cellular stress and the potential for immune responses. We sought to determine whether cellular stress and immune responses to the transgene are increased by overexpression of FVIII in mice by AAV vectors. Using AAV to deliver FVIII as a B-domain deleted single chain (single chain approach) or as two separate chains (light and heavy chains) (two chain approach), we analyzed dose-dependent FVIII synthesis and its cellular impact. We hypothesized that high levels of FVIII expression after AAV-mediated delivery of FVIII may induce cellular stress. HA mice were administered AAV serotype 8 (AAV8) vector in 3 groups: (1) two-chain approach (2) single chain approach or (3) AAV empty capsid (control). Three total AAV doses were administered: 1×1010 vector genomes/mouse (low dose), 5×1010 vg/mouse (mid-dose) and 2.5×1011 vg/mouse (high dose). FVIII antigen levels (heavy and light chain) and anti-cFVIII IgG antibodies were detected by ELISA at a series of time points post vector administration until the terminal time points (1, 2, 4, 8, 12 and 24 wks). As we previously observed, after single chain delivery we detect equivalent amounts of heavy chain and light chain in the circulation by ELISA that correlates with activity. However, in the two-chain approach we observe that the light chain is 〉2-fold higher than the heavy chain in the circulation and that the activity correlates with the amount of heavy chain. We observe a dose-dependent increase in FVIII expression and dose-dependent anti-cFVIII IgG antibody titers. The kinetics of FVIII expression and antibody formation are different for the two-chain delivery compared to the single chain delivery. For the two-chain delivery, we observe AAV dose-dependent peak FVIII expression within 1 week (30, 100, or 400 ng/ml, respective of dose) followed by the onset of anti-FVIII IgG2 antibodies by 4 weeks (16, 27, 52 μg/ml, respective of dose). For single chain delivery, we observe FVIII expression within 1 week and low level antibody titers by 4 weeks that increase dramatically by 12 weeks (14, 21, 38 μg/ml, respective of dose). Thus, we observe a more gradual increase in antibody titers in the single chain compared to the two-chain delivery but at late time points the immunogenicity of both approaches is indistinguishable. RNA was isolated from the livers of the mice at the terminal time points for quantitative PCR analysis of two key components of the unfolded protein response signaling network, BiP and CHOP. We compared the two-chain and single chain AAV delivery of FVIII alongside animals injected with AAV empty capsid that did not express FVIII. A tunicamycin treated positive control had an increase in BiP (54-fold) and CHOP (74-fold) expression compared to untreated HA mice. There were mild elevations (2–3-fold) of BiP and/or CHOP in some animals observed at week 1 in all three treatment groups. Only at 12 weeks post vector administration were significant increases in BiP (〉70 fold) and CHOP (〉4 fold) observed in the single chain treated group at the high dose expressing 〉300% but not the two-chain approach or empty capsid. These studies suggest that supraphysiological expression (〉100%) of BDD-cFVIII may increase the likelihood of an immune response to FVIII and cellular stress. Further studies may determine if there is a relationship between high FVIII expression, cellular stress and immune responses to FVIII. Thus, there is a threshold of FVIII expression levels that may prove unsafe. Moreover, these data are in agreement with the lack of evidence of cellular toxicity and immunogenicity in HA dogs expressing therapeutic levels of FVIII by AAV vectors. Disclosures: Lange: Bayer Healthcare: Research Funding. Altynova:Bayer Healthcare: Research Funding. Sabatino:Bayer Healthcare: Research Funding.

Description: Abstract 4184 Introduction: FACT-MM was developed with the aim to create a disease-specific, patient-reported outcomes (PRO) measure as part of the FACT measurement system to assess multiple myeloma (MM)-related symptoms. Literature review identified 52 MM specific symptoms and concerns. 13 MM expert clinicians rated these 52 items on relevance to health-related quality of life (HRQL) for MM patients and added 11 items for comprehensive PRO assessment in MM. These 63 candidate items were rated by 13 MM patients recruited through the International Myeloma Foundation website. Disease-related symptoms and concerns were described and provided by patients through free-text comments. Information from both the MM expert clinician and MM patient surveys including free-text items was analyzed and 14 highest ranked items were selected for the FACT-MM. The FACT-MM subscale (score 0–56), the FACT-G physical and functional well-being subscales (score 0–28), and the FACT-Neurotoxicity (FACT-Ntx) subscale (score 0–44) was administered to 48 E1A05 participants to assess disease and treatment-related symptoms of patients with newly diagnosed MM receiving 8 cycles of VRd versus Vd. Methods: In this study, the first three of seven sequential assessments were evaluated: baseline, cycle 5 and end of treatment (after cycle 8 or early diconstinuation). Instruments were scored as per the FACT scoring guidelines. Descriptive statistics were provided for each timepoint and changes in scores from baseline. The Wilcoxon rank sum test was used to assess differences in scores by ISS stage and ECOG PS. Pearson correlation coefficients were obtained between the scores. Cronbach's alpha was used to evaluate internal consistency of the FACT-MM. Results: At baseline and end of treatment, 46 and 41 patients, respectively, completed assessments. The FACT-MM demonstrated good to very good internal consistency (Cronbach alpha 0.79 – 0.89). The FACT-MM subscale was significantly correlated with the FACT physical and functional well-being subscales at baseline (r = 0.72), cycle 5 (r = 0.64; r = 0.49) and end of treatment (r = 0.72; r = 0.66). All scores declined modestly by cycle 5 and end of treatment from baseline. While sample size was limited, there appeared association with ECOG PS with higher scores for patients with PS 0 status. Conclusions: The 14-item FACT-MM scale is feasible for use to measure myeloma-related symptoms and has demonstrated acceptable psychometric properties based on findings from E1A05, an ECOG myeloma trial. Disclosures: Cella: Novartis: Research Funding. Fonseca:Consulting:Genzyme, Medtronic, BMS, Amgen, Otsuka, Celgene, Intellikine, Lilly Research Support: Cylene, Onyz, Celgene: Consultancy, Research Funding.

Description: Abstract 5071 Background: Multiple myeloma (MM) is de second most common haematological malignancy in adults worldwide. MM is common in the elderly. While the general life expectancy is increasing an ever increasing number of patients has to be expected. In the last decade treatment strategies are changed continuously because of the introduction of novel agents and allogeneic and autologous stem cell transplantation. In randomized controlled trials, overall survival in patients up to 65 years is improving convincing. In contrast with results in older patients. We studied data of patients treated outside clinical trials derived from the population based registry in the Netherlands to recognize trends in incidence en survival during 1989–2009. Patients and methods: We included all patients with newly diagnosed MM in the period 1989 – 2009 who were recorded in the Netherlands Cancer Registry (n =16.822 ). Follow-up was until January, 2010. We calculated the yearly age-standardised incidence rates for males and females and age-specific incidence rates in 10 year age groups for both sexes separately and combined. Changes in incidence were evaluated by calculating the estimated annual percentage change (EAPC). We then calculated relative survival, which may be interpreted as disease-specific survival within a cancer patient population. Traditional cohort-based, relative survival analysis was applied for patients diagnosed during 1989–2009. Since follow-up was available until 2010, 5 year relative survival for patients diagnosed in 2004–2009 was estimated using period-based relative survival analysis. Results: The number of newly diagnosed MM cases rose between 1989 and 2009 from 631 to 968 cases respectively. Significantly more males were diagnosed than females over time (p=0.01). Furthermore the proportion of male patients increased slightly over these time periods. However, the overall age standardised incidence rate for males and females remained stable over time but was higher among males than females. The median age at diagnosis was 71 years (p10-P90 range 53–84) and stable over time (p=0.07). Incidence was highest in the 70–79 age group for both sexes. However, because of the aging population the age-specific incidence rates (ASIR) were highest for patients aged 80+ years for both sexes. Within specific age groups significant changes were seen. In the population 50–59 years, the ASIR increased from 5.0 per 100,000 in 1989 to 6.9 in 2009 (EAPC 1989–2009 = + 0.7%; 95% CI: 0.0 –1.3). A decrease was seen in females aged 80+ years from 25.1 per 100,000 in 1989 to 22.4 in 2009 (EAPC 1989–2009 = −1,0; 95% CI: −1.8; −0.2). In the overall patient population the 1-year relative survival increased only slightly from 72% to 77% between 1989 and 2009. 5-year relative survival increased from 28% to 37%. Small improvements in survival were observed for all age groups in the past two decades except for patients aged 80+ years. Relative survival decreased with increasing age. In contrast, in the group aged 40–64 years improvements are already detectable from 1994 on. In 2004–2009 the highest 5-year relative survival, 62%, was seen in patients 40–49 year of age. However the strongest improvement over time was observed among the group 50–59 years. Conclusion: Although the average annual age-adjusted incidence rate remained stable from 1989–2009, the number of newly diagnosed MM patients increased because of the aging population. Relative survival increased slowly but continuously in time for patients until 80 years of age with strongest increase seen in patients up to 64 years of age. Improvement for these younger patients is most likely caused by the introduction of novel agents based regiments as well as by the introduction of high dose chemotherapy followed by stem cell transplantation. Our findings in trends of incidence and survival of MM are similar to those reported in other western populations. Disclosures: Sonneveld: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: Membership on an entity's Board of Directors or advisory committees.

Description: Abstract 475FN2 Background: An IMiDs® immunomodulatory agent, Len has a dual mechanism of action: its tumoricidal effect directly leads to tumor cell death, and its immunomodulatory effect may keep the tumor in remission. A phase 3, randomized, placebo (Pbo)-controlled trial, MM-015 compares MPR-R with fixed-duration MPR and MP induction in transplant-ineligible NDMM pts. Interim results showed unprecedented reduction in disease progression risk with MPR-R (Palumbo et al, IMW 2011); this analysis focuses on pts aged 65–75 yrs in whom the greatest benefit was observed. Methods: A total of 459 pts aged ≥ 65 yrs with NDMM were enrolled. Induction consisted of nine 28-day cycles of melphalan 0.18 mg/kg (D1-4), prednisone 2 mg/kg (D1-4), and Len 10 mg (D1-21) (MPR-R and MPR) or melphalan and prednisone with Pbo (MP). After induction, MPR-R pts received Len 10 mg (D1-21) maintenance until progression; MPR and MP pts received Pbo. Pts with progressive disease (PD) could enroll in an open-label extension phase to receive Len 25 mg (D1-21) ± dexamethasone 40 mg (D1-4, 9–12, and 17–20). This analysis includes data up to Feb 28, 2011 (median follow-up, 30 mos). Results: There were 116/152 (76%), 116/153 (76%), and 116/154 (75%) of MPR-R, MPR, and MP pts, respectively, aged 65–75 yrs. Nearly 50% had ISS stage III disease, 〉 40% had β2-microglobulin 〉 5.5 mg/L, and 40% had CrCL 〈 60 mL/min. With a median follow-up of 30 mos, MPR-R reduced progression risk by 70% and significantly prolonged median PFS (31 mos) vs MP (12 mos; hazard ratio [HR]: 0.30 [95% CI, 0.20–0.45]; P

Description: POEMS syndrome is a rare clonal plasma cell disorder without standard treatment. Based on the efficacy and low toxicity of a combination of melphalan and dexamethasone (MDex) for light chain amyloidosis, we conducted a prospective study of MDex treatment for patients with newly diagnosed POEMS syndrome. Thirty-one patients (19 men) were enrolled and the median age at the time of diagnosis was 44 years (range, 32-68 years). All patients received 12 cycles of MDex treatment. Twenty-five patients (80.6%) achieved hematologic response including 12 (38.7%) complete remission and 13 (41.9%) partial remission. Of all 31 patients, the neurologic response rate was 100%, assessed by overall neuropathy limitation scale (ONLS). The initial neurologic response was observed in 24 patients (77.4%) at 3 months after treatment and the median time to maximal neurologic response was 12 months (range, 3-15 months). Moreover, MDex substantially improved the level of serum vascular endothelial growth factor and relieved organomegaly, extravascular volume overload, and pulmonary hypertension. Only 6 patients (19.3%) suffered from grade 3 adverse events during treatment. All patients are alive and free of neurologic relapse after the median follow-up time of 21 months. Therefore, MDex is an effective and well-tolerated treatment option for patients with newly diagnosed POEMS syndrome.

Description: Multiple mechanisms operate to ensure T-cell tolerance toward self-antigens. Three main processes have been described: clonal deletion, anergy, and deviation to CD4+ regulatory T cells (Tregs) that suppress autoreactive T cells that have escaped the first 2 mechanisms. Although it is accepted that dendritic cells (DCs) and B cells contribute in maintaining T-cell tolerance to self-antigens, their relative contribution and the processes involved under physiologic conditions remain only partially characterized. In this study, we used different transgenic mouse models to obtain chimeras where a neo self-antigen is expressed by thymic epithelium and/or by DCs or B cells. We found that expression of cognate ligand in the thymus enhances antigen-specific FoxP3+ cells independently of whether the self-antigen is expressed on thymic epithelium or only on DCs, but not on B cells. On the contrary, self-antigen expression by B cells was very efficient in inducing FoxP3+ cells in the periphery, whereas self-antigen expression by DC led mainly to deletion and anergy of antigen-specific FoxP3− cells. The results presented in this study underline the role of B cells in Treg induction and may have important implications in clinical protocols aimed at the peripheral expansion of Tregs in patients.

Description: Treatment-related mortality (TRM) is important in acute lymphoblastic leukemia and acute myeloid leukemia (AML); however, little is known about how TRM is defined across trials. Two major problems are related to what constitutes treatment versus disease-related cause of death and to TRM attribution (for example, death because of infection or hemorrhage). To address the former, we conducted a systematic review of randomized therapeutic pediatric acute leukemia and adult/pediatric acute promyelocytic leukemia trials and any study type focused on TRM in pediatric acute leukemia. We described definitions used for TRM. Sixty-six studies were included. Few therapeutic pediatric acute lymphoblastic leukemia studies (2/32, 6.3%) provided definitions for TRM, whereas more therapeutic pediatric AML studies (6/9, 66.7%) provided definitions. There was great heterogeneity in TRM classification. The authors of most studies relied on deaths during induction or in remission to delineate whether a death was TRM. However, 44.4% of therapeutic AML studies used death within a specific time frame to delineate TRM. We suggest that a consistent approach to defining and determining attribution for TRM in acute leukemia is an important future goal. Harmonization of definitions across the age spectrum would allow comparisons between pediatric and adult studies.

Description: Using proteins in a therapeutic context often requires engineering to modify functionality and enhance efficacy. We have previously reported that the therapeutic antileukemic protein macromolecule Escherichia coli L-asparaginase is degraded by leukemic lysosomal cysteine proteases. In the present study, we successfully engineered L-asparaginaseto resist proteolytic cleavage and at the same time improve activity. We employed a novel combination of mutant sampling using a genetic algorithm in tandem with flexibility studies using molecular dynamics to investigate the impact of lid-loop and mutations on drug activity. Applying these methods, we successfully predicted the more active L-asparaginase mutants N24T and N24A. For the latter, a unique hydrogen bond network contributes to higher activity. Furthermore, interface mutations controlling secondary glutaminase activity demonstrated the importance of this enzymatic activity for drug cytotoxicity. All selected mutants were expressed, purified, and tested for activity and for their ability to form the active tetrameric form. By introducing the N24A and N24A R195S mutations to the drug L-asparaginase, we are a step closer to individualized drug design.

Description: Shwachman-Diamond syndrome (SDS) results from mutations in the SBDS gene, characterized by exocrine pancreatic insufficiency and hematologic and skeletal abnormalities. Neutropenia and neutrophil dysfunction are hallmark features of SDS; however, causes for the bone defects are unknown. Dysfunction of bone-resorbing osteoclasts, formed by the fusion of monocytic progenitors derived from the same granulocytic precursors as neutrophils, could be responsible. We report that Sbds is required for in vitro and in vivo osteoclastogenesis (OCG). Sbds-null murine monocytes formed osteoclasts of reduced number and size because of impaired migration and fusion required for OCG. Phenotypically, Sbds-null mice exhibited low-turnover osteoporosis consistent with findings in SDS patients. Western blotting of Rho GTPases that control actin dynamics and migration showed a 5-fold decrease in Rac2, whereas Rac1, Cdc42, and RhoA were unchanged or only mildly reduced. Although migration was rescued on Rac2 supplementation, OCG was not. This was attributed to impaired signaling downstream of receptor activator of nuclear factor-κB (RANK) and reduced expression of the RANK-ligand-dependent fusion receptor DC-STAMP. We conclude that Sbds is required for OCG by regulating monocyte migration via Rac2 and osteoclast differentiation signaling downstream of RANK. Impaired osteoclast formation could disrupt bone homeostasis, resulting in skeletal abnormalities seen in SDS patients.

Description: Thrombin is a positive mediator of thrombus formation through the proteolytic activation of protease-activated receptors (PARs), fibrinogen, factor XI (fXI), and other substrates, and a negative regulator through activation of protein C, a natural anticoagulant with anti-inflammatory/cytoprotective properties. Protease-engineering studies have established that 2 active-site substitutions, W215A and E217A (fIIWE), result in dramatically reduced catalytic efficiency with procoagulant substrates while largely preserving thrombomodulin (TM)–dependent protein C activation. To explore the hypothesis that a prothrombin variant favoring antithrombotic pathways would be compatible with development but limit inflammatory processes in vivo, we generated mice carrying the fIIWE mutations within the endogenous prothrombin gene. Unlike fII-null embryos, fIIWE/WE mice uniformly developed to term. Nevertheless, these mice ultimately succumbed to spontaneous bleeding events shortly after birth. Heterozygous fIIWT/WE mice were viable and fertile despite a shift toward an antithrombotic phenotype exemplified by prolonged tail-bleeding times and times-to-occlusion after FeCl3 vessel injury. More interestingly, prothrombinWE expression significantly ameliorated the development of inflammatory joint disease in mice challenged with collagen-induced arthritis (CIA). The administration of active recombinant thrombinWE also suppressed the development of CIA in wild-type mice. These studies provide a proof-of-principle that pro/thrombin variants engineered with altered substrate specificity may offer therapeutic opportunities for limiting inflammatory disease processes.

Description: Thrombomodulin (TM) is a predominantly endothelial transmembrane glycoprotein that modulates hemostatic function through a domain that controls thrombin-mediated proteolysis and an N-terminal lectin-like domain that controls inflammatory processes. To test the hypothesis that TM is a determinant of malignancy and dissect the importance of these functional domains in cancer biology, metastatic potential was evaluated in TMPro mice expressing a mutant form of TM with reduced thrombin affinity and TMLeD mice lacking the N-terminal lectin-like domain. Studies of TMPro mice revealed that TM is a powerful determinant of hematogenous metastasis. TMPro mice exhibited a strongly prometastatic phenotype relative to control mice that was found to result from increased survival of tumor cells newly localized to the lung rather than any alteration in tumor growth. The impact of the TMPro mutation on metastasis was dependent on both tumor cell-associated tissue factor and thrombin procoagulant function. In contrast, expression of a mutant form of TM lacking the lectin-like domain had no significant impact on metastasis. These studies directly demonstrate for the first time that TM-mediated regulation of tumor cell-driven procoagulant function strongly influences metastatic potential and suggest that endothelial cell-associated modulators of hemostasis may represent novel therapeutic targets in limiting tumor dissemination.

Description: Whether long-term use of vitamin K antagonists (VKAs) might affect the incidence of cancer is a longstanding hypothesis. We conducted a population-based study including all cancer- and thromboembolism-free patients of our health area; study groups were defined according to chronic anticoagulant use to VKA-exposed and control groups. Cancer incidence and cancer-related and overall mortality was assessed in both groups. 76 008 patients (3231 VKA-exposed and 72 777 control subjects) were followed-up for 8.2 (± 3.2) years. After adjusting for age, sex, and time-to-event, the hazard ratio of newly diagnosed cancer in the exposed group was 0.88 (95% confidence interval [95% CI] 0.80-0.98; P 〈 .015). VKA-exposed patients were less likely to develop prostate cancer, 0.69 (95% CI 0.50-0.97; P = .008). The adjusted hazard ratio for cancer-related and overall mortality was 1.07 (95% CI 0.92-1.24) and 1.12 (95% CI 1.05-1.19), respectively. These results support the hypothesis that anticoagulation might have a protective effect on cancer development, especially prostate cancer.

Description: Patients referred to tertiary care centers occasionally may have their diagnostic procedures repeated and have a final diagnosis that differs from that of the referring center. The aim of this study was to evaluate discordance rates and their clinical implications in the diagnosis of patients with myelodysplastic syndrome (MDS) referred to a tertiary center. We analyzed 915 patients with MDS who were referred to M. D. Anderson Cancer Center between September 2005 and December 2009. Discordance in the diagnosis was documented in 109 (12%) patients, with a majority reclassified as having higher-risk disease by French-American-British (67%) or by International Prognostic Scoring System (77%) with implications for therapy selection and prognosis calculation. These results demonstrate the complexity of the diagnosis of MDS and highlight the need for confirmation of diagnosis when in doubt.

Description: Abstract 2283 Introduction: von Willebrand factor (VWF) acts as bridging molecule between platelets and vessel wall or as a career for plasma factor VIII (FVIII). Earlier reports using flow chamber had revealed that VWF acts as an “initiator” through its interactionwith glycoprotein (GP) Ib, whereas fibrinogen, via its binding to GP IIb-IIIa, acts as a “stabilizer” against high shear. VWD is categorized as partial or complete quantitative defect (type 1 or 3) or qualitative defect (type 2) of VWF based on laboratory tests, such as VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), and FVIII activity (FVIII:C). However, the diagnosis of VWD remains difficult because the clinical and laboratory phenotype has wide-spectrum. An assay reflecting clinical phenotypes of VWD and effect of the treatment would be helpful for clinicians. Methods: We evaluated a thrombus formation with a new applicative microchip flow chamber instrument, total-thrombus-formation analysis system (T-TAS®). Citrated or hirudin-added blood from healthy volunteers (n=5) or 3 patients with VWD [symptomatic type 1 (S-1), asymptomatic type 1 (AS-1), and symptomatic type 3 (S-3)] were utilized. Normal blood was mixed with inhibitor for GP Ib (OS-1), GP IIb-IIIa (abciximab) and monoclonal antibody against for VWF (mAb-VWF). Patients' blood was infused with FVIII/VWF concentrates in (ex) vivo. Re-calcified citrated blood (450 μl) added corn trypsin inhibitor (30 μg/ml) was infused to AR chip of T-TAS at constant flow rate (240–600 s−1), which surface was coated by collagen and tissue factor. Hirudin-added blood (350 μl) was infused to PL chip of T-TAS at higher shear (1,000–2,000 s−1) which surface was coated by collagen. Flow pressure curve was plotted and time to 10 kPa (T10) was evaluated. Furthermore, flow images were recorded with a micro camera. AR chip promoted white thrombus formation (WTF) which reflected both platelet aggregation and fibrin generation, whilst PL chip did only platelet thrombus formation (PTF). Rotational thromboerastometry (ROTEM) were simultaneously investigated. Results: Both abciximab (0.5–2.0 μg/ml) and OS-1 (100–400 nM) inhibited WTF in AR chip dose-dependently. OS-1 (200 nM) inhibited WTF partially at 240 s−1 (T10 15.4±1.6 min, control 8.9±0.4 min), but completely at 600 s−1 (T10 〉30 min, control 7.5±0.2 min). In PL chip, both agents inhibited PTF at much lower dose than those in AR chip. ROTEM parameters showed little change in both agents. Using mAb-VWF, similar results to OS-1 were obtained. These data showed that GP Ib-VWF interaction was shear-dependent, consistent with earlier reports. In in (ex) vivo assays, S-1 and AS-1 had comparable VWF levels (VWF:Ag/VWF:RCo 20%/6.4% and 5.8%/3.2%, respectively) in spite of clear difference of clinical phenotype. ROTEM parameters of S-1 were rather better than those of AS-1 likely reflecting FVIII:C (60% and 7.2%). Interestingly, however, PTF in PL chip (at 1,000 s−1) showed significant delay of T10 in S-1 (9.0 min, control 4.1±0.5 min) compared to AS-1 (5.2 min) and after in vivo infusion of FVIII/VWF concentrates, T10 in S-1 was improved (5.0 min). In S-3, ROTEM parameters declined reflecting low FVIII:C (1.2%), and prolonged T10 (〉30 min) in AR chip likely reflected complete defect of VWF:Ag (

Description: Gene expression profiling (GEP) of purified plasma cells 48 hours after thalidomide and dexamethasone test doses showed these agents' mechanisms of action and provided prognostic information for untreated myeloma patients on Total Therapy 2 (TT2). Bortezomib was added in Total Therapy 3 (TT3), and 48 hours after bortezomib GEP analysis identified 80 highly survival-discriminatory genes in a training set of 142 TT3A patients that were validated in 128 patients receiving TT3B. The 80-gene GEP model (GEP80) also distinguished outcomes when applied at baseline in both TT3 and TT2 protocols. In context of our validated 70-gene model (GEP70), the GEP80 model identified 9% of patients with a grave prognosis among those with GEP70-defined low-risk disease and 41% of patients with favorable prognosis among those with GEP70-defined high-risk disease. PMSD4 was 1 of 3 genes common to both models. Residing on chromosome 1q21, PSMD4 expression is highly sensitive to copy number. Both higher PSMD4 expression levels and higher 1q21 copy numbers affected clinical outcome adversely. GEP80 baseline-defined high risk, high lactate dehydrogenase, and low albumin were the only independent adverse variables surviving multivariate survival model. We are investigating whether second-generation proteasome inhibitors (eg, carfilzomib) can overcome resistance associated with high PSMD4 levels.

Description: Factor XI deficiency is associated with a bleeding diathesis, but factor XII deficiency is not, indicating that, in normal hemostasis, factor XI must be activated in vivo by a protease other than factor XIIa. Several groups have identified thrombin as the most likely activator of factor XI, although this reaction is slow in solution. Although certain nonphysiologic anionic polymers and surfaces have been shown to enhance factor XI activation by thrombin, the physiologic cofactor for this reaction is uncertain. Activated platelets secrete the highly anionic polymer polyphosphate, and our previous studies have shown that polyphosphate has potent procoagulant activity. We now report that polyphosphate potently accelerates factor XI activation by α-thrombin, β-thrombin, and factor XIa and that these reactions are supported by polyphosphate polymers of the size secreted by activated human platelets. We therefore propose that polyphosphate is a natural cofactor for factor XI activation in plasma that may help explain the role of factor XI in hemostasis and thrombosis.

Description: Several studies have found that high levels of reactive oxidative species (ROS) are associated with stem cell dysfunction. In the present study, we investigated the role of nuclear factor erythroid-2–related factor 2 (Nrf2), a master regulator of the antioxidant response, and found that it is required for hematopoietic stem progenitor cell (HSPC) survival and myeloid development. Although the loss of Nrf2 leads to increased ROS in most tissues, basal ROS levels in Nrf2-deficient (Nrf2−/−) BM were not elevated compared with wild-type. Nrf2−/− HSPCs, however, had increased rates of spontaneous apoptosis and showed decreased survival when exposed to oxidative stress. Nrf2−/− BM demonstrated defective stem cell function, as evidenced by reduced chimerism after transplantation that was not rescued by treatment with the antioxidant N-acetyl cysteine. Gene-expression profiling revealed that the levels of prosurvival cytokines were reduced in Nrf2−/− HSPCs. Treatment with the cytokine G-CSF improved HSPC survival after exposure to oxidative stress and rescued the transplantation defect in Nrf2−/− cells despite increases in ROS induced by cytokine signaling. These findings demonstrate a critical role for Nrf2 in hematopoiesis and stem cell survival that is independent of ROS levels.

Description: Reactive oxygen species (ROS) are highly destructive toward cellular macromolecules. However, moderate levels of ROS can contribute to normal cellular processes including signaling. Herein we evaluate the consequence of a pro-oxidant environment on hematopoietic homeostasis. The NF-E2 related factor 2 (Nrf2) transcription factor regulates genes related to ROS scavenging and detoxification. Nrf2 responds to altered cellular redox status, such as occurs with loss of antioxidant selenoproteins after deletion of the selenocysteine-tRNA gene (Trsp). Conditional knockout of the Trsp gene using Mx1-inducible Cre-recombinase leads to selenoprotein deficiency and anemia on a wild-type background, whereas Trsp:Nrf2 double deficiency dramatically exacerbates the anemia and increases intracellular hydrogen peroxide levels in erythroblasts. Results indicate that Nrf2 compensates for defective ROS scavenging when selenoproteins are lost from erythroid cells. We also observed thymus atrophy in single Trsp-conditional knockout mice, suggesting a requirement for selenoprotein function in T-cell differentiation within the thymus. Surprisingly, no changes were observed in the myelomonocytic or megakaryocytic populations. Therefore, our results show that selenoprotein activity and the Nrf2 gene battery are particularly important for oxidative homeostasis in erythrocytes and for the prevention of hemolytic anemia.

Description: Human induced pluripotent stem cells (iPSCs) bearing monogenic mutations have great potential for modeling disease phenotypes, screening candidate drugs, and cell replacement therapy provided the underlying disease-causing mutation can be corrected. Here, we report a homologous recombination-based approach to precisely correct the sickle cell disease (SCD) mutation in patient-derived iPSCs with 2 mutated β-globin alleles (βs/βs). Using a gene-targeting plasmid containing a loxP-flanked drug-resistant gene cassette to assist selection of rare targeted clones and zinc finger nucleases engineered to specifically stimulate homologous recombination at the βs locus, we achieved precise conversion of 1 mutated βs to the wild-type βA in SCD iPSCs. However, the resulting co-integration of the selection gene cassette into the first intron suppressed the corrected allele transcription. After Cre recombinase-mediated excision of this loxP-flanked selection gene cassette, we obtained “secondary” gene-corrected βs/βA heterozygous iPSCs that express at 25% to 40% level of the wild-type transcript when differentiated into erythrocytes. These data demonstrate that single nucleotide substitution in the human genome is feasible using human iPSCs. This study also provides a new strategy for gene therapy of monogenic diseases using patient-specific iPSCs, even if the underlying disease-causing mutation is not expressed in iPSCs.

Description: The Ldb1/GATA-1/TAL1/LMO2 complex mediates long-range interaction between the β-globin locus control region (LCR) and gene in adult mouse erythroid cells, but whether this complex mediates chromatin interactions at other developmental stages or in human cells is unknown. We investigated NLI (Ldb1 homolog) complex occupancy and chromatin conformation of the β-globin locus in human erythroid cells. In addition to the LCR, we found robust NLI complex occupancy at a site downstream of the Aγ-globin gene within sequences of BGL3, an intergenic RNA transcript. In cells primarily transcribing β-globin, BGL3 is not transcribed and BGL3 sequences are occupied by NLI core complex members, together with corepressor ETO2 and by γ-globin repressor BCL11A. The LCR and β-globin gene establish proximity in these cells. In contrast, when γ-globin transcription is reactivated in these cells, ETO2 participation in the NLI complex at BGL3 is diminished, as is BCL11A occupancy, and both BGL3 and γ-globin are transcribed. In these cells, proximity between the BGL3/γ-globin region and the LCR is established. We conclude that alternative NLI complexes mediate γ-globin transcription or silencing through long-range LCR interactions involving an intergenic site of noncoding RNA transcription and that ETO2 is critical to this process.

Description: Megakaryocytes generate platelets by remodeling their cytoplasm first into proplatelets and then into preplatelets, which undergo fission to generate platelets. Although the functions of microtubules and actin during platelet biogenesis have been defined, the role of the spectrin cytoskeleton is unknown. We investigated the function of the spectrin-based membrane skeleton in proplatelet and platelet production in murine megakaryocytes. Electron microscopy revealed that, like circulating platelets, proplatelets have a dense membrane skeleton, the main fibrous component of which is spectrin. Unlike other cells, megakaryocytes and their progeny express both erythroid and nonerythroid spectrins. Assembly of spectrin into tetramers is required for invaginated membrane system maturation and proplatelet extension, because expression of a spectrin tetramer–disrupting construct in megakaryocytes inhibits both processes. Incorporation of this spectrin-disrupting fragment into a novel permeabilized proplatelet system rapidly destabilizes proplatelets, causing blebbing and swelling. Spectrin tetramers also stabilize the “barbell shapes” of the penultimate stage in platelet production, because addition of the tetramer-disrupting construct converts these barbell shapes to spheres, demonstrating that membrane skeletal continuity maintains the elongated, pre-fission shape. The results of this study provide evidence for a role for spectrin in different steps of megakaryocyte development through its participation in the formation of invaginated membranes and in the maintenance of proplatelet structure.

Description: Myeloid-derived suppressor cells (MDSCs) inhibit adaptive and innate immunity and accumulate in the blood of persons with cancer, chronic inflammation, trauma, infection, and stress. Some of the factors inducing their accumulation are known; however, mechanisms regulating their turnover have not been identified. Mass spectrometry showed prominent expression of apoptosis pathway proteins, suggesting that MDSC turnover may be regulated by Fas-FasL–mediated apoptosis. This hypothesis was confirmed by showing that blood MDSCs induced by 3 mouse tumors were Fas+ and apoptosed in response to Fas agonist in vitro and to activated FasL+ T cells in vivo. FasL-deficient mice contained significantly more blood MDSCs than FasL+/+ mice, and after removal of primary tumors MDSCs regressed in STAT6−/− and CD1−/− mice but not in STAT6−/−FasL−/− or CD1−/−FasL−/− mice. Fas+ macrophages and dendritic cells did not apoptose in response to activated T cells, indicating that Fas-FasL regulation of myeloid cells was restricted to MDSCs. These results identify a new mechanism regulating MDSC levels in vivo and show a retaliatory relationship between T cells and MDSCs in that MDSCs suppress T-cell activation; however, once activated, T cells mediate MDSC apoptosis.

Description: Abstract 41 Transfusion-related acute lung injury (TRALI) is a leading cause of transfusion-related death with a majority of the reported cases secondary to the infusion of antibodies (Abs) contained within the plasma/blood component. An experimental filter that removes IgG was developed. We hypothesize that filtration of plasma with antibodies to leukocyte antigens will decrease both antibody-mediated priming of PMNs and antibody-mediated TRALI in a two-event in vivo model. Methods: Human plasma was drawn from healthy volunteers and IgG concentrations were measured before and after filtration. Plasma was obtained from two multiparous female donors: one with antibodies to HLA-A2 and to DR7 and the other with antibodies against HNA-3a. These plasma samples were filtered (F-Plas) or left as an unmodified control (Plas) and the anti-leukocyte antibodies were measured in a blinded fashion in referral labs using flow cytometry and Luminex™ beads or standard granulocyte antibody detection assays. These plasma samples were then used to prime the fMLP-activated respiratory burst, measured as the SOD-inhibitable reduction of cytochrome c (nmol O2−/min), of PMNs from HNA-3a+ donors or donors homozygous donors for HLA-A2, respectively. For the two-event in vivo modeling rats were incubated with 2 μg/ml endotoxin (LPS, S. enteritides) or saline (NS) for 2 hours (first event) and then were transfused with heat-treated human plasma that contained 25 μg/ml of an antibody against the MHC class I antigen OX27 that was either filtered (or left unmodified) prior to infusion (second event) followed by Evans Blue dye (EBD). ALI was measured as %EBD leak from the plasma into the bronchoalveolar lavage fluid. Statistical differences were measured via paired (PMN priming) or independent (in vivo TRALI) ANOVA, and data are reported as the mean ± the standard error of the mean. *=p

Description: Clinical trials have demonstrated that rituximab improves overall survival in non-Hodgkin lymphoma (NHL), except in mantle cell lymphoma (MCL). We used Surveillance Epidemiology and End Results (SEER)–Medicare data to compare survival in older MCL patients who began chemotherapy with or without rituximab within 180 days of diagnosis. Patients were followed from diagnosis (January 1999 to December 2005) until death or the end of observation (December 2007). Medicare administrative and claims data were used to identify the date and cause of death and the immunochemotherapy regimen. Of 638 patients, the mean age at diagnosis was 75 years, 75% had stage III/IV disease, 67% had extranodal involvement, and 64% received rituximab. The average length of first-line treatment was 21 weeks, with no difference between the 2 groups (P = .76). Median survival was 27 months for chemotherapy alone, compared with 37 months for chemotherapy plus rituximab (P 〈 .001). In multivariate analysis of 2-year survival, rituximab plus chemotherapy was associated with lower all-cause (hazard ratio [HR] 0.58; 95% confidence interval [CI] 0.41-0.82; P 〈 .01), and cancer-specific (HR 0.56; 95% CI 0.37-0.84; P 〈 .01) mortality. Results were similar when using the entire observation period, propensity score analysis, and limiting chemotherapy to CHOP/CHOP-like. We conclude that first-line chemotherapy including rituximab is associated with significantly improved survival in older patients diagnosed with MCL.

Description: Abstract 4127 In the age of novel targeted agents, autologous stem cell transplant (ASCT) remains the standard of care for younger patients with newly diagnosed multiple myeloma (MM), offering similar treatment responses and overall survivals as standard chemotherapeutic agents but with the added benefit of a prolonged treatment-free period. Nevertheless, a standard of care for stem cell mobilization for ASCT has yet to be determined. Even in the era of new mobilization agents such as Plerixafor, Cyclophosphamide (Cy) and G-CSF combination remains the preferred mobilizing approach for patients with MM. Several studies have shown that Cy improves the stem cell yield at the expense of increased toxicity, but whether the administration of this chemotherapeutic agent pre-transplant has any impact on the long-term event-free and/or overall survival of myeloma patients remains controversial. In this study, we present a retrospective analysis of 186 patients with newly diagnosed MM who underwent ASCT with high-dose melphalan 200 mg/m2 (HDM) between December of 2000 and 2008 at our Institution. Eighty-three patients were mobilized with single agent G-CSF and 103 patients received high dose Cy (4 gm/m2) and G-CSF combination. Patient characteristics were similar between the treatment groups, including: age, gender, disease stage, and disease status prior to transplant. However, toxicity post-mobilization with Cy/G-CSF was significantly higher compared with G-SCF alone, including: febrile neutropenia (23%), hemorrhagic cystitis (8%), GI toxicity (57%), re-hospitalization due to complications and transplant delay (14%). The overall post-transplant toxicity was similar in the 2 groups, though the treatment related mortality was slightly higher in the Cy/G-CSF arm (4% versus 2%). Post transplant responses were not significantly different in the 2 groups, with 60% of patients achieving a VGPR or better after ASCT in the G-CSF group and 49% in the Cy/G-CSF group (p = 0.33). The median event-free survivals (EFS) for the Cy/G-CSF and G-CSF cohorts were 21.6 and 22.6 months, respectively, (p = 0.62) yielding no significant difference (Figure 1). Similarly, with a median follow up for surviving patients of 34.3 and 32.7 months, the median overall survivals were 68.2 and 62.3 months (p = 0.23) for the Cy/G-CSF and G-CSF cohorts, respectively (Figure 2). This retrospective analysis confirms that the addition of high dose Cy as part of the mobilizing regimen offers no improvement on the transplant outcome for patients with newly diagnosed myeloma and should therefore only be used in cases of difficult stem cell mobilization. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1753 Background: Ruxolitinib (INC424), a potent and selective oral JAK1 and JAK2 inhibitor, has demonstrated rapid and durable reductions in splenomegaly and improved disease-related symptoms, role functioning, and quality of life (QoL) in 2 phase 3 studies in patients with myelofibrosis (MF) (the COMFORT studies). These studies compared ruxolitinib with either placebo or best available therapy (BAT). This analysis compares the efficacy outcomes between the placebo arm from COMFORT-I and the BAT arm from COMFORT-II. Methods: COMFORT-I is a randomized (1:1), double-blind, multicenter study comparing ruxolitinib 15 or 20 mg twice daily (bid) with placebo, and COMFORT-II is a randomized (2:1), open-label, multicenter study comparing ruxolitinib 15 or 20 mg bid with BAT (investigator-selected therapy, including no treatment). Both studies met their primary end points with statistical significance (ruxolitinib vs control): the percentage of patients achieving ≥35% reduction in spleen volume at week 24 (COMFORT-I, P

Description: Abstract 1768 B-cell chronic lymphocytic leukemia (CLL) is characterized by deregulated expression of microRNAs (miRNAs). These post-transcriptional regulators of gene expression play a crucial role in controlling multiple cellular processes. By microarray analysis and quantitative RT-PCR we observed significantly lower levels of miR-126, miR-130a, miR-143, miR-181a and miR-326 in primary CLL cells compared to normal B cells. Transfection of synthetic miR-130a or miR-143 induced a significant reduction in cell viability of both primary CLL cells and the CLL cell line MEC-1. As autophagy is connected to cancer cell survival and resistance to apoptosis, we investigated the effect of these two miRNAs on autophagy by following the specific autophagosome marker LC3 (microtubule-associated protein 1 light chain 3). Therefore, we generated MEC-1 cells stably expressing GFP-tagged LC3 and analyzed autophagosome formation by using an imaging flow cytometer quantifying GFP-positive dots. These experiments revealed that autophagy is induced in these cells upon starvation, and that introduction of miR-130a, but not miR-143, resulted in a reduction of autophagosome formation (see Figure). These findings were verified by LC3 Western blot analysis, and extended to primary CLL cells, showing for the first time that autophagy is an active process in these cells and that miR-130a inhibits autophagy in primary CLL cells as well. To further elucidate the molecular mechanism of miR-130a-mediated CLL cell survival and autophagy, we aimed at identifying putative target genes of this miRNA and identified ATG2B, an autophagy-related gene, as well as DICER1 and AGO4, two components of the miRNA processing machinery, as direct target genes of miR-130a in CLL cells. The relevance and role of these three novel target genes in miR-130a-regulated cell death/cell survival programs is under current investigation. Figure: Analysis of autophagy using MEC-1 cell line stably expressing GFP-tagged LC3 protein. Green dots representing autophagosomes were quantified in MEC-1/GFP-LC3 cells under starvation by imaging flow cytometry (Image Stream, Amnis). Transfection with synthetic miR-130a reduced the autophagic flux in these cells compared to scrambled negative control miRNA (NC). Figure:. Analysis of autophagy using MEC-1 cell line stably expressing GFP-tagged LC3 protein. Green dots representing autophagosomes were quantified in MEC-1/GFP-LC3 cells under starvation by imaging flow cytometry (Image Stream, Amnis). Transfection with synthetic miR-130a reduced the autophagic flux in these cells compared to scrambled negative control miRNA (NC). Disclosures: No relevant conflicts of interest to declare.

Description: The Blood and Marrow Transplant Clinical Trials Network conducted 2 parallel multicenter phase 2 trials for individuals with leukemia or lymphoma and no suitable related donor. Reduced intensity conditioning (RIC) was used with either unrelated double umbilical cord blood (dUCB) or HLA-haploidentical related donor bone marrow (Haplo-marrow) transplantation. For both trials, the transplantation conditioning regimen incorporated cyclophosphamide, fludarabine, and 200 cGy of total body irradiation. The 1-year probabilities of overall and progression-free survival were 54% and 46%, respectively, after dUCB transplantation (n = 50) and 62% and 48%, respectively, after Haplo-marrow transplantation (n = 50). The day +56 cumulative incidence of neutrophil recovery was 94% after dUCB and 96% after Haplo-marrow transplantation. The 100-day cumulative incidence of grade II-IV acute GVHD was 40% after dUCB and 32% after Haplo-marrow transplantation. The 1-year cumulative incidences of nonrelapse mortality and relapse after dUCB transplantation were 24% and 31%, respectively, with corresponding results of 7% and 45%, respectively, after Haplo-marrow transplantation. These multicenter studies confirm the utility of dUCB and Haplo-marrow as alternative donor sources and set the stage for a multicenter randomized clinical trial to assess the relative efficacy of these 2 strategies. The trials are registered at www.clinicaltrials.gov under NCT00864227 (BMT CTN 0604) and NCT00849147 (BMT CTN 0603).

Description: Various combinations of antibodies directed to cell surface markers have been used to isolate human and rhesus macaque hematopoietic stem cells (HSCs). These protocols result in poor enrichment or require multiple complex steps. Recently, a simple phenotype for HSCs based on cell surface markers from the signaling lymphocyte activation molecule (SLAM) family of receptors has been reported in the mouse. We examined the possibility of using the SLAM markers to facilitate the isolation of highly enriched populations of HSCs in humans and rhesus macaques. We isolated SLAM (CD150+CD48−) and non-SLAM (not CD150+CD48−) cells from human umbilical cord blood CD34+ cells as well as from human and rhesus macaque mobilized peripheral blood CD34+ cells and compared their ability to form colonies in vitro and reconstitute immune-deficient (nonobese diabetic/severe combined immunodeficiency/interleukin-2 γc receptornull, NSG) mice. We found that the CD34+ SLAM population contributed equally or less to colony formation in vitro and to long-term reconstitution in NSG mice compared with the CD34+ non-SLAM population. Thus, SLAM family markers do not permit the same degree of HSC enrichment in humans and rhesus macaques as in mice.

Description: T-cell therapy with genetically modified T cells targeting CD19 or CD20 holds promise for the immunotherapy of hematologic malignancies. These targets, however, are only present on B cell–derived malignancies, and because they are broadly expressed in the hematopoietic system, their targeting may have unwanted consequences. To expand T-cell therapies to hematologic malignancies that are not B cell–derived, we determined whether T cells can be redirected to CD70, an antigen expressed by limited subsets of normal lymphocytes and dendritic cells, but aberrantly expressed by a broad range of hematologic malignancies and some solid tumors. To generate CD70-specific T cells, we constructed a chimeric antigen receptor (CAR) consisting of the CD70 receptor (CD27) fused to the CD3-ζ chain. Stimulation of T cells expressing CD70-specific CARs resulted in CD27 costimulation and recognition of CD70-positive tumor cell lines and primary tumor cells, as shown by IFN-γ and IL-2 secretion and by tumor cell killing. Adoptively transferred CD70-specific T cells induced sustained regression of established murine xenografts. Therefore, CD70-specific T cells may be a promising immunotherapeutic approach for CD70-positive malignancies.

Description: Abstract 2542 Background: Patients with high-risk chronic lymphocytic leukemia (CLL) uniformly relapse after conventional chemo-immunotherapy, but roughly half achieve long-term disease-free survival (DFS) following allogeneic hematopoietic cell transplantation (allo-HCT). Quantification of minimal residual disease (MRD) following allo-HCT predicts post-transplant relapse (PTR) when CLL disease burden remains greater than 10e-4 (ie, 1 leukemic cell in 10,000 peripheral blood mononuclear cells [PBMC]) when quantified by allele-specific oligonucleotide quantitative polymerase chain reaction (ASO-PCR) or flow cytometry. We previously demonstrated the feasibility of MRD quantification using consensus primers to amplify all immunoglobulin heavy chain (IGH) genes in a mixture of PBMC, followed by high-throughput sequencing (HTS) and clonotypic quantification. In our prior work, we used 454 pyrosequencing technology which enabled 10e-5 sensitivity. Here, we report 10e-6 MRD sensitivity using novel Illumina-based HTS that provides better prediction of disease recurrence than ASO-PCR. Methods: We amplified IGH loci from genomic DNA extracted from PBMC (median input 2.4×10e6 cells; range 1.1–23.7×10e6) using V and J segment consensus primers. Amplified IGH molecules were then sequenced with one million or more dedicated reads using Illumina HiSeq and clones were quantified using Sequenta HTS bioinformatics. To verify 10e-6 sensitivity using this system, a clonal B cell population was diluted to 10e-6 in PBMC from a healthy donor with successful clonal detection. Disease-bearing samples (either pre-treatment or after PTR) were sequenced to verify applicability of consensus primers for each patient and to determine each patient's unique clonal IGH sequence. Thirty-seven PBMC samples from 14 patients which were either negative (n=30) by ASO-PCR or detectable below the linear limit of detection (n=7) were subjected to MRD quantification by HTS. The integrity of all samples was determined by preliminary IGH quantitative PCR. Results: CLL-specific IGH clonotypes from all 14 patients amplified successfully from samples with known disease burden, confirming the acceptability of consensus primers for all patients. Concordant MRD negativity by ASO-PCR and IGH-HTS was only observed in 14/37 samples (38%), while 16 samples (43%) were negative by ASO-PCR but detectable at the 10e-6 level using IGH-HTS (range 0.1–11 CLL IGH sequences per 10e6 PBMC genomes). Two of 37 samples (5%) exhibited concordant low-level positivity in the 10e-5 range. 4/37 samples (11%) were concordantly positive, but quantified more than or equal to 0.5log higher with ASO-PCR than HTS. One sample was positive below the linear limit of detection by ASO-PCR but negative by HTS. With median clinical follow-up of 1072 days (range 522–1986 days), one of 7 patients (14%) who exhibited MRD negativity by both ASO-PCR and IGH-HTS relapsed. All 5 patients found to have MRD negativity by ASO-PCR with concurrent MRD positivity using IGH-HTS relapsed. The association between IGH-HTS negativity and long-term DFS was highly significant (p=0.005), whereas ASO-PCR negativity was not significantly associated with DFS (p=0.47). In patients found to be MRD negative by ASO-PCR but positive by IGH-HTS, the HTS result predicted clinical relapse by a median 321 days (range 38–644 days). Conclusions: Massively parallel immunoglobulin gene sequencing using Illumina HiSeq provides a heretofore unachievable level of MRD sensitivity in peripheral blood samples from patients with CLL. Samples found to be negative or below the linear limits of detection for CLL MRD using ASO-PCR were more accurately quantified using IGH-HTS. Quantification of CLL MRD using IGH-HTS has tremendous prognostic value since achievement of MRD negativity with 10e-6 sensitivity is highly associated with long-term DFS. To further validate the performance of Illumina-based HTS, we are currently sequencing 289 archived post-transplant PBMC from 42 CLL patients. This scalable and cost-effective platform for ultra-sensitive MRD quantification using consensus primers will broadly expand the availability and utility of post-transplant MRD assessment. Disclosures: Faham: Sequenta, Inc.: Employment, Equity Ownership. Carlton:Sequenta, Inc.: Employment, Equity Ownership. Zheng:Sequenta, Inc.: Employment, Equity Ownership. Moorhead:Sequenta, Inc.: Employment, Equity Ownership. Willis:Sequenta, Inc.: Employment, Equity Ownership.

Description: The anti-CD20 mAb rituximab has substantially improved the clinical outcome of patients with a wide range of B-cell malignancies. However, many patients relapse or fail to respond to rituximab, and thus there is intense investigation into the development of novel anti-CD20 mAbs with improved therapeutic efficacy. Although Fc-FcγR interactions appear to underlie much of the therapeutic success with rituximab, certain type II anti-CD20 mAbs efficiently induce programmed cell death (PCD), whereas rituximab-like type I anti-CD20 mAbs do not. Here, we show that the humanized, glycoengineered anti-CD20 mAb GA101 and derivatives harboring non-glycoengineered Fc regions are type II mAb that trigger nonapoptotic PCD in a range of B-lymphoma cell lines and primary B-cell malignancies. We demonstrate that GA101-induced cell death is dependent on actin reorganization, can be abrogated by inhibitors of actin polymerization, and is independent of BCL-2 overexpression and caspase activation. GA101-induced PCD is executed by lysosomes which disperse their contents into the cytoplasm and surrounding environment. Taken together, these findings reveal that GA101 is able to potently elicit actin-dependent, lysosomal cell death, which may potentially lead to improved clearance of B-cell malignancies in vivo.

Description: Although sickle cell disease (SCD) has a variable clinical course, many patients develop end-organ complications that are associated with significant morbidity and early mortality. Myeloablative allogeneic HSCT (allo-HSCT) is curative but has been historically performed only in children younger than 16 years of age. Modest modifications in the conditioning regimen and supportive care have improved outcome such that the majority of children with a suitable HLA-matched sibling donor can expect a cure from this approach. However, adult patients have been excluded from myeloablative allo-HSCT because of anticipated excess toxicity resulting from accumulated disease burden. Efforts to use nonmyeloablative transplantation strategies in adults logically followed but were initially met with largely disappointing results. Recent results, however, indicate that nonmyeloablative allo-HSCT in adult patients with SCD allows for stable mixed hematopoietic chimerism with associated full-donor erythroid engraftment and normalization of blood counts, and persistence in some without continued immunosuppression suggests immunologic tolerance. The attainment of tolerance should allow extension of these potentially curative approaches to alternative donor sources. Efforts to build on these experiences should increase the use of allo-HSCT in patients with SCD while minimizing morbidity and mortality.

Description: Abstract 2708 Background: The simplified Mantle Cell Lymphoma International Prognostic Index (MIPI) has been shown to be a good predictor of patient survival (Blood 2008;111:558–65; Blood 2010;115:1530–1533). This post hoc study analyzed data from a randomized, phase III clinical trial investigating temsirolimus (TEM) in relapsed/refractory mantle cell lymphoma (MCL) in which TEM 175/75 (175 mg for first 3 weeks then 75 mg weekly) demonstrated significantly longer progression-free survival (PFS) vs investigator's choice of therapy (INV; 4.8 vs 1.9 months, respectively; hazard ratio [HR]=0.44; P=.0009; J Clin Oncol 2009;27:3822–9). Patients receiving TEM 175/25 (175 mg for first 3 weeks then 25 mg weekly) also had longer PFS vs INV, but this difference was not significant (3.4 vs 1.9 months; respectively; HR=0.65; P=.06). During the trial, baseline prognostic risk classification was not recorded; thus, patients were retrospectively assigned baseline prognostic scores, and outcomes were analyzed according to risk category. Methods: All patients (N=162) were classified as low, intermediate, or high risk using the simplified MIPI. The MIPI scores were based on 4 independent prognostic markers: age, Eastern Cooperative Oncology Group (ECOG) performance status, lactate dehydrogenase level, and white blood cell count. Median PFS and overall survival (OS) were calculated using Kaplan-Meier estimates, and treatment effect was assessed using log-rank statistics. P values of ≤.05 indicated significance of the treatment effect between the 2 treatment groups. As the phase III study was not powered to analyze patients according to MIPI risk categorization, statistical analyses shown are for explanatory purposes. Results: Distribution was relatively even across MIPI risk categories (55 patients low, 59 patients intermediate, 48 patients high). MIPI distributions in the 2 TEM arms were: 175/75 (n=54: 28% low, 43% intermediate, 30% high); 175/25 (n=54: 28% low, 33% intermediate, 39% high). Relative to the TEM arms, the INV arm (n=54) had a higher proportion of low-risk patients (46% low; 33% intermediate; 20% high). TEM 175/75 resulted in significant improvement in median PFS (independent assessment) vs INV in high-risk patients (P=.003) (Table); trends toward improvement were observed for intermediate-risk and low-risk patients (P=.06 in each group). By investigator assessment, TEM 175/75 improved median PFS vs INV by 7.9 months in the low-risk category (P=.0007) and by 2.8 months (P=.06) and 1.1 month (P=.001) in the intermediate-risk and high-risk categories, respectively. A trend toward longer OS was observed in the low-risk patients treated with TEM 175/75 vs INV (P=.0502). In the low-risk category, maintenance of stable disease or better response was achieved in more patients receiving TEM 175/75 (9/15 [60%]) vs INV (5/25 [20%]); objective responses were observed in 5 patients with TEM 175/75 and in no patients with INV. Patients in the low-risk category treated with TEM 175/75 received a longer duration of therapy vs INV (30.7 vs 9.0 wk, respectively). Across the duration of treatment, the average frequency of delay was once per 5.6 wk with TEM 175/75 vs once per 6.4 wk with INV. TEM was generally well tolerated. Grade 3/4 anemia, thrombocytopenia, and infection also were analyzed by patient risk category. In both the TEM 175/75 and INV groups, the selected grade 3/4 events occurred more commonly in high-risk than low-risk patients. In the low-risk category, a higher incidence of grade 3/4 thrombocytopenia and anemia was observed with TEM 175/75 vs INV. Conclusions: Retrospective risk analysis of patients according to the simplified MIPI demonstrated that TEM 175/75 was effective across patient risk categories. The greatest benefit trend was observed in low-risk patients. In this study of relapsed/refractory MCL patients, MIPI was a good predictor of survival outcome. Disclosures: Hess: Pfizer: Consultancy, Honoraria, Research Funding. Off Label Use: Torisel is licensed for treatment of relapsed and/or refractory mantle cell lymphoma and renal cell carcinoma in Europe. Torisel is licensed in the US for renal cell carcinoma. Kang:Pfizer: Employment. Moran:Pfizer: Employment.

Description: A long outstanding problem is the resolution of the full potential of hematopoietic precursors. The commonly used allotypic marker Ly5 permits the tracing of lymphoid and granulocyte-macrophage (GM) output. Here we present a novel eGFP allele that allows the quantitative analysis of red blood cell (RBC) origin at the single-cell level. The miR-144/451 locus is required for erythroid development and homeostasis. Taking advantage of the fact that miR-451 is specifically and highly expressed in the erythroid lineage, we inserted an eGFP expression cassette into the miR-144/451 locus. In miR-144/451+/eGFP animals, accumulation of eGFP is exclusively observed during terminal erythroid differentiation. Expression of miR-144/451eGFP ignites immediately before the CFU-E stage and results in strong and complete labeling of all mature RBCs in circulation. Using competitive reconstitution experiments in the Ly5 transplant model, we show that eGFP linearly correlates with Ly5 expression. Thus, the miR-144/451eGFP allele represents a novel tool for the resolution of erythroid potential.

Description: CD36 modulates platelet function via binding to oxidized LDL (oxLDL), cell-derived microparticles, and thrombospondin-1. We hypothesized that the level of platelet CD36 expression may be associated with inheritance of specific genetic polymorphisms and that this would determine platelet reactivity to oxLDL. Analysis of more than 500 subjects revealed that CD36 expression levels were consistent in individual donors over time but varied widely among donors (200-14 000 molecules per platelet). Platelet aggregometry and flow cytometry in a subset of subjects with various CD36 expression levels revealed a high level of correlation (r2 = 0.87) between platelet activation responses to oxLDL and level of CD36 expression. A genome-wide association study of 374 white subjects from the Cleveland Clinic ASCLOGEN study showed strong associations of single nucleotide polymorphisms in CD36 with platelet surface CD36 expression. Most of these findings were replicated in a smaller subset of 25 black subjects. An innovative gene-based genome-wide scan provided further evidence that single nucleotide polymorphisms in CD36 were strongly associated with CD36 expression. These studies show that CD36 expression on platelets varies widely, correlates with functional responses to oxLDL, and is associated with inheritance of specific CD36 genetic polymorphisms, and suggest that inheritance of specific CD36 polymorphisms could affect thrombotic risk.

Description: A multifaceted immunotherapeutic strategy that includes hematopoietic stem cell (HSC) transplantation, T-cell adoptive transfer, and tumor vaccination can effectively eliminate established neuroblastoma tumors in mice. In vivo depletion of CD4+ T cells in HSC transplantation recipients results in increased antitumor immunity when adoptively transferred T cells are presensitized, but development of T-cell memory is severely compromised. Because increased percentages of regulatory T (Treg) cells are seen in HSC transplantation recipients, here we hypothesized that the inhibitory effect of CD4+ T cells is primarily because of the presence of expanded Treg cells. Remarkably, adoptive transfer of presensitized CD25-depleted T cells increased tumor vaccine efficacy. The enhanced antitumor effect achieved by ex vivo depletion of CD25+ Treg cells was similar to that achieved by in vivo depletion of all CD4+ T cells. Depletion of CD25+ Treg cells resulted in elevated frequencies of tumor-reactive CD8 and CD4+ T cells and increased CD8-to-Treg cell ratios inside tumor masses. All mice given presensitized CD25-depleted T cells survived a tumor rechallenge, indicating the development of long-term CD8+ T-cell memory to tumor antigens. These observations should aid in the future design of immunotherapeutic approaches that promote the generation of both acute and long-term antitumor immunity.

Description: The microvasculature assumes an inflammatory and procoagulant state in a variety of different diseases, including sickle cell disease (SCD), which may contribute to the high incidence of ischemic stroke in these patients. This study provides evidence for accelerated thrombus formation in arterioles and venules in the cerebral vasculature of mice that express hemoglobin-S (βs mice). Enhanced microvascular thrombosis in βs mice was blunted by immunologic or genetic interventions that target tissue factor, endothelial protein C receptor, activated protein C, or thrombin. Platelets from βs mice also exhibited enhanced aggregation velocity after stimulation with thrombin but not ADP. Neutropenia also protected against the enhanced thrombosis response in βs mice. These results indicate that the cerebral microvasculature is rendered vulnerable to thrombus formation in βs mice via a neutrophil-dependent mechanism that is associated with an increased formation of and enhanced platelet sensitivity to thrombin.

Description: Megakaryocytes transfer a diverse and functional transcriptome to platelets during the final stages of thrombopoiesis. In platelets, these transcripts reflect the expression of their corresponding proteins and, in some cases, serve as a template for translation. It is not known, however, if megakaryocytes differentially sort mRNAs into platelets. Given their critical role in vascular remodeling and inflammation, we determined whether megakaryocytes selectively dispense transcripts for matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) into platelets. Next-generation sequencing (RNA-Seq) revealed that megakaryocytes express mRNA for 10 of the 24 human MMP family members. mRNA for all of these MMPs are present in platelets with the exception of MMP-2, 14, and 15. Megakaryocytes and platelets also express mRNA for TIMPs 1-3, but not TIMP-4. mRNA expression patterns predicted the presence and, in most cases, the abundance of each corresponding protein. Nonetheless, exceptions were observed: MMP-2 protein is present in platelets but not its transcript. In contrast, quiescent platelets express TIMP-2 mRNA but only traces of TIMP-2 protein. In response to activating signals, however, platelets synthesize significant amounts of TIMP-2 protein. These results demonstrate that megakaryocytes differentially express mRNAs for MMPs and TIMPs and selectively transfer a subset of these into platelets. Among the platelet messages, TIMP-2 serves as a template for signal-dependent translation.

Description: Abstract 794FN2 LBH589 is a novel pan-deacetylase inhibitor (DACi) that has demonstrated clinical activity in phase I/II studies in patients with a variety of hematologic malignancies. Our group has previously presented preliminary results of a phase I study of LBH589 in patients with myelofibrosis (MF) (Mascarenhas et al, ASH 2009, a308) while a phase II trial using higher doses of LBH589 has also been reported (DeAngelo et al, ASH 2010,a630). Both studies identified reversible thrombocytopenia as the DLT and reported evidence of clinical responses. The final results of our phase I study and the effects of extended treatment with LBH589 are reported here. We enrolled 18 patients at 3 dose levels. Fifty-five percent of these patients had PMF, 28% Post-PV MF and 17% Post ET MF; all were intermediate/high risk based on Lille classification. Twenty-five mg PO TIW was determined to be the recommended phase II dose. All patients experienced resolution of their systemic symptoms and 10/11 patients with baseline palpable splenomegaly, who were evaluable after 1 month of therapy, had a median reduction of 30%, range 0–100%. Five patients entered into an extension phase of the trial and received 〉 6 months of therapy with a mean dose of 20mg PO TIW at time of optimal response (Table 1). Of these patients, 2 were initially enrolled in the 20 mg PO TIW cohort, 1 in the 30 mg PO TIW cohort and 2 in the 25 mg PO TIW cohort. Both patients at the lowest dose achieved clinical improvement (CI) by IWG-MRT response criteria at 6 months as did one patient at the 25 mg dose. The remaining 2 patients had SD at 30 and 25 mg. A mean reduction in palpable splenomegaly at 3 and 6 months of 55% and 83%, respectively, was observed in this group. Two of these patients had marked and durable improvement in anemia (patients 1 and 4). Patient 4 achieved a near CR at 16 months with resolution of palpable splenomegaly, elimination of peripheral blood dacrocytes and leukoerythroblastosis, a 4g/dL increase in hemoglobin, improvement in overall marrow cellularity and megakaryocyte atypia with an increase in erythroid precursors and a significant reduction of reticulin/collagen fibrosis. Patient 1 was heavily transfusion dependent requiring RBC transfusions weekly to maintain a mean hemoglobin of 6.5g/dL and after 6 months on LBH589 achieved 〉50% reduction in transfusion dependence maintaining a mean hemoglobin of 9g/dL. Patient 2 had resolution of palpable splenomegaly and leukoerythroblastosis by cycle 6 and the bone marrow at cycle 26 was characterized by a reduction in marrow fibrosis from grade 4 to 1. A phase II study is ongoing, 14 patients are currently enrolled, with a planned goal of 22 patients. Pharmacokinetic and pharmacodynamic studies as well as cytokine profiling of the phase I patients are being analyzed and will be presented at the meeting. We conclude that low doses of LBH589 delivered for greater than 6 months in patients with MF are capable of ameliorating symptoms, improving clinical features and reversing pathologic marrow changes. Disclosures: Off Label Use: Oral histone deacetylase inhibitor that targets epigenetic changes in malignant myelofibrosis cells with an goal to modify the disease process.

Description: Abstract 1506 BACKGROUND: A wide range of neuropsychological sequelae, noted in long-term survivors of ALL, have been in part ascribed to cranial irradiation (CRT), especially in children who are more vulnerable than adults. We had attempted to abandon prophylactic CRT by intensifying systemic chemotherapy and protracted triple IT (TIT) in ALL-02 trial; whereas one third of patients received CRT for CNS directed therapy in the former study (ALL-97). We present here the results of JACLS ALL-02 trials especially about the effect of the CNS-directed therapy. PATIENTS AND METHODS: Between 2002 and 2008, 1139 children with newly diagnosed non-T ALL with 1–18 years of age were enrolled to JACLS ALL-02 trial. Patients with Ph+ALL and T-ALL were registered to independent protocol. The criteria for diagnosis of CNS disease of the JACLS ALL-02 trial were defined as a CSF pleocytosis of 〉 5 cells /microL and the presence of recognizable blast cells on a well-stained cytospin preparation, or the presence of cranial-nerve palsies at a diagnosis of leukemia. Treatment group was divided into 3 groups according to the BFM-style initial prednisolone (PSL) response and the modified National Cancer Institute (NCI) workshop criteria. PSL good responders were divided into standard risk (SR) and high risk (HR) by WBC 10,000 and age 10. BCP-ALL with PSL poor responders and acute mixed lineage leukemia/ acute unclassified leukemias were treated in extremely high risk (ER). The SR patients with unequivocal CNS involvement (CNS3) at diagnosis were treated as an HR group. The patients in M1 marrow at day 33 with M2/M3 at the day 15 bone marrow (BM) were assigned to shift higher risk after induction therapy. Treatment of ALL-02 consists of early phase and maintenance phase. Early phase includes induction, consolidation, sanctuary (2 courses of high-dose MTX at a dose of 3g /m2), and re-induction therapy for 15 to 26 weeks depending on risk group. Prophylactic CRT was replaced by protracted TIT (MTX, CA, HDC) in all except the patients with CNS3 at diagnosis in ALL-02 trial. The patients with CNS3 at diagnosis received CRT at a dose of 12 Gy. Depending on the risk group, protracted TIT was given during induction, intensification and maintenance in ALL-02 (12 doses in SR and 15 in HR/ER). Accumulative doses of DEX were 120 mg/m2 in SR, whereas 170 mg/m2 in HR and 670 mg/m2 in ER. Treatment duration is 24 months for any risk. The patients assigned ER were candidate for allogeneic stem cell transplantation (SCT) by the end of early phase, provided HLA-matched siblings were available. The probability of event-free survival (EFS) and overall survival were constructed using the Kaplan-Meier method. Events in the analysis of EFS included induction failure, death, relapse and secondary malignant neoplasm. All statistical analyses were done according to intent-to treat methods. RESULTS: The numbers of patients at each risk in ALL-02 were 457 (40%) for SR, 543 (48%) for HR, and 139 (12%) for ER. The number of patients with CNS3 at diagnosis was 31 (2.7%). Of 1139 patients, 16 patients (1.4%) had CNS relapses of the leukemia (an isolated CNS relapse: 10 pts, a combined CNS and BM relapse: 6 pts). The 5-year EFS rate ALL-02 was 83.7% (SE=1.1), slightly better than for ALL-97 (79.3%, SE=1.7, p =0.054). The 5-year cumulative risk of an isolated and total CNS relapse was lower in ALL-02 than in ALL-97 (isolated CNS relapse: 0.9% vs 2.7%, p=0.017, total CNS relapse: 4.5% vs 1.4%, p =0.01). The proportion of CRT and SCT were 2%, 8% in ALL-02 respectively, whereas 31%, 11% in ALL-97 respectively. CONCLUSIONS: The ALL-02 trial found that CRT was abolished successfully, in all except those with CNS3 at diagnosis, with substitution of more intensive systemic chemotherapy and protracted TIT. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 255 Introduction: Standard treatment of acute myeloid leukemia (AML) comprises one or two cycles of chemotherapy to induce complete remission (CR) followed by postremission treatment in order to prevent relapse of the disease (consolidation therapy). In 2003, we initiated a prospective multicenter randomized trial to investigate the impact of different consolidation strategies on long-term outcome in AML patients ≤ 60 years. Consolidation options comprised upfront allogeneic stem cell transplantation (allo SCT) in aplasia after induction therapy, autologous SCT, and three cycles of standard high-dose-cytarabine-based consolidation. For patients receiving high-dose cytarabine, the main study aim was to evaluate the benefit of adding additional mitoxantrone and amsacrine to cytarabine consolidation. Design: From 2003 to 2009, 1182 patients (median age, 48 years; range 16–60 years) with untreated AML were randomly assigned at diagnosis to different consolidation strategies after classical 7+3 induction. According to the risk-adapted treatment strategy of the trial, cytogenetically or molecular intermediate-risk (IR) and adverse-risk (AR) patients should receive an allo SCT as consolidation treatment if an HLA-identical-sibling donor (IR) or HLA-matched related or unrelated donor (AR) was available. IR and AR patients with no available donor should receive autologous SCT. All favorable risk patients and patients with no available donor were scheduled for high-dose cytarabine based consolidation. Half of the patients were randomized for high dose cytarabine based consolidation. Half of the patients were randomized for high dose cytarabine alone while the other half received high dose cytarabine with the addition of amsacrine and mitoxantrone. Standard chemotherapy consisted of three cycles with high dose cytarabine (2 × 3 g/m2, day 1,3,5) whereas combined consolidation contained two cycles of MAC (cytarabine 2 × 1g/m2, day 1–6, mitoxantrone 10 mg/m2, day 4–6) plus one cycle of MAMAC (cytarabine 2 × 1 g/m2, day 1–5, amsacrine 100 mg/m2, day 1–5). In order to evaluate the effect of the two cytarabine based consolidation strategies, we determined overall survival (OS) and event free survival (EFS) using the method of Kaplan Meyer. Survival distributions were compared using the log rank test. Results: 1182 patients were randomized for further intervention (Arm A+B: n=582, 49.3%; Arm C+D: n=600, 50.7 %). Median follow-up was 41.4 months (95%-CI 39.3–43.6). A total number of 375 patients received allogeneic (n=322) or autologous SCT (n=53) and 807 patients were consolidated with cytarabine. Of these patients, 407 were randomized for cytarabine alone and 400were randomized to receive cytarabine plus mitoxantrone and amsacrine (MAC/MAC/MAMAC). Complete remission rate (CR) after second induction therapy was 59.1% (n=698). Between the four arms, there were no significant differences of the CR rates. Five-year OS of patients receiving high dose cytarabine alone was 47.1% (95%-CI 42.0–52.2%), for patients receiving MAC/MAMAC as consolidation therapy it was 46.8% (95%-CI 42.3–51.3%; p = 0.610). Three-year event free survival (EFS) was also not significant with 30.5% (95%-CI 26.6–34.4%) for patients receiving high dose cytarabine alone and 35.6% (95%-CI 31.7–39.5%; p = 0.059) for patients receiving MAC/MAMAC. Conclusions: According to our data, the addition of mitoxantrone and amsacrine to high dose cytarabine consolidation confers no benefit for treatment outcome in younger AML patients. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1122 The survival of replicating B cells, with DNA damage arising from oxidative stress and/or activation-induced cytosine deaminase (AID), appears in part to be under p53 control. Importantly, a common C〉G single nucleotide polymorphism (SNP) within codon 72 of p53 influences p53 function. Among other differences, p53-72R (CGC=Arginine) is notably more effective than p53-72P (CCC=Proline) at inducing apoptosis. The SNP has been linked to clinical outcome in multiple settings, including malignancy. Most individuals in the US population display heterozygosity. In this study, we have examined whether B cells, whose genomic DNA is heterozygous for the codon 72 SNP, exhibit allelic exclusion at the level of expressed RNA. This was suggested by reported evidence that p53 expression is strongly regulated by gene methylation status; mRNA of peripheral blood cells from p53-72P/R heterozygous individuals is skewed toward the representation of only one SNP, depending upon ethnic status; and a p53 intron 2 SNP, representing a potential methylation site, is in strong linkage disequilibrium with the codon 72 SNP (PLoS ONE 6:e15320, 2011). Evidence for allelic exclusion of this functionally relevant p53 SNP would suggest that not all identically-stimulated B cells have equal likelihoods of survival within p53-72P/R heterozygous individuals. To investigate this issue, we first confirmed that p53 was expressed in non-transformed human B cells replicating in response to surrogate C3d-bound antigen, IL4 and BAFF. These physiologically-relevant stimuli synergize to induce a burst of T cell-independent clonal expansion, followed by apoptosis of many of the divided progeny (J. Immunol. 175:6143, 2005). Expression of p53 was monitored by immunoblotting and flow cytometry. Consistent with a role in regulating clonal burst size, p53 protein and mRNA/protein of p53-regulated pro-apoptotic genes were significantly elevated in blasts, prior to apoptosis. This contrasted with undectable p53 protein in non-stimulated B cells. To assess whether expressed p53 within a single lymphoblast derives from one allele, i.e. demonstrates allelic exclusion, we first identified tonsil donors heterozygous for the codon 72 polymorphism. This involved PCR-restriction fragment length polymorphism (RFLP) analysis of genomic DNA, as described by others (Leukemia Research 30:1113, 2006). Subsequently, purified resting B2 cells from cryopreserved tonsil cell suspensions, determined to be heterozygous for p53-72P/R, were labeled with CFSE and stimulated as above. At d5, single B lymphoblasts were sorted into 96 well PCR plates containing lysis buffer and cDNA prepared using random hexamer primers. A p53 sequence comprising exons 2a, 3, and 4 was subjected to two rounds of PCR amplification with the following primers, Forward: 5-cagccagactgccttccg-3 & Reverse: 5-gcaagtcacagacttggctg-3. Nucleotide sequencing of PCR-amplified p53 was performed commercially (Applied Biosystems Big Dye Terminator v3.1 cycle sequencing) and analyzed by chromasPro software. In two experiments, only 39 cells of a total of 236 assayed were positive for p53. By contrast, β-actin cDNA from two rounds of PCR amplification yielded 88% of the single cell-containing wells positive for actin. These yields indicate that p53 mRNA levels within a single cell are significantly more limiting than those for actin and are consistent with quantitative PCR of cDNA obtained from activated cell pools. Interestingly, analysis of the cDNA p53 chromatogram sequence of the p53-positive single cells (n=39), whose genomic DNA was heterozygous for the p53 codon 72 SNP, showed only a single homogenous sequence: either P (n=21) or R (n=18), but never both. This was in marked contrast to the chromatogram of cDNA derived from a pool of activated B cells within each experiment. In the latter cases, two overlapping peaks indicating co-expression of p72-P and p72-R were noted. Taken together, our findings suggest that p53 mRNA expression from a single non-transformed human B lymphocyte arises from the transcriptional activation of a single allele, i.e. shows allelic exclusion. Although the mechanism for this phenomenon requires further investigation, these results imply that B cells within individuals heterozygous for the functionally important p53-p72 polymorphism might vary considerably in their resistance to apoptosis. Disclosures: No relevant conflicts of interest to declare.

Description: HSCs are rare cells that have the unique ability to self-renew and differentiate into cells of all hematopoietic lineages. The lack of donors and current inability to rapidly and efficiently expand HSCs are roadblocks in the development of successful cell therapies. Thus, the challenge of ex vivo human HSC expansion remains a fertile and critically important area of investigation. Here, we show that either SALL4A- or SALL4B-transduced human HSCs obtained from the mobilized peripheral blood are capable of rapid and efficient expansion ex vivo by 〉10 000-fold for both CD34+/CD38− and CD34+/CD38+ cells in the presence of appropriate cytokines. We found that these cells retained hematopoietic precursor cell immunophenotypes and morphology as well as normal in vitro or vivo potential for differentiation. The SALL4-mediated expansion was associated with enhanced stem cell engraftment and long-term repopulation capacity in vivo. Also, we demonstrated that constitutive expression of SALL4 inhibited granulocytic differentiation and permitted expansion of undifferentiated cells in 32D myeloid progenitors. Furthermore, a TAT-SALL4B fusion rapidly expanded CD34+ cells, and it is thus feasible to translate this study into the clinical setting. Our findings provide a new avenue for investigating mechanisms of stem cell self-renewal and achieving clinically significant expansion of human HSCs.

Description: Abstract 2873 International staging system (ISS) and cytogenetics are the main prognostic factors of Multiple Myeloma (MM) but they reflect biologic characteristics of disease without taking into account individual host features. On the contrary, clinical characteristics of single patient could be substantial as to various points of view. For instance, in elderly MM patients, novel therapies reduction or interruption due to toxicity represent the major cause of unsatisfactory outcome. Therefore, it was empirically suggested different schedule of drugs in these “frail” patients but, how the “frailty” should be assessed in every single patient, is still unsettled. Advanced age, poor performance status (PS) and comorbidities are usually applied to recognize the “frailty” but it is not well known which of them are really prominent and whether these parameters, adjusted for conventional prognostic factors, still affect final outcome. We analyzed a population of symptomatic MM diagnosed from 2007 to 2010 included in the Marche Region MM Registry, to assess the frequency of “frailty” features, such as age, PS, comorbidities, cytopenias, renal insufficiency (RI) and lytic bone lesions, and their role on the overall survival (OS) when adjusted for prognostic factors. Comorbidities were scored according to Charlson Comorbidity Index (CCI) that split patients in 4 categories according to number and type of comorbidity. Patients were treated with transplant or standard therapy according to their eligibility. Overall, 88% of patients were treated with new drug-based therapies and 12% with MP. Median age of the 266 patients analyzed was 73 years (range 38–90). Twenty-four percent of patients had IgA MM, fifty patients (23%) had ISS stage=3 and 29/166 (17.5%) had unfavourable cytogenetics. Regarding “frailty” measures, 38% of patients had 〉 75 years, 39% had PS=2–4, 34% had 1 or more comorbidities. The most frequent comorbidities were hypertension (35%), heart diseases (22%), diabetes (15%), neurological diseases (16%), COBP (8%), secondary malignancies (8%) and chronic renal failure (6%). CCI ≥1 was detected in 51%. Increasing comorbitities number and CCI were associated with increased age although 37% of patients aged less than 65 years had CCI ≥2. Moreover, 35% had at least 2 cytopenias, 76% had bone disease and 14% had RI. Fifty patients (19%) died during follow-up. OS at 3 years was 74%. Univariate analysis performed on the total population determined age 〉 65 years (p=0.065), PS=2–4 (p

Description: Xanthomas are a common manifestation of lipid metabolism disorders. They include hyperlipemic xanthoma, normolipemic xanthoma, and a related condition, necrobiotic xanthogranuloma (NXG). All 3 forms can be associated with monoclonal immunoglobulin (MIg). In an attempt to improve diagnosis, understanding, and treatment of this association, we retrospectively analyzed a personal series of 24 patients (2 hyperlipemic xanthoma, 11 normolipemic xanthoma, and 11 NXG) and 230 well-documented reports from the literature. With the exception of the nodules and plaques featured in NXG, the clinical presentation of xanthomatous lesions usually resembled that seen in common hyperlipidemic forms and could not be used to suspect MIg-associated xanthomas. Extracutaneous sites were not rare. The MIg was an IgG in 80% of cases. Myeloma was diagnosed in 35%. Hypocomplementemia with low C4 fraction was present in 80% of studied patients. Low C1 inhibitor serum levels were found in 53%. Cryoglobulinemia was detected in 27%. These abnormalities suggest immune complex formation because of interactions between the MIg and lipoproteins and argue in favor of a causal link between MIg and xanthomas. Monoclonal gammopathy therapy could thus be an option. Indeed, among the patients who received chemotherapy, hematologic remission was accompanied by improvement in xanthoma lesions in several cases.

Description: Abstract 223 A coding variant form of GFI1 (GFI136N) increases the risk to develop AML by 60% and is present in about 10–15 % of all Caucasian AML patients. To determine the underlying molecular mechanism and potentially develop new therapeutic approaches, we generated “knockin” mouse strains wherein the endogenous murine Gfi1 gene was replaced either by the human GFI1 variant (GFI136N, the form predisposing to AML) or by the more common form of GFI1 (GFI136S). In most hematopoietic compartments no difference was observable between GFI136N and GFI136S expressing mice; however, there was a 3–5 fold increase in the number of granulocytic monocytic progenitors (GMPs) and common myeloid progenitors (CMPs) in Gfi136N expressing (either homozygous or heterozygous) mice compared to wild-type or Gfi136S expressing mice(p≤0.01). Interestingly, both human and murine AML leukemic cells are thought to originate from GMPs and CMPs. To assess functional differences, we seeded GMPs from GFI136N or GFI136S knockin mice on methylcelluose or transplanted them into into syngenic animals. We found that GFI136N expressing GMPs proliferate faster and have an increased self-renewal capacity both in-vitro and in-vivo compared to GMPs carrying Gfi136S alleles. A gene expression array analysis showed that GFI136N GMPs have a stem cell-like gene signature with elevated levels of Hoxa9 expression and a deregulation of a number of oncogenes involved in the development of human AML such as Trib2, Tet2 or Idh2. It is of particular interest that Hoxa9, a known GFI1 target gene, was up-regulated 3–4 fold in GFI136N GMPs compared to in GFI136S GMPs (p≤0.01). It is known that high levels of Hoxa9 accelerate AML development in mice and are associated with a poor prognosis in AML patients. GFI1 is a transcriptional repressor and exerts its function by recruiting different histone modifying enzymes, in particular LSD1, which de-methylates histone 3 (H3) at lysine 4 (K4), or histone deacetylases (HDACs), which remove acetyl groups from H3K9 residues and G9a, which initiates dimethylation of H3K9. Both H3K4 methylation and H3K9 acetylation correlate with actived gene expression, whereas H3K9dimethyl correlates with repession. Chromatin-immuno-precipitation (ChIP) of Gfi1-bound chromatin from Lin−Sca1−c-Kit+ cells, which contains the GMP population, showed that GFI136N binds to a lesser degree to the Hoxa9 locus than GFI136S. This diminished binding of Gfi136N correlated with an increased H3K4 dimethylation and H3K9 acetylation as well as diminished H3K9 dimethylation across the Hoxa9 locus in GFI136N cells. It is likely that these epigenetic changes lead to the increased Hoxa9 expression observed in GFI136N GMPs. A more exhaustive ChIP-Seq analysis with antibodies recognizing H3K4dimethyl in Lin−Sca1−c-Kit+ cells from Gfi136N or Gfi136S mice showed significant epigenetic alterations throughout the Hoxa9 locus genome and at other GFI1 target genes. It is conceivable that these epigenetic alterations explain, at least in part, the changed gene expression signatures in GFI136N GMPs. To investigate the role of GFI136N in myeloid leukemogenesis, we induced the expression of a mutated form of KRAS (K12D) in both GFI136N and GFI136S mice. All mice developed a deadly myelo-proliferative disorder, but animals carrying the GFI136N allele succumbed to the disease within a significantly shorter latency period (17 against 31 days, p≤0.01) than GFI136S mice. We also transduced GFI136N and GFI136S GMPs with retroviral vectors directing the expression of either the AML1-Eto9a or the MLL-AF9 onco-fusion proteins typically found in human AML. We observed that GFI136N GMPs expressing MLL-AF9 or AML1-Eto9a generated 5–10 fold more colonies (p≤0.01) on methylcellulose and exhibited a higher replating efficiency than the respective GFI136S GMPs. Finally, AML blast cells from GFI136N heterozygous patients expressed higher levels of HOXA9 compared to AML blasts from GFI136S homozygous patients, suggesting that our mouse model reflects the disease predisposition in human patients. Our knockin mice are, to our knowledge, the first animal model for a human genetic variation that predisposes to leukemia. Based on the findings with this model, we propose that the human GFI136N variant predisposes to AML by inducing epigenetic changes affecting the expression of important regulators with oncogenic potential such as Hoxa9. Disclosures: No relevant conflicts of interest to declare.

Description: We prove that the SH2-containing tyrosine phosphatase 1 (SHP-1) plays a prominent role as resistance determinant of imatinib (IMA) treatment response in chronic myelogenous leukemia cell lines (sensitive/KCL22-S and resistant/KCL22-R). Indeed, SHP-1 expression is significantly lower in resistant than in sensitive cell line, in which coimmunoprecipitation analysis shows the interaction between SHP-1 and a second tyrosine phosphatase SHP-2, a positive regulator of RAS/MAPK pathway. In KCL22-R SHP-1 ectopic expression restores both SHP-1/SHP-2 interaction and IMA responsiveness; it also decreases SHP-2 activity after IMA treatment. Consistently, SHP-2 knocking-down in KCL22-R reduces either STAT3 activation or cell viability after IMA exposure. Therefore, our data suggest that SHP-1 plays an important role in BCR-ABL–independent IMA resistance modulating the activation signals that SHP-2 receives from both BCR/ABL and membrane receptor tyrosine kinases. The role of SHP-1 as a determinant of IMA sensitivity has been further confirmed in 60 consecutive untreated patients with chronic myelogenous leukemia, whose SHP-1 mRNA levels were significantly lower in case of IMA treatment failure (P 〈 .0001). In conclusion, we suggest that SHP-1 could be a new biologic indicator at baseline of IMA sensitivity in patients with chronic myelogenous leukemia.

Description: Abstract 3986 Background: Multiple Myeloma (MM) is an incurable plasma cell neoplasm. Although current lenalidomide (R) and bortezomib containing up-front regimens can now achieve overall response rates approaching 100%, patients eventually relapse with progressively refractory disease. Histone deacetylase inhibitors (HDACi), in Phase I clinical trials in patients with multiple myeloma, have shown promising activity when combined with other agents such as bortezomib. Vorinostat, (suberoylanilide hydroxamic acid; SAHA) is an oral HDACi, currently FDA approved in the United States for the treatment of cutaneous T-cell lymphoma. Here we report the findings of the combination of vorinostat (Zolinza®), lenalidomide and dexamethasone (ZRD) in multiple myeloma patients who were refractory to RD. Methods: Patients received oral vorinostat 300 mg or 400 mg once daily (days 1–7 and days 15–21), lenalidomide 10–25 mg (days 1–21) and dexamethasone 20–40 mg weekly (days 1, 8, 15, 22) in a 28-day cycle Subjects: Twenty-nine patients were treated and all were refractory to RD; 76% were refractory to at least one bortezomib containing regimen and 48% were refractory to the combination of VRD. Twenty-six patients (90%) had undergone prior high dose therapy with autologous stem cell transplant. The median number of prior therapies was 4 (range 2–13). Results: The overall response rate (ORR) was 24 % with 1 VGPR and 6 PR. The clinical benefit rate (ORR + MR) was 51% including 8 MR. Nine patients (31%) had stable disease. The median duration of response (DOR) was 4 months (range, 0–36). The median overall survival (OS) was 11 months (range, 4–36). Common toxicities including diarrhea and fatigue (all grades) were 41% and 34% respectively. The incidence of grade 3/4 neutropenia was 45 % and grade 3/4 thrombocytopenia was 34%. Conclusion: The combination of ZRD showed significant activity in patients with RD relapsed/refractory multiple myeloma. ZRD was well tolerated and is a viable option for patients who do not respond to lenalidomide-based therapy. Further, since all 3 agents are available in oral formulations, ZRD provides an additional option for those patients wishing to avoid intravenous therapy. Formal phase II studies of this combination are in preparation. Disclosures: Off Label Use: Vorinostat is an oral HDAC inhibitor and is being evaluated in the treatment of Multiple Myeloma. Bilotti:Celgene: Consultancy, Speakers Bureau; Millenium: Consultancy, Speakers Bureau. McNeill:Celgene: Consultancy, Speakers Bureau; Millenium: Consultancy, Speakers Bureau. Graef:Merck: Employment. Vesole:Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millennium: Speakers Bureau. Siegel:Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Millenium: Consultancy, Honoraria, Research Funding, Speakers Bureau; Merck: Consultancy.

Description: We performed nonmyeloablative HSCT in 6 patients with a newly described genetic immunodeficiency syndrome caused by mutations in GATA2—a disease characterized by nontuberculous mycobacterial infection, monocytopenia, B- and NK-cell deficiency, and the propensity to transform to myelodysplastic syndrome/acute myelogenous leukemia. Two patients received peripheral blood stem cells (PBSCs) from matched-related donors, 2 received PBSCs from matched-unrelated donors, and 2 received stem cells from umbilical cord blood (UCB) donors. Recipients of matched-related and -unrelated donors received fludarabine and 200 cGy of total body irradiation (TBI); UCB recipients received cyclophosphamide in addition to fludarabine and TBI as conditioning. All patients received tacrolimus and sirolimus posttransplantation. Five patients were alive at a median follow-up of 17.4 months (range, 10-25). All patients achieved high levels of donor engraftment in the hematopoietic compartments that were deficient pretransplantation. Adverse events consisted of delayed engraftment in the recipient of a single UCB, GVHD in 4 patients, and immune-mediated pancytopenia and nephrotic syndrome in the recipient of a double UCB transplantation. Nonmyeloablative HSCT in GATA2 deficiency results in reconstitution of the severely deficient monocyte, B-cell, and NK-cell populations and reversal of the clinical phenotype. Registered at www.clinicaltrials.gov as NCT00923364.

Description: In multiple myeloma (MM) pathogenesis, hyperdiploidy and nonhyperdiploidy are recognized as 2 major cytogenetic pathways. Here, we assessed the role of hyperdiploidy in 426 patients with monoclonal plasma cell disorders, among them 246 patients with AL amyloidosis (AL), by interphase fluorescence in situ hybridization. Hyperdiploidy was defined by a well-established score requiring trisomies for at least 2 of the 3 chromosomes 5, 9, and 15. The hyperdiploidy frequency in AL was a mere 11% compared with 30% in monoclonal gammopathy of undetermined significance (P 〈 .001) and 46% in AL with concomitant MM I (P 〈 .001). Overall, hyperdiploidy was associated with an intact immunoglobulin, κ light chain restriction, higher age, and bone marrow plasmacytosis, but was unrelated to the organ involvement pattern in AL. Clustering of 6 major cytogenetic aberrations in AL by an oncogenetic tree model showed that hyperdiploidy and t(11;14) were almost mutually exclusive, whereas gain of 1q21 favored hyperdiploidy. Deletion 13q14 and secondary IgH translocations were equally distributed between ploidy groups. We conclude that the interphase fluorescence in situ hybridization–based hyperdiploidy score is also a feasible tool to delineate hyperdiploid patients in early-stage monoclonal gammopathies and that the cytogenetic pathogenetic concepts developed in MM are transferable to AL.

Description: Abstract 98 Background: Advanced follicular lymphomas (FL) are incurable with conventional chemotherapy and there is no consensus on the best treatment approach. The SWOG cancer research cooperative group and Cancer and Leukemia Group B (CALGB) compared the safety and efficacy of two immunochemotherapy regimens for FL in a Phase III randomized intergroup protocol (S0016) that enrolled 554 patients with previously untreated, advanced stage FL between 3/1/2001 and 9/15/2008. Methods: Pts were eligible if they had advanced stage (bulky stage II, III or IV) evaluable FL of any grade (1, 2, or 3) and had not received any prior therapy. In one arm (CHOP-R), patients received 6 cycles of CHOP chemotherapy (750 mg/m2 cyclophosphamide, 50 mg/m2 doxorubicin, 1.4 mg/m2 vincristine, and 100 mg prednisone daily for 5 days) at 3 week intervals with 6 doses of rituximab anti-CD20 antibody (375 mg/m2 on days 1, 6, 48, 90, 134 and 141 according to the schedule described by Czuczman et al.[J.Clin.Oncol 17:268, 1999]). In the second arm of the protocol, patients received 6 cycles of CHOP, followed by a dosimetric infusion of tositumomab/iodine I-131 tositumomab and then 1–2 weeks later a therapeutic infusion of I-131-tositumomab labeled with sufficient I-131 (median 85 mCi) to deliver a total body dose of 75 cGy (CHOP-RIT). The study was designed to have 86% power to detect a hazard ratio (HR) of CHOP-RIT to CHOP-R of 0.67 for 2 yr PFS based on a one-sided.021 level test (accounting for 3 interim analyses). Results: Of the 554 enrolled pts, 532 were eligible and 526 were evaluable for toxicity (263 on each arm of the protocol). Pt characteristics (age, sex, race, stage, beta 2 microglobulin level, tumor bulk, B symptoms) were well-balanced in the two arms of the protocol. In general, both regimens were well-tolerated (Table I). Median follow-up time among patients still alive is 4.9 years. One hundred and six of 267 eligible pts on the CHOP-R arm have progressed or died compared to 86 of 265 eligible pts on the CHOP-RIT arm. The 2 year estimate of PFS was 76% on the CHOP-R arm and 80% on the CHOP-RIT arm (Figure 1). In multivariate Cox regression adjusting for the stratification factor (serum beta-2 microglobulin level), the hazard ratio for CHOP-RIT vs. CHOP-R was 0.79 (95% CI: 0.60–1.05, p=.11 [2-sided] or p=.06 [1-sided]). Twenty-six of 267 pts on the CHOP-R arm have died compared to 40 of 265 eligible pts on the CHOP-RIT arm. The 2-year estimate of overall survival was 97% on the CHOP-R arm and 93% on the CHOP-RIT arm. In multivariate Cox regression adjusting for the stratification factor serum beta-2 microglobulin, the hazard ratio for CHOP-RIT vs. CHOP-R was 1.55 (95% CI: 0.95–2.54, p=.08 [2-sided]). Conclusion: No statistically significant differences in PFS, OS, or serious toxicities are yet demonstrable with either regimen administered in this trial. However, PFS and OS are outstanding with either of the two regimens with median times to progression not yet reached for either treatment. Future studies will be needed to assess whether combining CHOP-R with RIT consolidation and with maintenance rituximab will confer additive benefit, as being evaluated in a follow-up trial (SWOG protocol S0801) that has recently completed accrual. Disclosures: Press: Spectrum: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria, Research Funding. Off Label Use: Front-line use of I-131-tositumomab for consolidation therapy in 1st remission of follicular lymphoma. Friedberg:Genentech: Consultancy, Honoraria. Czuczman:Glaxo Smith Kline: Consultancy, Research Funding; Genentech: Consultancy, Honoraria. Kaminski:Glaxo Smith Kline: Patents & Royalties. Maloney:Genentech/Roche: Consultancy, Honoraria, Speakers Bureau; Glaxo Smith Kline: Consultancy, Honoraria, Speakers Bureau. Cheson:Glaxo Smith Kline: Research Funding. Fisher:Roche: Consultancy, Honoraria.

Description: Abstract 1131 Humanin (HN), a 24-amino acid endogenous antiapoptotic peptide, was initially shown to protect against neuronal cell death by Alzheimer's disease-related insults. It has recently been found that an exogenous analog of HN (HNG) in which the 14th amino acid serine is replaced with glycine protected against cerebral and cardiac ischemia reperfusion (I/R) injury in cortical neurons and cardiomyocytes, respectively. Platelet activation and thrombus formation has been shown to play an important role during I/R injury by exacerbating the extent of the infarct size. However, it is presently unknown whether HNG affects platelet function and the subsequent arterial thrombus formation. We thus examined whether HNG affects platelet activation and thrombus formation both in vitro and in vivo. Human platelets were isolated from healthy adults. Preincubation of washed human platelets with HNG (4μM) reduced collagen- or convulxin-induced platelet aggregation by 56.8% (P

Description: Chronic GVHD is one of the most severe complications of allogeneic HSCT. The sclerotic skin manifestations of cGVHD (ScGVHD) result from inflammation and fibrosis of the dermis, subcutaneous tissue, or fascia, leading to significant functional disability. Risk factors and clinical markers associated with ScGVHD remain largely unexamined. By using a single-visit, cross-sectional design, we evaluated 206 patients with cGVHD at the National Institutes of Health. Most patients manifested severe (ie, 63% National Institutes of Health score “severe”), refractory disease (median treatments = 4). ScGVHD was detected in 109 (52.9%) patients. ScGVHD was associated with greater platelet count (P 〈 .001) and C3 (P 〈 .001), and decreased forced vital capacity (P = .013). Total body irradiation (TBI) was associated with development of ScGVHD (P = .002). TBI administered in reduced-intensity conditioning was most strongly associated with ScGVHD (14/15 patients, P 〈 .0001). Patients with ScGVHD had significant impairments of joint range of motion and grip strength (P 〈 .001). Greater body surface area involvement was associated with poorer survival (P = .015). We conclude that TBI, particularly in reduced-intensity regimens, may be an important risk factor for ScGVHD. Widespread skin involvement is associated with significant functional impairment, distressing symptoms, and diminished survival. This trial is registered at http://www.clinicaltrials.gov as NCT00331968.

Description: Abstract 1704 Background: Lenalidomide (LEN) is approved in the US for the treatment of RBC transfusion-dependent patients with IPSS Low- or Int-1-risk myelodysplastic syndromes (MDS) with del(5q), with or without other cytogenetic abnormalities. In a phase 3 trial, treatment with LEN 5 mg and 10 mg resulted in RBC transfusion independence (TI) for ≥ 26 weeks in 43% and 56% of such patients, cytogenetic response in 25% and 50%, and a significant improvement of health-related quality of life (p

Description: Abstract 1723 Background: Two multicenter studies (MDS-003/-004) found LEN leads to RBC transfusion independence (TI) in 〉 50% of pts with RBC transfusion dependent Low-/Int-1-risk MDS with del5q (List A et al. NEJM 2006;355: 1456–65; Fenaux P et al. Blood 2011;doi: 10.1182/blood-2011-01-330126). RBC-TI ≥ 8 wks with LEN was associated with significantly reduced risk of AML progression and death (Fenaux P et al. Blood 2011;doi: 10.1182/blood-2011-01-330126). Alternative therapy is required for pts failing LEN therapy. Aims: To assess predictive factors of LEN response and long term outcomes (especially after primary or secondary LEN failure) of pts 〈 65 yrs included in MDS-003/-004; ie, those in whom intensive therapies including allogeneic stem cell transplantation (ASCT) may be considered. Methods: LEN was administered as follows (all 28-d cycles): 5 mg/d on d 1–28 and 10 mg/d on d 1–21 or 1–28. RBC-TI ≥ 26 wks and cytogenetic response (CyR; IWG 2000) are reported. Overall survival (OS) and AML progression were assessed using Kaplan-Meier method. Response rates and outcomes in pts 〈 65 yrs were retrospectively compared with pts ≥ 65 yrs. Primary failure was defined as lack of RBC-TI with LEN treatment and secondary failure as relapse after achievement of RBC-TI ≥ 26 wks. Cox proportional hazards models were used to evaluate the effect of potential risk factors (ie, age, sex, time since diagnosis, FAB classification, LEN dose, IPSS risk, WPSS risk, cytogenetics, bone marrow blast %, transfusion burden, no. of cytopenias, hemoglobin level, platelet and neutrophil counts, RBC-TI ≥ 26 wks [time-dependent variable] and CyR [categorical variable]) on OS and AML progression. Logistic model was used to evaluate the effect of potential risk factors on achievement of RBC-TI ≥ 26 wks. Results: The trials included 97 (33.9%) pts 〈 65 yrs. Of these, 73.2% were female; 20.6% were IPSS Low-, 52.6% Int-1-, and 4.1% Int-2-risk; 30.9% had del5q with ≥ 1 additional cytogenetic abnormality (8.2% had complex cytogenetics). At baseline (BL), median time since diagnosis was 2.4 yrs (range 0.2–20.7) and median RBC transfusion requirement was 6 units/8 wks (range 1–15). In pts ≥ 65 yrs (n = 189) most BL characteristics were similar except IPSS risk, which was lower (36.5% Low-, 37.0% Int-1-, 5.8% Int-2-risk; p =.012). RBC-TI ≥ 26 wks was achieved by 54 (55.7%) pts 〈 65 yrs (vs 49.7% pts ≥ 65 yrs; p =.563). The median duration of RBC-TI ≥ 26 wks in responders was not estimable in pts 〈 65 yrs or ≥ 65 yrs (log-rank p =.879). None of the potential risk factors assessed was a significant predictor of RBC-TI ≥ 26 wks in pts 〈 65 yrs, possibly due to small pt number. In pts 〈 65 yrs with available follow-up cytogenetics (n = 71), CyR was achieved by 32 (45.1%) pts (vs 64.5% pts ≥ 65 yrs; p =.014). At time of data cutoff, 51 (52.6%) pts 〈 65 yrs were alive (vs 36.0% pts ≥ 65 yrs; p =.008); 29 (29.9%) pts progressed to AML (vs 20.1% pts ≥ 65 yrs; p =.077). The 1-, 2-, and 3-yr AML-progression rates were 9.7%, 15.4%, and 24.0% in pts 〈 65 yrs; and 6.0%, 17.9%, and 22.3% in pts ≥ 65 yrs (log-rank p =.308). The 1-, 2-, and 3-yr OS rates were 91.7%, 78.1%, and 66.4% in pts 〈 65 yrs; and 83.1%, 65.2%, and 49.9% in pts ≥ 65 yrs (log-rank p

Description: The “in situ” lymphomas are often incidental findings in an otherwise reactive-appearing lymph node. Notably, the risk of progression to clinically appreciable lymphoma is not yet fully known. The diagnosis of “in situ” lymphoma is feasible when immunohistochemical characterization is carried out and genetic abnormalities are assessed. “In situ” follicular lymphoma is characterized by the presence within the affected germinal centers of B cells that strongly express BCL2 protein, a finding that supports their neoplastic nature, in the absence of interfollicular infiltration. In “in situ” mantle cell lymphoma, the lymphoma involvement is typically limited to the inner mantle zone, where lymphoma cells are cyclin D1+ and weakly BCL2+, CD5+. A staging workup to exclude other site of involvement is highly recommended for the possible coexistence of an overt lymphoma. Biopsy of all sites of suspicious involvement should be mandatory. No evidence for starting therapy also in the presence of multifocal “in situ” lymphoma exists, and a “wait-and-see policy” is strongly suggested. A follow-up strategy reserving imaging evaluation only in the presence of disease-related symptoms or organ involvement appears to be a reasonable option. For patients with concomitant overt lymphoma, staging and treatment procedures must be done according to malignant counterpart.

Description: Abstract 382FN2 VWF is a multimeric plasma sialoglycoprotein essential for normal haemostasis. Although the biosynthesis, structure and functional properties of VWF have been well characterized, the molecular mechanism(s) underlying its clearance remain poorly understood. Nevertheless, enhanced VWF clearance is important in the pathophysiology of VWD. Moreover, emerging data suggest that variation in VWF glycosylation (notably ABO blood group) may constitute an important regulator of in vivo clearance rates. To define the role of VWF glycans in modulating clearance, VWF was purified from human plasma (pdVWF) by cryoprecipitation and gel filtration. Subsequently, VWF glycosylation was modified using exoglycosidases and quantified by specific lectin-binding ELISAs. Finally, the effect of altered glycosylation on VWF plasma half-life was characterized by administration of VWF glycan variants to VWF−/− mice. Wild type pdVWF was cleared in biphasic manner, characterized by a rapid initial phase followed by a slower secondary phase (t1/2 = 46.9 min). Enzymatic desialylation of VWF with α2–3,6,8,9 neuraminidase (Neu-VWF) markedly enhanced VWF clearance (t1/2 = 3.7 min; p

Description: Abstract 773 Peripheral T-cell lymphomas (PTCLs) are rare and heterogeneous tumors whose biology is largely unknown. Interestingly, the commonest subtypes (i.e. PTCL not otherwise specified, NOS; angioimmunoblastic T-cell lymphoma, AITL; and anaplastic large cell lymphoma, ALCL) present on one hand few disease-specific molecular features and, on the other hand, several apparently common abnormalities. So far, no data are available regarding miRNA expression in these tumors. In order to identify miRNA deregulated in PTCLs, we performed an extensive miRNA profiling (by studying 379 targets on the TaqMan Array MicroRNA Cards) of 44 PTCLs (including 23 PTCLs/NOS, 12 ALCLs, and 9 AITLs) and 13 sample representative of normal T-cell sub-populations (CD4+ and CD8+, both resting and activated). In addition, for all these cases, gene expression profiles (GEPs) were generated by the Ilumina whole genome DASL-assay. TaqMan Quantitative-PCR (qPCR) was then used for validation. First, we found that PTCLs and normal T-cells could be easily distinguished based on their miRNA profile, by both unsupervised and supervised analysis. Specifically, the latter identified 91 miRNA differentially expressed in PTCLs vs. T-cells with a fold change ≥2 and a pvalue

Description: Abstract 770 Background: Waldenstrom's macroglobulinemia (WM) is a B-cell lymphoma characterized by high serum monoclonal IgM and infiltration of lymphoplasmacytic cells into the bone marrow. As with many hematologic malignancies, cytokines within the tumor microenvironment play an important role in supporting the growth and survival of malignant WM cells. IL-21 is a pleiotropic cytokine involved in the differentiation of B cells into plasma cells. During malignancy, IL-21 has demonstrated diverse effects promoting the growth of myeloma and Hodgkin lymphoma cells while inducing apoptosis in chronic lymphocytic leukemia. However, the biologic significance of IL-21 has not been examined in WM. Our objective here was to assess the expression of IL-21 and its receptor in WM cells and to examine whether IL-21 contributes to the biology of WM. Results: When compared to normal bone marrows, immunohistochemistry revealed significant IL-21 staining in the bone marrow of patients with WM (n=5). To determine whether WM cells are susceptible to IL-21 in the microenvironment, expression of the IL-21 receptor (IL-21R) was assessed via PCR in CD19+CD138+ cells isolated by positive selection from patients with WM (n=8) and in the newly characterized WM cell line, MWCL-1. Nearly all (7/8) CD19+CD138+ WM cells expressed IL-21R, as did MWCL-1 cells. Using flow cytometry we detected expression of IL-21R protein on the surface of WM cells as well. The contribution of microenvironmental IL-21 to the biology of WM tumors was then examined. In MWCL-1 cells, IL-21 (100 ng/mL) increased proliferation by 37% (p=0.005) over untreated controls as determined by thymidine incorporation at 72 hr, and in primary WM cells, proliferation increased by nearly 50% (p=0.003). Interestingly, the immortalized B cell line, IM-9, responded to IL-21 with a significant decrease in proliferation, consistent with previous data indicating differential effects of IL-21 depending on the pathological status of the B-cell in question. IL-21 also significantly induced (p

Description: Abstract 877 Introduction: Patients with follicular lymphoma (FL) usually respond well to immuno-chemotherapy as initial treatment and can have long survival times. However, a small proportion of patients are refractory or have early relapses. Early identification of this subgroup of patients could lead to early therapeutic changes and, potentially, to a better prognosis. [18F]Fluorodeoxyglucose-Positron Emission Tomography (FDG-TEP) is widely used for the staging and restaging of aggressive lymphoma patients but little is known about the use of FDG-PET in patients with FL, except that FL is almost uniformly FDG-avid. FDG-PET is not recommended today as a routine procedure in FL patients (Cheson et al. J Clin Oncol 2007). In this first prospective, multicentric study, we evaluated the prognostic value of FDG-PET performed at mid-treatment and at the end of treatment in patients with high-tumor mass FL treated with immuno-chemotherapy in first line. Patients & methods: Patients with previously untreated FL (grades 1–3A) presenting with a high tumor burden as defined by the GELF criteria, were treated with 6 cycles of R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone) plus 2 cycles of rituximab, given every 21 days. Patients did not receive rituximab maintenance. A FDG-PET was performed before treatment, after 4 cycles of R-CHOP (interim FDG-PET: I-PET), and at the end of treatment (final PET: F-PET). FDG-PET scans were first interpreted in each centre, then centrally reviewed by 3 investigators blinded to clinical data. Positivity or negativity was rated according to the Deauville visual semi-quantitative criteria (Meignan M: Leuk Lymphoma 2009), positivity being defined as a fixation at level 4 (FDG uptake superior to that of the liver) or 5 (uptake clearly superior to liver and/or new sites of disease). Results: 121 patients were included. The median age was 57 years and the male-to-female ratio was 1.12. Ninety-three percent had Ann Arbor stage III–IV. Ninety-seven percent had a good performance status (ECOG score 0–1). The repartition according to the FLIPI was: 0–1 factors: 15%; 2 factors: 43%; 3–5 factors: 42%. The Kappa coefficient indicated a good degree of concordance between the 3 PET reviewers. The initial FDG-TEP was positive in all except 1 case out of 118 centrally reviewed cases. After central review, I-PET (n=111) was negative in 76% of patients and F-PET (n=106) was negative in 78%. With a median follow-up of 23 months, 2-year-progression-free survival rates for I-PET negative versus positive and for F-PET negative versus positive were I-PET 86% versus 61% (p=0.0046); F-PET 87% versus 51% (p

Description: Abstract 731 Paroxysmal Nocturnal Hemoglobinuria (PNH) is an acquired disorder of hemopoietic stem cells (HSCs). Affected individuals experience intravascular hemolysis and have a predisposition to thromboembolism, renal impairment and pulmonary hypertension. These symptoms vary in severity, but in general, the higher the proportion of PNH blood cells produced, the more severe the symptoms. The molecular pathogenesis in PNH is known to relate to a single gene mutation in the X-linked phosphatidylinositol glycan class A gene (PIG-A) which causes a complete or partial deficiency of glycophosphatidylinositol (GPI) anchored proteins leading to the symptoms of the disease. The recent development of eculizumab therapy in PNH has had a dramatic impact in reducing both morbidity and mortality in the disease but PNH remains incurable. A sub-optimal response to eculizumab, certainly when assessed by transfusion requirements, is often due to the underlying bone marrow failure that is considered to be universally present in PNH. The factors that lead from the development of a mutant clone to clonal expansion and symptomatic disease are poorly understood but are the key to improving responses and potentially to move towards a cure. Bone marrow failure appears to provide the environment necessary for expansion of PNH clones and small PNH clones are often detected in aplastic anemia and less commonly in myelodysplasia. There is no evidence that PNH HSCs have an intrinsic proliferative advantage compared to normal HSCs and an immune-mediated extrinsic suppression of normal hematopoiesis with a selective advantage for the PNH cells over residual normal stem cells is likely to explain the preferential development of PNH clones concurrent with bone marrow failure. To gain a better understanding of clonal expansion in PNH we have developed an in vitro PNH bone marrow culture model using a stromal cell line which allows PNH stem cells to be maintained in long term cultures and their capacity to form progenitor cells in myeloid colony forming assays to be assessed. We have evaluated bone marrow from 11 patients with PNH (median age 47 years, median granulocyte clone size 95.3%) and 10 normal controls (median age 42 years) within these long term bone marrow culture experiments. Unmanipulated bone marrow mononuclear cells (MNCs), CD34 selected cells and MNCs with their T-cell component depleted were used in this model. This in vitro model provides the environment for PNH stem cells to be maintained for up to eight weeks and, unlike previous studies, produce progenitor cells. When the patient's MNC's are used to seed the culture system there is poor growth of the culture which is only maintained for a median of 2 weeks. However if CD34 selected cells are used then the cultures are maintained for up to 8 weeks (similar to the normal controls) suggesting that there is a component within the MNC's that is responsible for suppressing the marrow culture which is removed by CD34 select. We next selectively removed the T-cells from the PNH MNC's and demonstrated that the marrow cultures now survived to the extent of the controls (see Figure). This demonstrates that the immune suppression in PNH resides in the T-cells. The progenitor cells produced in both the CD34 selected or the T-cell depleted MNCs over the course of the long term culture experiments show an increase in the proportion of normal progenitors the longer the cultures are maintained. This supports the hypothesis that PNH stem cells have no intrinsic proliferative advantage over normal HSCs and that T-cells are the critical cell suppressing the normal hematopoiesis in PNH. We are now examining the specific cell type that causes the myelosuppression in PNH and which will facilitate a targeted approach to the treatment of bone marrow failure in PNH and related disorders. In conclusion we have developed an in vitro PNH bone marrow culture model that allows the immune insult in PNH to be evaluated. Furthermore, this work confirms the important role that T-cells play in the etiology of PNH and provides a model with which to define the exact mechanism of suppression of hematopoiesis in PNH (and aplastic anemia). This information is essential to develop targeted therapies for the marrow failure seen in PNH and aplastic anemia. Disclosures: Kelly: Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Richards:Alexion Pharmaceuticals: Honoraria, Speakers Bureau. Arnold:Alexion Pharmaceuticals: Honoraria. Hill:Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Hillmen:Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.