ALBERT

Beschreibung: The Aegean water masses and circulation structure are studied via two large-scale surveys performed during the late winters of 1988 and 1990 by the R/V Yakov Gakkel of the former Soviet Union. The analysis of these data sheds light on the mechanisms of water mass formation in the Aegean Sea that triggered the outflow of Cretan Deep Water (CDW) from the Cretan Sea into the abyssal basins of the eastern Mediterranean Sea (the so-called Eastern Mediterranean Transient). It is found that the central Aegean Basin is the site of the formation of Aegean Intermediate Water, which slides southward and, depending on their density, renews either the intermediate or the deep water of the Cretan Sea. During the winter of 1988, the Cretan Sea waters were renewed mainly at intermediate levels, while during the winter of 1990 it was mainly the volume of CDW that increased. This Aegean water mass redistribution and formation process in 1990 differed from that in 1988 in two major aspects: (i) during the winter of 1990 the position of the front between the Black Sea Water and the Levantine Surface Water was displaced farther north than during the winter of 1988 and (ii) heavier waters were formed in 1990 as a result of enhanced lateral advection of salty Levantine Surface Water that enriched the intermediate waters with salt. In 1990 the 29.2 isopycnal rose to the surface of the central basin and a large volume of CDW filled the Cretan Basin. It is found that, already in 1988, the 29.2 isopycnal surface, which we assume is the lowest density of the CDW, was shallower than the Kassos Strait sill and thus CDW egressed into the Eastern Mediterranean.

Beschreibung: Published

Beschreibung: 1841-1859

Beschreibung: JCR Journal

Beschreibung: reserved

Beschreibung: A land surface model (LSM) has been included in the ECMWF Hamburg version 4 (ECHAM4) atmospheric general circulation model (AGCM). The LSM is an early version of the Organizing Carbon and Hydrology in Dynamic Ecosystems (ORCHIDEE) and it replaces the simple land surface scheme previously included in ECHAM4. The purpose of this paper is to document how a more exhaustive consideration of the land surface–vegetation processes affects the simulated boreal summer surface climate. To investigate the impacts on the simulated climate, different sets of Atmospheric Model Intercomparison Project (AMIP)-type simulations have been performed with ECHAM4 alone and with the AGCM coupled with ORCHIDEE. Furthermore, to assess the effects of the increase in horizontal resolution the coupling of ECHAM4 with the LSM has been implemented at different horizontal resolutions. The analysis reveals that the LSM has large effects on the simulated boreal summer surface climate of the atmospheric model. Considerable impacts are found in the surface energy balance due to changes in the surface latent heat fluxes over tropical and midlatitude areas covered with vegetation. Rainfall and atmospheric circulation are substantially affected by these changes. In particular, increased precipitation is found over evergreen and summergreen vegetated areas. Because of the socioeconomical relevance, particular attention has been devoted to the Indian summer monsoon (ISM) region. The results of this study indicate that precipitation over the Indian subcontinent is better simulated with the coupled ECHAM4–ORCHIDEE model compared to the atmospheric model alone.

Beschreibung: Published

Beschreibung: 255–278

Beschreibung: 3.7. Dinamica del clima e dell'oceano

Beschreibung: JCR Journal

Beschreibung: partially_open

Beschreibung: The Indian Summer Monsoon (ISM) is one of the main components of the Asian summer monsoon. It is well known that one of the starting mechanisms of a summer monsoon is the thermal contrast between land and ocean and that sea surface temperature (SST) and moisture are crucial factors for its evolution and intensity. The Indian Ocean, therefore, may play a very important role in the generation and evolution of the ISM itself. A coupled general circulation model, implemented with a high resolution atmospheric component, appears to be able to simulate the Indian summer monsoon in a realistic way. In particular, the features of the simulated ISM variability are similar to the observations. In this study, the relationships between ISM and Tropical Indian Ocean (TIO) SST anomalies are investigated, as well as the ability of the coupled model to capture those connections. The recent discovery of the Indian Ocean Dipole Mode (IODM) may suggest new perspectives in the relationship between ISM and TIO SST. A new statistical technique, the Coupled Manifold, is used to investigate the TIO SST variability and its relation with the Tropical Pacific Ocean (TPO). The analysis shows that the SST variability in the TIO contains a significant portion that is independent from the TPO variability. The same technique is used to estimate the amount of Indian rainfall variability that can be explained by the Tropical Indian Ocean SST. Indian Ocean SST anomalies are separated in a part remotely forced from the Tropical Pacific Ocean variability and a part independent from that. The relationships between the two SSTA components and the Indian monsoon variability are then investigated in detail.

Beschreibung: Published

Beschreibung: 3083-3105

Beschreibung: 3.7. Dinamica del clima e dell'oceano

Beschreibung: JCR Journal

Beschreibung: reserved

Beschreibung: An assessment of the present European operational marine monitoring and forecasting systems shows how observations, atmospheric forcing fields and ocean models combine to make useful oceanographic products possible.

Beschreibung: Published

Beschreibung: 1081-1090

Beschreibung: open

Beschreibung: Ensemble experiments are performed with five coupled atmosphere–ocean models to investigate the potential for initial-value climate forecasts on interannual to decadal time scales. Experiments are started from similar model-generated initial states, and common diagnostics of predictability are used. We find that variations in the ocean meridional overturning circulation (MOC) are potentially predictable on interannual to decadal time scales, a more consistent picture of the surface temperature impact of decadal variations in the MOC is now apparent, and variations of surface air temperatures in the North Atlantic Ocean are also potentially predictable on interannual to decadal time scales, albeit with potential skill levels that are less than those seen for MOC variations. This intercomparison represents a step forward in assessing the robustness of model estimates of potential skill and is a prerequisite for the development of any operational forecasting system.

Beschreibung: Published

Beschreibung: 1195-1203

Beschreibung: JCR Journal

Beschreibung: reserved

Beschreibung: This paper is not subject to U.S. copyright. The definitive version was published in Journal of Atmospheric and Oceanic Technology 21 (2004): 1448–1461, doi:10.1175/1520-0426(2004)021〈1448:AOAPAD〉2.0.CO;2.

Beschreibung: The accuracy of velocities measured by a pulse-coherent acoustic Doppler profiler (PCADP) in the bottom boundary layer of a wave-dominated inner-shelf environment is evaluated. The downward-looking PCADP measured velocities in eight 10-cm cells at 1 Hz. Velocities measured by the PCADP are compared to those measured by an acoustic Doppler velocimeter for wave orbital velocities up to 95 cm s−1 and currents up to 40 cm s−1. An algorithm for correcting ambiguity errors using the resolution velocities was developed. Instrument bias, measured as the average error in burst mean speed, is −0.4 cm s−1 (standard deviation = 0.8). The accuracy (root-mean-square error) of instantaneous velocities has a mean of 8.6 cm s−1 (standard deviation = 6.5) for eastward velocities (the predominant direction of waves), 6.5 cm s−1 (standard deviation = 4.4) for northward velocities, and 2.4 cm s−1 (standard deviation = 1.6) for vertical velocities. Both burst mean and root-mean-square errors are greater for bursts with ub ≥ 50 cm s−1. Profiles of burst mean speeds from the bottom five cells were fit to logarithmic curves: 92% of bursts with mean speed ≥ 5 cm s−1 have a correlation coefficient R2 〉 0.96. In cells close to the transducer, instantaneous velocities are noisy, burst mean velocities are biased low, and bottom orbital velocities are biased high. With adequate blanking distances for both the profile and resolution velocities, the PCADP provides sufficient accuracy to measure velocities in the bottom boundary layer under moderately energetic inner-shelf conditions.

Beschreibung: This work was funded by the U.S. Geological Survey as part of the Southwest Washington Coastal Erosion Study

Beschreibung: When a forecast is assessed, a single value for a verification measure is often quoted. This is of limited use, as it needs to be complemented by some idea of the uncertainty associated with the value. If this uncertainty can be quantified, it is then possible to make statistical inferences based on the value observed. There are two main types of inference: confidence intervals can be constructed for an underlying “population” value of the measure, or hypotheses can be tested regarding the underlying value. This paper will review the main ideas of confidence intervals and hypothesis tests, together with the less well known “prediction intervals,” concentrating on aspects that are often poorly understood. Comparisons will be made between different methods of constructing confidence intervals—exact, asymptotic, bootstrap, and Bayesian—and the difference between prediction intervals and confidence intervals will be explained. For hypothesis testing, multiple testing will be briefly discussed, together with connections between hypothesis testing, prediction intervals, and confidence intervals.

Beschreibung: Studies using International Satellite Cloud Climatology Project (ISCCP) data have reported decreases in cloud optical depth with increasing temperature, thereby suggesting a positive feedback in cloud optical depth as climate warms. The negative cloud optical depth and temperature relationships are questioned because ISCCP employs threshold assumptions to identify cloudy pixels that have included partly cloudy pixels. This study applies the spatial coherence technique to one month of Advanced Very High Resolution Radiometer (AVHRR) data over the Pacific Ocean to differentiate overcast pixels from the partly cloudy pixels and to reexamine the cloud optical depth–temperature relationships. For low-level marine stratus clouds studied here, retrievals from partly cloudy pixels showed 30%–50% smaller optical depths, 1°–4°C higher cloud temperatures, and slightly larger droplet effective radii, when they were compared to retrievals from the overcast pixels. Despite these biases, retrievals for the overcast and partly cloudy pixels show similar negative cloud optical depth–temperature relationships and their magnitudes agree with the ISCCP results for the midlatitude and subtropical regions. There were slightly negative droplet effective radius–temperature relationships, and considerable positive cloud liquid water content–temperature relationships indicated by aircraft measurements. However, cloud thickness decreases appear to be the main reason why cloud optical depth decreases with increasing temperature. Overall, cloud thickness thinning may explain why similar negative cloud optical depth–temperature relationships are found in both overcast and partly cloudy pixels. In addition, comparing the cloud-top temperature to the air temperature at 740 hPa indicates that cloud-top height generally rises with warming. This suggests that the cloud thinning is mainly due to the ascending of cloud base. The results presented in this study are confined to the midlatitude and subtropical Pacific and may not be applicable to the Tropics or other regions.

Beschreibung: A statistical model to analyze different time scales of the variability of extreme high sea levels is presented. This model uses a time-dependent generalized extreme value (GEV) distribution to fit monthly maxima series and is applied to a large historical tidal gauge record (San Francisco, California). The model allows the identification and estimation of the effects of several time scales—such as seasonality, interdecadal variability, and secular trends—in the location, scale, and shape parameters of the probability distribution of extreme sea levels. The inclusion of seasonal effects explains a large amount of data variability, thereby allowing a more efficient estimation of the processes involved. Significant correlation with the Southern Oscillation index and the nodal cycle, as well as an increase of about 20% for the secular variability of the scale parameter have been detected for the particular dataset analyzed. Results show that the model is adequate for a complete analysis of seasonal-to-interannual sea level extremes providing time-dependent quantiles and confidence intervals.

Beschreibung: The interannual and intraseasonal variability of West African vegetation over the period 1982–2002 is studied using the normalized difference vegetation index (NDVI) from the Advanced Very High Resolution Radiometer (AVHRR).The novel independent component analysis (ICA) technique is applied to extract the main modes of the interannual variability of the vegetation, among which two modes are worth describing. The first component (IC1) describes NDVI variability over the Sahel from August to October. A strong photosynthetic activity over the Sahel is related to above-normal convection and rainfall within the intertropical convergence zone (ITCZ) in summertime and is partly associated with colder (warmer) SST in the eastern tropical Pacific (the Mediterranean). The second component (IC2) depicts a dipole pattern between the Sahelian and Guinean regions during the northern summer followed by a southward-propagating signal from October to December. It is associated with a north–south dipole in convection and rainfall induced by variations in the latitudinal location of the ITCZ as a response to the occurrence of the tropical Atlantic dipole.The analysis of the intraseasonal variability of the Sahelian vegetation relies on the analysis of the seasonal marches and their main phenological stages. Green-up usually starts in early July and shows a very low year-to-year variability, while senescence ends by mid-November and is prone to larger interannual variability. Six types of vegetative seasonal marches are discriminated according to variations in the timing of phenological stages as well as in the greening intensity. These types appear to be strongly dependent on rainfall distribution and amount, particularly those recorded in late August. Finally, year-to-year memory effects are highlighted: NDVI recorded during the green-up phase in year j appears to be strongly related to the maximum NDVI value recorded at year j − 1.

Beschreibung: Based on the data of optimum interpolation sea surface temperature (OISST), the temporal correlations of the sea surface temperature anomaly (SSTA) in the South China Sea (SCS) are studied by using the rescaled range analysis (R/S) and detrended fluctuation analysis (DFA). The results show that the scaling exponents of SSTAs are larger than 0.8. This finding indicates that the SSTAs in the SCS exhibit persistent long-range time correlation of the fluctuations and the interval spreads over a wide period, from about 1 month to 4.5 yr (4∼235 weeks). In addition, the “degree” of the correlations depends very much on the geographic locations: near to the coastal regions, the value is small, while far from the coastline, the value is relatively larger. This means that SSTAs in the central SCS are smoother than those of the coastal regions. The persistence of SST in the SCS may be used as a “minimum skill” to assess the ocean models and to evaluate their performance.

Beschreibung: Winds at the Salt Lake City International Airport (SLC) during the April–October period from 1948 to 2003 have been observed to shift to the north (up-valley direction) between late morning and afternoon on over 70% of the days without precipitation. Lake-breeze fronts that develop as a result of the differential heating between the air over the nearby Great Salt Lake and that over the lake’s surroundings are observed at SLC only a few times each month. Fewer lake-breeze fronts are observed during late July–early September than before or after that period. Interannual fluctuations in the areal extent of the shallow Great Salt Lake contribute to year-to-year variations in the number of lake-breeze frontal passages at SLC. Data collected during the Vertical Transport and Mixing Experiment (VTMX) of October 2000 are used to examine the structure and evolution of a lake-breeze front that moved through the Salt Lake Valley on 17 October. The onset of upslope and up-valley winds occurred within the valley prior to the passage of the lake-breeze front. The lake-breeze front moved at roughly 3 m s−1 up the valley and was characterized near the surface by an abrupt increase in wind speed and dewpoint temperature over a distance of 3–4 km. Rapid vertical mixing of aerosols at the top of the 600–800-m-deep boundary layer was evident as the front passed.

Beschreibung: This study combines the experimental measurements with large-eddy simulation (LES) data of a neutral planetary boundary layer (PBL) documented by a 60-m tower instrumented with eight sonic anemometers, and a high-resolution Doppler lidar during the 1999 Cooperative Atmospheric and Surface Exchange Study (CASES-99) experiment. The target of the paper is (i) to investigate the multiscale nature of the turbulent eddies in the surface layer (SL), (ii) to explain the existence of a −1 power law in the velocity fluctuation spectra, and (iii) to investigate the different nature of turbulence in the two sublayers within the SL, which are the eddy surface layer (ESL; lower sublayer of the SL lying between the surface and about 20-m height) and the shear surface layer (SSL; lying between the ESL top and the SL top). The sonic anemometers and Doppler lidar prove to be proper instruments for LES validation, and in particular, the Doppler lidar because of its ability to map near-surface eddies.This study shows the different nature of turbulence in the ESL and the SSL in terms of organized eddies, velocity fluctuation spectra, and second-order moment statistics. If quantitative agreement is found in the SSL between the LES and the measurements, only qualitative similarity is found in the ESL due to the subgrid-scale model, indicating that the LES captures part of the physics of the ESL. The LES helps explain the origin of the −1 power-law spectral subrange evidence in the velocity fluctuation spectra computed in the SL using the CASES-99 dataset: strong interaction between the mean flow and the fluctuating vorticities is found in the SL, and turbulent production is found to be larger than turbulent energy transfer for k1z 〉 1 (k1 being the longitudinal wavenumber and z the height), which is the condition of the −1 power-law existence.

Beschreibung: The effects of natural and anthropogenic heterogeneity on a hydrological simulation are evaluated using a distributed biosphere hydrological model (DBHM) system. The DBHM embeds a biosphere model into a distributed hydrological scheme, representing both topography and vegetation in a mesoscale hydrological simulation, and the model system includes an irrigation scheme. The authors investigated the effects of two kinds of variability, precipitation variability and the variability of irrigation redistributing runoff, representing natural and anthropogenic heterogeneity, respectively, on hydrological processes. Runoff was underestimated if rainfall was placed spatially uniformly over large grid cells. Accounting for precipitation heterogeneity improved the runoff simulation. However, the negative runoff contribution, namely, the situation that mean annual precipitation is less than evapotranspiration, cannot be simulated by only considering the natural heterogeneity. This constructive model shortcoming can be eliminated by accounting for anthropogenic heterogeneity caused by irrigation water withdrawals. Irrigation leads to increased evapotranspiration and decreased runoff, and surface soil moisture in irrigated areas increases because of irrigation. Simulations performed for the Yellow River basin of China indicated streamflow decreases of 41% due to irrigation effects. The latent heat flux in the peak irrigation season [June–August (JJA)] increased 3.3 W m−2 with a decrease in the ground surface temperature of 0.1 K for the river basin. The maximum simulated increase in the latent heat flux was 43 W m−2, and the ground temperature decrease was 1.6 K in the peak irrigation season.

Beschreibung: A method for routinely verifying numerical weather prediction surface marine winds with satellite scatterometer winds is introduced. The marine surface winds from the Australian Bureau of Meteorology’s operational global and regional numerical weather prediction systems are evaluated. The model marine surface layer is described. Marine surface winds from the global and limited-area models are compared with observations, both in situ (anemometer) and remote (scatterometer). A 2-yr verification shows that wind speeds from the regional model are typically underestimated by approximately 5%, with a greater bias in the meridional direction than the zonal direction. The global model also underestimates the surface winds by around 5%–10%. A case study of a significant marine storm shows that where larger errors occur, they are due to an underestimation of the storm intensity, rather than to biases in the boundary layer parameterizations.

Beschreibung: The applicability of axisymmetric theory of angular momentum conserving circulations to the large-scale steady monsoon is studied in a general circulation model with idealized representations of continental geometry and simple physics. Results from an aquaplanet setup with localized subtropical forcing are compared with a continental case. It is found that the meridional circulation that develops is close to angular momentum conserving for cross-equatorial circulation cells, both in the aquaplanet and in the continental cases. The equator proves to be a substantial barrier to boundary layer meridional flow; flow into the summer hemisphere from the winter hemisphere tends to occur in the free troposphere rather than in the boundary layer. A theory is proposed to explain the location of the monsoon; assuming quasiequilibrium, the poleward boundary of the monsoon circulation is collocated with the maximum in subcloud moist static energy, with the monsoon rains occurring near and slightly equatorward of this maximum. The model results support this theory of monsoon location, and it is found that the subcloud moist static energy distribution is determined by a balance between surface forcing and advection by the large-scale flow.

Beschreibung: This paper concerns the calculation of the probability of exceedance of wave crest elevation. The statistics have been calculated for broadbanded, unidirectional, deep-water sea states by incorporating a fully nonlinear wave model into a spectral response surface method. This is a novel approach to the calculation of statistics and, as all of the calculations are performed in the probability domain, avoids the need for long time-domain simulations. Furthermore, in contrast to theoretical distributions, the broadbanded, fully nonlinear nature of the sea state can be considered. The results have been compared with those of fully nonlinear time-domain simulations and are shown to be in good agreement. The results indicate that in unidirectional seas the crest elevations of the largest waves can be much higher than would be predicted by linear or second-order theory. Hence, the occurrence of locally long crested sea states offers a possible explanation for the formation of freak or rogue waves.

Beschreibung: Nearly one-half of the earth’s terrestrial surface is susceptible to drought, which can have significant social, economic, and environmental impacts. Therefore, it is important to develop better descriptions and models of the processes linking the land surface and atmosphere during drought. Using data collected during the International H2O Project, the study presented here investigates the effects of variations in the environmental factors driving the latent heat flux (λE) during drought conditions at a rangeland site located in the panhandle of Oklahoma. Specifically, this study focuses on the relationships of λE with vapor pressure deficit, wind speed, net radiation, soil moisture content, and greenness fraction. While each of these environmental factors has an influence, soil moisture content is the key control on λE. The role of soil moisture in regulating λE is explained in terms of the surface resistance to water vapor transfer. The results show that λE transitioned between being water or energy limited during the course of the drought. The implications of this on the ability to understand and model drought conditions and transitions into or out of droughts are discussed.

Beschreibung: In March 2003 several autonomous underwater vehicle (AUV) missions were carried out under sea ice in the western Bellingshausen Sea. Data from the upward-looking acoustic Doppler current profiler (ADCP) on the “Autosub” AUV indicate a strongly oscillating horizontal velocity of the ice due to ocean swell. Swell period, height, direction, and directional spread have been computed every 800 m from the ice edge to 10 km inward for three missions. Exponential, period-dependent attenuation of waves propagating through sea ice was observed. Mean period increased with distance from the ice edge. The wave field refracted during propagation. The directional wave spread does not seem to relate to distance from the ice edge, although higher frequencies tended to be more spread. If suitably deployed, an ordinary ADCP may be used with this technique to study both scalar and directional properties of waves in open or ice-covered water.

Beschreibung: Wind-sea generation was observed during two experiments off the coast of North Carolina. One event with offshore winds of 9–11 m s−1 directed 20° from shore normal was observed with eight directional stations recording simultaneously and spanning a fetch from 4 to 83 km. An opposing swell of 1-m height and 10-s period was also present. The wind-sea part of the wave spectrum conforms to established growth curves for significant wave height and peak period, except at inner-shelf stations where a large alongshore wind-sea component was observed. At these short fetches, the mean wave direction θm was observed to change abruptly across the wind-sea spectral peak, from alongshore at lower frequencies to downwind at higher frequencies. Waves from another event with offshore winds of 6–14 m s−1 directed 20°–30° from shore normal were observed with two instrument arrays. A significant amount of low-frequency wave energy was observed to propagate alongshore from the region where the wind was strongest. These measurements are used to assess the performance of some widely used parameterizations in wave models. The modeled transition of θm across the wind-sea spectrum is smoother than that in the observations and is reproduced very differently by different parameterizations, giving insights into the appropriate level of dissipation. Calculations with the full Boltzmann integral of quartet wave–wave interactions reveal that the discrete interaction approximation parameterization for these interactions is reasonably accurate at the peak of the wind sea but overpredicts the directional spread at high frequencies. This error is well compensated by parameterizations of the wind input source term that have a narrow directional distribution. Observations also highlight deficiencies in some parameterizations of wave dissipation processes in mixed swell–wind-sea conditions.

Beschreibung: Seven sets of 2D particle image velocimetry data obtained in the bottom boundary layer of the coastal ocean along the South Carolina and Georgia coast [at the South Atlantic Bight Synoptic Offshore Observational Network (SABSOON) site] are examined, covering the accelerating and decelerating phases of a single tidal cycle at several heights above the seabed. Additional datasets from a previous deployment are also included in the analysis. The mean velocity profiles are logarithmic, and the vertical distribution of Reynolds stresses normalized by the square of the free stream velocity collapse well for data obtained at the same elevation but at different phases of the tidal cycle. The magnitudes of 〈u′u′〉, 〈w′w′〉, and −〈u′w′〉 decrease with height above bottom in the 25–160-cm elevation range and are consistent with the magnitudes and trends observed in laboratory turbulent boundary layers. If a constant stress layer exists, it is located below 25-cm elevation. Two methods for estimating dissipation rate are compared. The first, a direct estimate, is based on the measured in-plane instantaneous velocity gradients. The second method is based on fitting the resolved part of the dissipation spectrum to the universal dissipation spectrum available in Gargett et al. Being undervalued, the direct estimates are a factor of 2–2.5 smaller than the spectrum-based estimates. Taylor microscale Reynolds numbers for the present analysis range from 24 to 665. Anisotropy is present at all resolved scales. At the transition between inertial and dissipation range the longitudinal spectra exhibit a flatter than −5/3 slope and form spectral bumps. Second-order statistics of the velocity gradients show a tendency toward isotropy with increasing Reynolds number. Dissipation exceeds production at all measurement heights, but the difference varies with elevation. Close to the bottom, the production is 40%–70% of the dissipation, but it decreases to 10%–30% for elevations greater than 80 cm.

Beschreibung: Scalar exchange between San Francisco Bay and the coastal ocean is examined using shipboard observations made across the Golden Gate Channel. The study consists of experiments during each of the following three “seasons”: winter/spring runoff (March 2002), summer upwelling (July 2003), and autumn relaxation (October 2002). Within each experiment, transects across the channel were repeated approximately every 12 min for 25 h during both spring and neap tides. Velocity was measured from a boat-mounted ADCP. Scalar concentrations were measured at the surface and from a tow-yoed SeaSciences Acrobat. Net salinity exchange rates for each season are quantified with harmonic analysis. Accuracy of the net fluxes is confirmed by comparison with independently measured values. Harmonic results are then decomposed into flux mechanisms using temporal and spatial correlations. In this study, the temporal correlation of cross-sectionally averaged salinity and velocity (tidal pumping flux) is the largest part of the dispersive flux of salinity into the bay. From the tidal pumping flux portion of the dispersive flux, it is shown that there is less exchange than was found in earlier studies. Furthermore, tidal pumping flux scales strongly with freshwater flow resulting from the density-driven movement of a tidally trapped eddy and stratification-induced increases in ebb–flood frictional phasing. Complex bathymetry makes salinity exchange scale differently with flow than would be expected from simple tidal asymmetry and gravitational circulation models.

Beschreibung: As part of a program aimed at developing a long-duration, subsurface mooring, known as Ultramoor, several modern acoustic current meters were tested. The instruments with which the authors have the most experience are the Aanderaa RCM11 and the Nortek Aquadopp, which measure currents using the Doppler shift of backscattered acoustic signals, and the Falmouth Scientific ACM, which measures changes in travel time of acoustic signals between pairs of transducers. Some results from the Doppler-based Sontek Argonaut and the travel-time-based Nobska MAVS are also reported. This paper concentrates on the fidelity of the speed measurement but also presents some results related to the accuracy of the direction measurement. Two procedures were used to compare the instruments. In one, different instruments were placed close to one another on three different deep-ocean moorings. These tests showed that the RCM11 measures consistently lower speeds than either a vector averaging current meter or a vector measuring current meter, both more traditional instruments with mechanical velocity sensors. The Aquadopp in use at the time, but since updated to address accuracy problems in low scattering environments, was biased high. A second means of testing involved comparing the appropriate velocity component of each instrument with the rate of change of pressure when they were lowered from a ship. Results from this procedure revealed no depth dependence or measurable bias in the RCM11 data, but did show biases in both the Aquadopp and Argonaut Doppler-based instruments that resulted from low signal-to-noise ratios in the clear, low scattering conditions beneath the thermocline. Improvements in the design of the latest Aquadopp have reduced this bias to a level that is not significant.

Beschreibung: The energy pathways in geostrophic turbulence are explored using a two-layer, flat-bottom, f-plane, quasigeostrophic model forced by an imposed, horizontally homogenous, baroclinically unstable mean flow and damped by bottom Ekman friction. A systematic presentation of the spectral energy fluxes, the mean flow forcing, and dissipation terms allows for a comprehensive understanding of the sources and sinks for baroclinic and barotropic energy as a function of length scale. The key new result is a robust inverse cascade of kinetic energy for both the baroclinic mode and the upper layer. This is consistent with recent observations of satellite altimeter data over the South Pacific Ocean. The well-known forward cascade of baroclinic potential and total energy was found to be very robust. Decomposing the spectral fluxes into contributions from different terms provided further insight. The inverse baroclinic kinetic energy cascade is driven mostly by an efficient interaction between the baroclinic velocity and the barotropic vorticity, the latter playing a crucial catalytic role. This cascade can be further enhanced by the baroclinic mode self-interaction, which is only present with nonuniform stratification (unequal layer depths). When model parameters are set such that modeled eddies compare favorably with observations, the inverse baroclinic kinetic energy cascade is actually much stronger than the well-known inverse cascade in the barotropic mode. The upper-layer kinetic energy cascade was found to dominate the lower-layer cascade over a wide range of parameters, suggesting that the surface cascade and time mean density stratification may be sufficient for estimating the depth-integrated cascade from ocean observations. This may find useful application in inferring the kinetic to gravitational potential energy conversion rate from satellite measurements.

Beschreibung: In order to establish an efficient gd T cell-mediated immunotherapy for hematological malignancies, we tried to clarify whether γδ T cells could be expanded from blood cells of patients with myeloma, lymphoma and acute leukemia by culture with zoledronate and a low dose of IL-2 and whether the expanded patients’ γδ T cells could kill tumor cells including self tumor cells with sparing normal clone cells. In addition, we explored the methods to enhance the anti-tumor cytotoxicity of the expanded γδ T cells by activating them with type I IFN, monocyte-derived dendritic cells (mo-DCs), or ab T cells. Although γδ T cells could be expanded in patients with myeloma, lymphoma and leukemia as well as normal persons, the amplification rates of gd T cells before and after the culture were varied from patient to patient in the patients with hematological malignancies. γδ T cells generated in patients with myeloma and lymphoma showed a potent cytotoxic ability against myeloma/lymphoma cell lines (RPMI8226, Daudi) as shown in γδ T cells generated in normal persons. In addition, γδ T cells generated in a patient with myeloma and acute leukemia showed a cytotoxic ability against self myeloma or leukemia cells freshly prepared from bone marrow. However, the same γδ T cells were not cytotoxic to normal lymphocytes of the patients. Then the expanded γδ T cells were stimulated with type I IFN, mo-DCs, or αβ T cells and the activation (CD69 expression) and cytotoxicity against tumor cells were examined. By the stimulation with type I IFN, the expression of CD69 and Trail of γδ T cells was increased and the cytotoxic ability of γδ T cells was enhanced at dose-dependent manner of type I IFN. CD69 expression on γδ T cells was enhanced by co-culture with both immature and mature mo-DCs in a cell-number-dependent fashion. CD69 expression was enhanced after the addition of mo-DCs of either autologous or allogeneic origin. Activation of γδ T cells with mo-DCs enhanced anti-tumor cytotoxicity of γδ T cells against RPMI8226 and CML blastic crisis cell line (C2F8) in an effector-to-target ratio-dependent manner. Although CD69 expression of γδ T cells was enhanced by the co-culture with allogeneic ab T cells, autologous ab T cells couldn’t activate γδ T cells. However, autologous ab T cells stimulated with IL-2 or PHA could induce the activation of γδ T cells. The activation of γδ T cells with stimulated αβ T cells required cell-to-cell interaction. These findings suggested that αβ T cells stimulated by allogeneic γδ T cells could activate the same allogeneic γδ T cells. The present data demonstrated that γδ T cells, which could be expanded in vitro from blood cells of the patients with myeloma, lymphoma and leukemia by culture with zoledronate and IL-2, possess an enough cytotoxic ability against tumor cells including self tumor cells with sparing normal cells. These findings suggested that in vitro generated patients’ γδ T cells could be applied to γδ T cell-mediated immunotherapy for hematological malignancies. Besides, potent γδ T cells activated by type I IFN, mo-DCs or activated αβ T cells were considered to be applicable for γδ T cell-mediated immunotherapy.

Beschreibung: Vascular endothelial growth factor (VEGF), which induces angiogenesis and increases vascular permeability, is a major growth factor mediating tumor progression. In this study, we employed immunohistochemical-staining method to detect the expression of VEGF in lymph nodes taken from39 non-Hodgkin’s lymphomas patients and analyzed the relation of the expression levels to malignant aggressiveness, treatment response, histological grade, clinical stage and prognosis. The patients had been observed for at least 5 years or until death. 9 patients with benign lymphadenopathy were acted as control. The expression of VEGF was assessed according to the percentage of immunoreactive cells in a total of 1000 neoplastic cells (quantitative analysis). Immunoreactivity was graded positive, more than 10% of carcinoma cells stained and negative, no detectable staining or less than 10% of carcinoma cells stained. Furthermore, the qualitative intensity of staining for VEGF was assessed using a scale of 0–3+. The expression analysis of VEGF revealed that in 31 out of 39 (79.49%) specimens VEGF staining was positive. The VEGF staining was always cell membrane. Significant associations were found between the expression of VEGF and histological grade, Ann Arbor stage, prognosis (according to International Prognostic Index, IPI) and chemotherapy response. Among 8 cases of low grade, 7 had lower-level expression and 1 had higher-level expression, but among 31 cases of intermediate and high grade, 13 had lower-level expression and18 had higher-level expression (P=0.044

Beschreibung: We examined a large cohort (N=2,457) of chronic lymphocytic leukemia (CLL) patients evaluated by the CLL Research Consortium (CRC) and found 63 (2.6%) used IGHV3-21. Comparing the Ig heavy chain third complementarity determining region (HCDR3) of the IGHV3-21 cases: 25/63 cases (39.7%) had a conserved amino acid motif (motif 1: DANGMDV) in the otherwise highly variable Ig HCDR3, as described by Tobin et al. Blood 2003. All but one of these Ig heavy chains (IgH) were paired with a lambda light chain encoded by IGLV3-21. In addition, we found that 3/63 cases (4.8%) had a previously unrecognized conserved HCDR3 amino acid motif (motif 2: DPSFYSSSWTLFDY). In contrast, these IgH invariably were paired with kappa immunoglobulin light chains (IgL) encoded by IGKV3-20. Similarly to that noted for CLL cases that use IgH encoded by unmutated IGHV1-69 (Widhopf et al. Blood Epub First Edition 2007), the pairing of IgH encoded by IGHV3-21 with IgL appears governed by the HCDR3. The non-stochastic pairing of IgH with IgL argues strongly that antigen plays a role in selecting the Ig expressed in CLL. To examine for the antigen(s) recognized by the most common Ig encoded by IGHV3-21, we isolated IgH and IgL genes expressed by IGHV3-21/IGLV3-21 CLL cases and generated recombinant antibodies, which we examined for binding to antigen(s) present on microarray of self or environmental antigens. We found that Ig encoded by IGHV3-21/IGLV3-21 had apparent specific binding for protein L, a multi-domain cell-wall protein isolated from Peptostreptococcus magnus, a Gram-positive commensal bacteria that comprise a large portion of the human bacterial gut flora. Prior studies identified that protein L is a superantigen capable of binding human Ig kappa light chains encoded by IGKV genes of the I, III, and IV subgroups, but not human Ig lambda light chains. The specific binding of IGHV3-21/IGLV3-21 to protein L suggested that protein L might play a role in the development of CLL cells that express such Ig. To test this hypothesis, we examined the capacity of various recombinant antibodies to bind protein L by ELISA. We found that lambda IgL encoded by IGLV3-21 could bind to protein L with similar activity, independent of whether this lambda IgL paired with the native IgH, IgH encoded by IGHV3-21 lacking the DANGMDV HCDR3 motif, or even irrelevant IgH encoded by IGHV4-39 that are not found paired with IGLV3-21 in the Ig expressed in CLL. Moreover, Ig formed by pairing IgH encoded by IGHV3-21 that has the DANGMDV HCDR3 motif with an IgL encoded by an IGLV that was irrelevant to IGLV3-21 did not bind protein L. These results reveal a previously unrecognized capacity of human IgL encoded by IGLV3-21 to bind the protein L superantigen of Peptostreptococcus magnus, a bacteria commonly found in the human gastrointestinal tract. However, because the binding of IGLV3-21 does not depend upon the non-stochaistic pairing of IgH and IgL observed in CLL, we reason that the capacity of IGLV3-21 to bind protein L cannot account for the selected Ig repertoire expressed in CLL, suggesting that it actually does not play a role in CLL leukemogenesis. This finding suggests that caution should be exercised when defining an antigen that is found capable of binding the restricted Ig expressed in CLL as the driving factor responsible for leukemogenesis.

Beschreibung: Background: Heavy chain disease (HCD) is a rare lymphoproliferative disorder characterized by a monoclonal heavy chain (HC) unattached to a light chain (LC). IgGHCD or γHCD typically presents as a lymphoproliferative disorder with lymphadenopathy and hepatosplenomegaly. Myeloma has been described associated with γHCD but only with a second intact Ig paraprotein. This report describes a unique presentation of multiple myeloma with monoclonal free γ3HC and kappa free light chains. Case: A 34 year old gentleman presented with mild persistent neutropenia following two episodes of pneumonia, 18 months previously. He admitted to persistent night sweats but no other significant history. Baseline investigations revealed a mild anaemia, neutropenia and a large IgG paraprotein with no associated light chain. Bone marrow aspirate and trephine confirmed myeloma. The patient was treated with cyclophosphamide, thalidomide and dexamethasone and has had a very good partial remission. He is awaiting a sibling allogeneic peripheral blood stem cell transplant. Investigations and results: Serum Electrophoresis confirmed a large IgG paraprotein (23g/l) with no associated light chain in the serum and identified as γ3 subclass by radial immunodiffusion. Western blot showed the γ3HC was truncated with a large deletion. Markedly elevated free kappa (κ) LC (503.58 mg/l [3.30–19.4]) were found in the serum with gross skewing of the kappa/lambda ratio. Urine electrophoresis revealed separate γHC and κ LC paraproteins. Western blot of the fractionated urine protein demonstrated different sized κLC aggregates. Flow cytometry of the marrow aspirate revealed an unusual staining pattern; CD5,19,38,45+ve and CD20,22,23,34,56,138 –ve plasma cells. Cytoplasmic staining revealed 2 distinct populations of plasma cells, the first producing γ3HC and the second only free κLC. Cytogenetics and FISH analysis for 14q, p53 and c-myc abnormalities were normal. Discussion: This is the first description of a Biclonal Myeloma with separate plasma cell populations producing γ3HC and κLC paraproteins. The biclonality confirms the free HC occurs as a result of abnormal synthesis not cleavage. The clinical and immunological findings are clearly different to typical findings in both γ3HCD and Myeloma. HCD has an appalling prognosis and this case is likely to have been ‘smouldering’ for 18 months, evidenced by the 2 pneumonias and persistent night sweats. There is no lymphadenopathy or organomegaly associated with γ3HCD. The immunophenotype of the malignant plasma cells is unique. Other atypical features include frank proteinuria, with a HC in the urine, but normal renal function and no radiological or biochemical evidence of bone involvement. We propose that this unique biclonal myeloma has distinct immunological and clinical features.

Beschreibung: Background: Recent advances in the therapy of multiple myeloma (MM) have greatly increased the treatment options for this uniformly fatal plasma cell malignancy. It is not clear if introduction of novel therapies and the increased use of high dose therapy (HDT) in the past decade have translated into better outcome for patients (pts) with MM. Methods: We examined the outcome of two cohorts of pts seen at our institution. The first cohort consisted of 387 pts who relapsed after HDT and was examined for potential improvement in survival following relapse after HDT. These pts were divided into two groups; those who relapsed before or after December 31, 2000. The second cohort consisted of 2981 patients with newly diagnosed MM seen between January 1971 and December 2006 and was used to examine the trends in overall survival (OS). Results: Among those relapsing after HDT, there were 245 males (63%); median (range) age at HDT was 57 years (32.8–75.4) and the median time to HDT was 8.1 months (1–90 months) from diagnosis. The median time to relapse was 13.2 months (1.1 months-10.3 years) from HDT. In this cohort, a clear improvement in OS from time of relapse was seen over the past decade, with those relapsing after 2000 having a median OS of 23.9 months (95% CI; 19.8, 27.6) compared to 11.8 months (95% CI; 8.7, 14.9) for the rest (P 〈 0.001) (Figure 1). In a multivariate analysis, the effect of the date of relapse on survival was independent of other prognostic factors such as relapse 5.5 mg/dL. Pts who were treated with one or more of the newer drugs (thalidomide, lenalidomide, bortezomib) had longer survival from relapse (30.9 months (95% CI; 23.6, 38.2) compared to 14.8 months (95% CI; 11.3, 18.4); P 〈 0.001) for others. Among the newly diagnosed MM cohort, the median age at diagnosis was 66 years (20.2 – 97 years), and 1,770 (59.4%) were males. The median follow up for the entire group was 27.4 months (0–29.4 years); and at the time of analysis 558 patients (18.7%) were alive with a median follow up of 32.7 months (0–29.4 years). Pts diagnosed in the last decade had an improved OS (44.8 months) compared to those diagnosed before this period (29.9 months; P 〈 0.001) (Figure 2). The improvement in survival seen in the last decade among newly diagnosed patients was predominantly among those younger than 65 years (60.3 mos vs. 33.3 mos improvement in median survival); compared to those over 65 at diagnosis (26.5 mos vs. 32 mos). Conclusion: In this study, for the first time, we demonstrate definite proof for improved outcome in patients with myeloma, both in the relapsed setting as well as at diagnosis, a change that is likely to continue with increased use of these drugs. Figure Figure Figure Figure

Beschreibung: Absence of shear stress due to disturbed blood flow at arterial bifurcations and curvatures leads to endothelial dysfunction and proinflammatory gene expression, ultimately resulting in atherogenesis. KLF2 has recently been implicated as a transcription factor involved in mediating the anti-inflammatory effects of flow. We investigated the effect of shear on basal and TNF-α–induced genomewide expression profiles of human umbilical vein endothelial cells (HUVECs). Cluster analysis confirmed that shear stress induces expression of protective genes including KLF2, eNOS, and thrombomodulin, whereas basal expression of TNF-α–responsive genes was moderately decreased. Promoter analysis of these genes showed enrichment of binding sites for ATF transcription factors, whereas TNF-α–induced gene expression was mostly NF-κB dependent. Furthermore, human endothelial cells overlying atherosclerotic plaques had increased amounts of phosphorylated nuclear ATF2 compared with endothelium at unaffected sites. In HUVECs, a dramatic reduction of nuclear binding activity of ATF2 was observed under shear and appeared to be KLF2 dependent. Reduction of ATF2 with siRNA potently suppressed basal proinflammatory gene expression under no-flow conditions. In conclusion, we demonstrate that shear stress and KLF2 inhibit nuclear activity of ATF2, providing a potential mechanism by which endothelial cells exposed to laminar flow are protected from basal proinflammatory, atherogenic gene expression.

Beschreibung: Tumor cell–associated tissue factor (TF) is a powerful determinant of metastatic potential. TF may increase metastasis by supporting thrombin-mediated proteolysis, through intracellular signaling events mediated by the TF cytoplasmic domain, through TF/fVIIa/fXa–mediated activation of protease-activated receptors, or through a combination of these processes. To better define the relationship between tumor cell-associated TF and circulating hemostatic factors in malignancy, we generated a set of C57Bl/6-derived tumor lines genetically lacking TF, expressing wild-type murine TF, or expressing a mutant TF lacking the cytoplasmic domain. Comparison of the metastatic potential of these cells in immunocompetent mice with genetic deficits in prothrombin, platelet function, or fibrinogen revealed that TF supports metastasis through mechanisms independent of the cytoplasmic domain, but dependent on each of these distal hemostatic factors. TF was neither required for primary tumor growth nor necessary for initial localization of embolized tumor cells within the lungs. Rather, tumor cell fate studies indicated TF supports metastasis by increasing the survival of micrometastases. One mechanism linking TF to metastasis is through a fibrin(ogen)-dependent and platelet-dependent restriction in natural killer cell–mediated clearance of micrometastases. However, TF also supported the early success of micrometastases through an additional mechanism independent of natural killer cells, but coupled to circulating prothrombin.

Beschreibung: GA101 is a novel monoclonal antibody of IgG1 type which binds with high affinity and selectivity to the extracellular domain of the human CD20 antigen on B cells. In contrast to rituximab which is a chimeric antibody and recognizes a type I epitope, GA101 is humanized and recognizes a type II epitope which is also localized in the extracellular loop of CD20. The recognition of the type II epitope together with a modification of the elbow hinge region results in enhanced direct non-caspase dependent cell death induction, and concomitant reduction in CDC upon binding to CD20. In addition, using GlycoMab technology, the Fc-region of GA101 was glycoengineered to contain bisected, afucosylated carbohydrates. As a result GA101 has increased affinity for the low and high affinity FcγRIIIa receptor expressed on natural killer cells, macrophages and monocytes. Consequently, GA101 mediated a 5–50 fold enhanced induction of effector cell mediated ADCC. In B-cell depletion assays with whole blood from healthy donors, an assay combining all mechanisms of action, GA101 was significantly more potent and efficacious in depleting B cells than rituximab. In preclinical NHL testing these properties translated into superior anti-tumoral efficacy of GA101 in direct comparison to rituximab against a number of aggressive NHL xenograft models. In cynomolgus monkeys the induction of B cell depletion mediated by GA101 and subsequent B cell recovery were investigated. GA101 induced complete, rapid and long-lasting B cell depletion both in peripheral blood and in lymphoid tissue e.g. spleen and lymph nodes. The efficacy of GA101 (10 and 30 mg/kg) at depleting B cells in different lymphoid tissues of cynomolgus monkeys was compared with that of rituximab (10 mg/kg) following 2 i.v. doses administered on days 0 and 7. Notably, GA101 showed statistically superior depletion of total B cells from lymph nodes compared to Rituximab from day 9 to 35 onwards with B cell numbers decreased by over 95%. These results demonstrated that GA101 was more efficacious at depleting B cells from lymph nodes and spleen of cynomolgus monkeys compared to rituximab. Compared to existing antibodies, GA101 constitutes the first type II CD20 antibody engineered for increased ADCC with significantly enhanced efficacy in a variety of preclinical models. Based on these data it is assumed that the combination of the recognition of a type II epitope together with improved ADCC potency might translate into superior efficacy in the clinical treatment of CD20 positive malignant diseases.

Beschreibung: Polycythemia vera (PV) and essential thrombocythemia (ET) are two common types of myeloproliferative disorders (MPD). The prevalence of PV and ET in the United States (US) has not been well documented. Recent breakthroughs in the molecular etiology of these disorders and the accelerated development of targeted pharmacotherapeutics to treat the MPD have significantly increased the need to accurately define the affected population. In the present study, we obtained detailed demographic and health claims data from major commercial insurance payers in Connecticut and the Center for Medicare and Medicaid Services to estimate the prevalence of PV and ET. Health claims data from one payer and the actual diagnoses made by physicians who submitted claims with MPD-related ICD-9 codes were utilized to develop claim-based statistical algorithms to predict the probability that an individual with claims suggestive of MPD truly has PV or ET. Specifically, logistic regression was used to develop the algorithms, and area under the receiver operating characteristics curve (AUC) was used as the measure of goodness-of-fit for each model. Different models were fit, and the model with the highest AUC was selected. For PV, the best-fitting model included age as a continuous variable and the frequency of two PV-related ICD-9 codes (238.4 and 289.6 combined) as a continuous variable, and the AUC was 0.95. For ET, the best-fitting model included age as a continuous variable and the frequency of code 289.9 as a categorical variable (=0, =1, or ≥2), and the AUC was 0.72. For both models, the addition of gender or the frequency of the non-specific code 238.7 did not improve AUC. Subsequently, the algorithms were applied to health claims from multiple payers to estimate the number of PV and ET patients in Connecticut. The total number of Connecticut residents included in the study was close to 2,900,000, which represented about 83.2% of the estimated population of the entire state in July 2003. As of 2003, the age-standardized prevalence of PV and ET in Connecticut was 22 per 100,000 and 24 per 100,000, respectively. Applying the age-specific prevalence of PV and ET to the entire US population resulted in an estimated total of 65,243 patients with PV and 71,078 patients with ET in the US in 2003. This study is the first to assess the prevalence of PV and ET in a very large US population. Given the large number of individuals afflicted with these diseases and the fact that demographic changes alone will further increase the burden of these diseases in the foreseeable future, it is imperative to conduct more systematic research into the etiology and treatment of PV and ET.

Beschreibung: Background: Neurologic complications of multiple myeloma (MM) are numerous, however little is known about strokes occurring in the course of MM and its treatment. Methods: 2877 MM patients were seen for an initial evaluation at our institution over a 5-year period (1998–2002). Patients were identified using discharge summaries by combining ICD-9 codes for MM and stroke. They were included if they had clinical and radiological features of acute stroke. Ischemic stroke was defined as a new acute neurologic deficit lasting 〉 24 hours, irrespective of diffusion-weighted MRI results. Diagnosis of hemorrhagic stroke was made in patients with new acute neurologic deficit plus evidence of hemorrhage on CT scan. We retrospectively reviewed medical records for demographics, type of MM and treatment, stroke type, clinical features, relevant imaging and laboratory data, and outcomes. Results: The 11 patients that we identified included eight with ischemic strokes and three with subarachnoid hemorrhages (SAH). There were no patients with intraparenchymal hemorrhage. The overall incidence of stroke in this cohort was 76/100,000 per year, whereas the incidence of ischemic stroke was 56/100,000 per year. The mean age of patients with ischemic stroke was 59 years. Seven had one or more stroke risk factors and an equal number had received thalidomide. The latter were given small doses of coumadin as part of the treatment protocol; at the time of the stroke, INR range was 0.9–1.4. Based on clinical, radiological and other laboratory features, of the eight patients with ischemic strokes, two were presumed cardio-embolic (one had infective endocarditis), five were thrombotic, and one was caused by hypoperfusion. Two patients had findings of severe stenosis/occlusion in vessels corresponding to the infracted brain territory (internal carotid and basilar arteries). Two patients had documented normal plasma viscosity and three had evidence of extra-cranial thrombosis at the time of the stroke. In the three patients with SAH, hemorrhage occurred in the setting of trauma and thrombocytopenia, although one patient had an incidental anterior communicating artery aneurysm. Seven patients were left with minor or no deficits and four died, two in each of the groups. Conclusion: Overall, strokes did not appear to be more common in MM patients than in the general population, and the pathophysiology was likewise, not different than in patients without MM, with the exception of one patient with infective endocarditis, which is probably directly related to MM or its treatment. The highly selected referral population at our myeloma institute, and the retrospective nature of the study probably underestimate the incidence of stroke. The role of thalidomide in stroke remains unknown. Thrombocytopenia and trauma are probable risk factors for subarachnoid hemorrhage in MM patients. Fatal outcomes were frequent, but when death did not occur, the deficits were minor.

Beschreibung: Recent advances in the diagnosis, molecular pathogenesis, classification and therapy have been made in the field of myelodysplastic syndrome (MDS) and juvenile myelomonocytic leukemia (JMML) in childhood. We report a retrospective analysis of children with MDS and JMML diagnosed between 2001 and 2006 in Korea. In total, 135 patients were enrolled from 19 major hospitals with pediatric hematology oncology clinics: MDS, 96 (primary MDS, 77; constitutional anomalies with MDS, 13; treatment-related MDS, 6) and JMML, 39. The incidence of MDS/JMML was around 22.5/year, which is about 6% of childhood leukemia. Various classification systems including FAB, WHO, IPSS, CCC system, and pediatric adjustment of the WHO classification were applied. The median ages at diagnosis were 68 and 10 months in MDS, and JMML, respectively. Males dominated in JMML. Cytogenetic abnormalities were observed in 43% of MDS (monosomy 7, 5; trisomy 8, 3) and in 10% of JMML. Treatment was chosen by each institute’s preference: 34 patients with MDS received AML-type intensive chemotherapy, with complete remission rate of 82.0%. The 5-year Kaplan-Meier overall survival rate was 54% each for MDS and JMML. Survival of MDS patients was influenced by the marrow blast % (P = 0.007) and disease category (P= 0.006). Stem cell transplantations (SCT) were undertaken in 56 patients (MDS, 29; JMML, 27). The sources of stem cells were: bone marrow, 36; umbilical cord, 18; peripheral blood, 2). Matched related transplants were 9 cases. Conditioning was various, but BuCy based regimen was used in 68.4%. Acute GvHD ≥ Grade II was found in 43.8% and chronic GvHD in 35.1%. The 5-year Kaplan-Meier overall survival rate was 55% for MDS, and 57% for JMML. Survival after unrelated transplant was comparable with that of matched related transplants. This analysis inspired the necessity of nation-wide prospective studies in Korea, including morphologic study by a central pathology review board, epidemiologic study, molecular pathophysiologic study, and therapeutic trials incorporating SCTs, or new drugs.

Beschreibung: The somatic mutation JAK2 V617F has been identified as a pathogenic factor in typical chronic myeloproliferative diseases (MPD) such as polycythemia vera (PV), essential thrombocythemia (ET), and myelofibrosis with myeloid metaplasia (MMF). In typical forms of myelodysplastic syndromes (MDS), JAK2 V617F mutation is rarely present (2–5%); on the contrary, it has been found with higher prevalence in patients with RARS-T (i.e. MDS/MPD-U with platelet count 〉600×109/L and ringed sideroblasts more than 15%) and in a subgroup of MDS patients with isolated 5q deletion and a proliferative bone marrow. In this study we analysed the JAK2 V617F mutational status in 53 MDS patients (26 males, 27 females; median age at the time of the study 76 years, range 45–91). Patients were classified as follows: 4 cases 5q- syndrome, 3 RCMD, 5 MDS/MPD, 1 MDS-U, 23 RA, 12 RARS, 5 RAEB. DNA was extracted from purified granulocytes; all samples were analyzed by allele-specific polymerase chain reaction (PCR), according to Baxter el al (2005). DNA samples were further subjected to direct sequencing for confirmatory testing. The JAK2 V617F mutation was present in 3 cases, with an overall frequency of 5%. With respect to MDS subtype, 1 patient had RA and 2 RARS. Among the 12 RARS patients, the two V617F postive displayed thrombocytosis (680×109/L and 649×109/L), whereas none of the 10 RARS V617F negative patients showed high platelet counts (median Plt 157×109/L, range 5–422×109/L). In one JAK2 mutant case, thrombocytosis required treatment with hydroxyurea. Moreover, the two V617F positive RARS patients displayed higher WBC count (6.2×109/L and 8.5×109/L) than the V617F negatives (median WBC 4.05×109/L); no difference was observed in Hb levels. The JAK2 positive RA patient had 10% of sideroblasts in bone marrow, normal platelet and WBC count and no proliferative characteristics; since the occurrence of the mutation may be an early event and preceed the classical manifestations of MDS/MPD, a longer follow-up is necessary to determine its possible prognostic significance. Considering the V617F negative MDS cases, only one patient, diagnosed as MDS/MPD, showed a platelet count 〉600×109/L. In conclusion, we confirmed recent reports showing that JAK2 V617F is present with low prevalence (about 5%) in MDS; in particular, the JAK2 mutation identifies a subset of MDS patients with and “overlap” syndrome, characterised by proliferative bone marrow morphology and frequent thrombocytosis and leucocytosis, who may benefit from JAK2 specifically targeted therapies.

Beschreibung: The simultaneous manifestation of different lymphomas in the same patient or in the same tissue is defined composite lymphoma. Although reports of synchronous or metachronous Hodgkin’s lymphoma (HL) and Non Hodgkin’s lymphoma (NHL) are not uncommon in the literature, the biologic relationship of the 2 malignancies is often unclear. Primary cutaneous B-cell lymphomas (pCBCLs) have been recognized as distinct clinicopathologic entities; they represent a wide spectrum of lymphoproliferative disorders separated from of B-cell NHL secondarily involving the skin and cutaneous B-cell pseudolymphomas. As regarding pCBCLs, a concomitant diagnosis of HL has been described very rarely. This is the case of a caucasian man affected by primary cutaneous follicolar B cell lymphoma (pCFCL). He presented grouped red plaques located on the nape, abdomen, shoulders, arms and even some little tumors surrounded by erythematous papules. Complete staging procedures did show no evidence of extracutaneous disease. A subcutaneous interferon therapy was started, in some months the patient reached a complete remission, and a maintainance therapy was continued for about 2 years. After 15 years, at the age of 58, the patient presented a red to violaceous infiltrated solitary plaque on the back, appeared about 2 months before. The lesion was completely excised and the biopsy showed a diffuse dermal infiltrate, not involving the epidermis, structured in follicle with reactive germinal centers; they were surrounded by small sized monomorphic lymphocytes with irregular nuclei and pale cytoplasm, showing the following immunophenotype pattern: CD20+, CD3−, IRTA+, CD10−, BCL6−, BCL2+, low proliferative index. A plasmacellullar CD138+ and CD79a+ population was at the periphery of the infiltrates, with monotypic expression of cytoplasmic k chain. The whole picture was interpreted as primary cutaneous marginal zone B-cell lymphoma (pCMZL). The association with Borrelia burgdorferi infection, sometimes described in pCMZL, wasn’t demonstrated. The patient presented a large right axillary lymph node that was excised and found to be unexpectly infiltrated by HL, mixed cellularity subtype. The patient underwent a standard baseline staging procedure with total body CT scan and bone marrow trephine biopsy; the latter resulted negative; the t(11;14) and t(14;18) rearrangements weren’t demonstrated in bone marrow; the CT did show no other suspected masses nor lymphoadenopathy, besides the clinically evident right axillary lymph node. A 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) revealed sites of hyperactivity in the same right axillary region, but extending to the subclavian region and the thoracic region. The patient started chemoterapy (ABVD regimen), and at the end of 4 courses a whole body CT scan and FDG-PET resulted both negative. Now he is being treated with radiotherapy. If the nodal malignancy would have been diagnosed first, the skin lesion probably could have been misinterpreted as a secondary localization of HL; if the node biopsy wouldn’t have been performed we could diagnose B-cell NHL secondarily involving the skin (stage IV). Nevertheless the cutaneous and nodal infiltrates had a completely different general picture and phenotype. This case probably reflects a HL after 15-year remission of a pCBCL of low grade and the relationship between HL and the preceding pCFCL is not clear: casual or related to genetic predisposition for oncogenic events or favoured by an immunodeficiency state related to the first disease and the previous immunomodulatory therapy.

Beschreibung: Tyrosine kinase inhibitors (TKI) selective for Bcr-Abl, such as dasatinib, imatinib, and nilotinib have had remarkable success in the clinic, potentially shifting the prognosis of chronic myelogenous leukemia (CML) to a manageable chronic disease. With the increase in longevity of CML patients, there is rising concern of co-morbidities that may be influenced by chemotherapy (Force et al., Nature Rev.2007;7:332–340). Recently, congestive heart failure (CHF) and direct cellular cardiotoxicity have been reported in CML patients on imatinib therapy (Kerkela et al., Nature Medicine2006;12:908–916). Ultrastructural mitochondrial abnormalities in cardiomyocytes were observed in CML patients with severe CHF and, interestingly, similar abnormalities were observed in cardiomyocytes of imatinib-treated mice, thus providing a prospective in vivo animal model for imatinib-induced cardiotoxicity. Furthermore, correlative findings of mitochondrial membrane potential loss, decreased cell viability, and increased apoptosis resulted from an array of cell-based assays in imatinib-treated primary rat cardiomyocytes, consequentially affording a supportive, if not predictive, in vitro cardiomyocyte toxicity model. Since imatinib-induced inhibition of the native form of c-Abl kinase was speculated to cause the observed cardiotoxicity and c-Abl is a shared target of dasatinib, imatinib, and nilotinib, the in vitro cardiotoxicity potential of dasatinib and nilotinib at pharmacologically relevant concentrations (0.09 μM and 5 μM, respectively) and up to 10-fold higher concentrations were compared side-by-side with imatinib in primary rat cardiomyocytes. Dasatinib did not significantly affect mitochondrial membrane potential, cell viability, apoptosis, or cellular ultrastructure in vitro, whereas imatinib significantly affected these parameters. Nilotinib at pharmacologically relevant concentration demonstrated decreased cell viability, but differed from imatinib in that mitochondrial membrane potential integrity was not affected under identical experimental conditions. Results suggest that at pharmacologically relevant concentrations, dasatinib does not induce cardiotoxicity, as does imatinib and nilotinib, and the molecular mechanisms of the observed cardiotoxicities may differ between imatinib and nilotinib. Of indirect relation, results from assessing another cardiovascular liability, namely hERG K+ channel blockade, demonstrated that dasatinib, imatinib and nilotinib differentially inhibited the hERG currents in vitro with IC50 of 14.3, 15.6 and 0.66 μM, respectively. These in vitro findings occurred at concentration levels approximately 150, 3 and 0.1-fold the expected human Cmax for the three TKIs, respectively. Thus, although TKI therapies may share similar targeting and clinical indications, differentiating specific toxicity profiles may be predictive of differences in potential clinical adversities.

Beschreibung: Factors regulating which patients become alloimmunized to red blood cell (RBC) antigens are poorly understood. Using a murine model of transfusion, we recently reported that viral-like inflammation with polyinosinic polycytidylic acid [poly (I:C)] significantly enhances RBC alloimmunization. Herein, we tested the hypothesis that poly (I:C) exerts this effect, at least in part, at the level of antigen-presenting cells (APCs). Using a novel in vivo method, we report that in the noninflamed state, most transfused RBCs were consumed by splenic macrophages, with only trace consumption by splenic dendritic cells (DCs). To a lesser extent, RBCs were also consumed by APCs in the liver. However, unlike soluble antigens, no RBCs were consumed by APCs in the lymph nodes. Inflammation with poly (I:C) induced significant consumption of transfused RBCs by splenic DCs, with a concomitant increase in costimulatory molecule expression. Moreover, this resulted in increased proliferation of CD4+ T cells specific for the mHEL RBC alloantigen. Finally, splenectomy abrogated the enhancing effects of poly (I:C) on RBC alloimmunization. Together, these data provide additional insight into the nature of transfused RBCs as an immunogen and provide a mechanism by which viral-like inflammation enhances alloimmunization to transfused RBCs.

Beschreibung: Dietary plant polyphenols are known to have antitumor, antiinflammatory, and antioxidant activity and as such may sensitize tumor cells to chemotherapeutic agents. We have evaluated the effects of the green tea polyphenol epigallocatechin gallate (EGCG) alone and in combination with other drugs on human myeloma cells. The compound is currently under investigation in several phase I/II clinical trials including for treatment of patients with early stage chronic lymphocytic leukemia. EGCG inhibited the in vitro growth of human myeloma cell lines in a time and dose-dependent manner. IC50 concentrations were between 12.5 μM and 50 μM as measured in a colorimetric tetrazolium (MTS) based assay and by trypan blue exclusion. Excess amounts of IL-6, bone marrow stromal cells, or overexpression of Mcl-1 and Bcl-xL could not protect from EGCG induced cytotoxicity. Pretreatment of INA-6 cells with EGCG resulted in a dose-dependent inhibition of IL-6 induced STAT3 tyrosine phosphorylation. In accordance with the essential role of STAT3 for INA-6 cell survival, EGCG induced apoptosis as determined by flow cytometry upon 7-amino-actinomycin D/annexin-V staining. In cell lines not dependent on exogenous IL-6, EGCG induced growth inhibition was abolished by pretreating the cells with 200 U/ml catalase, an enzyme which reduces reactive oxygen species (ROS). The combination of EGCG with doxorubicin, dexamethason, or rapamycin did not result in increased growth inhibition. In contrast, growth inhibition by bortezomib was antagonized with EGCG at concentrations that were not inhibitory when used alone (1–10 μM). In conclusion, EGCG exerts its effects on myeloma cells through several mechanisms including inhibition of IL-6 mediated signalling pathways via STAT3 and induction of oxidative stress. Notably, at pharmacologically achievable concentrations, EGCG antagonized bortezomib activity. Thus, the intake of natural polyphenols (high consumption of green tea or taking green tea extracts) may be critical during therapy with bortezomib.

Beschreibung: PU.1 (Sfpi1) is an ets family transcription factor required for the proper generation of both myeloid (macrophages and neutrophils) and lymphoid lineages (B and T lymphocytes)(Scott 1994, McKercher 1996). Graded expression of exogenous PU.1 in murine PU.1-deficient fetal liver hematopoietic progenitors demonstrated that increased levels of PU.1 are required to initiate development of macrophages (DeKoter, 2000). We have studied the effects of graded expression of PU.1 on its occupancy in chromatin and on the development of myeloid cells in vitro. We measured changes in gene expression, PU.1 occupancy and histone modifications in PU.1-null hematopoietic progenitor cells stably expressing PU.1 fused to the ligand-binding domain of the estrogen receptor (PU.1-ER) (Walsh 2002). The level of active PU.1-ER was regulated with graded levels of the ER inducer tamoxifen. In vitro, intermediate levels of tamoxifen produced cells with granulocyte characteristics in the suspension cell fraction and macrophage-like characteristics in the attached fraction, whereas high levels of PU.1 produced mostly attached macrophage-like cells. Expression of granulocyte-specific PU.1 target mRNAs including gelatinase B (Mmp9) and myeloperoxidase (Mpo) were observed to be expressed only with intermediate levels of tamoxifen. In contrast, expression of macrophage PU.1 target mRNAs including Cd14, F4/80 and Cd68 mRNAs were observed to be gradually upregulated upon PU.1-ER activation, with the maximum expression at the highest levels of tamoxifen. Thus, the expression levels of PU.1 target genes and phenotypic characteristics of the cells are dependent on PU.1 levels. Interestingly, macrophage-like cells can be produced from granulocytic-like cells by changing tamoxifen levels and vice versa. Chromatin immunoprecipitation analysis revealed specific PU.1 occupancy within regulatory regions of the genes predominantly expressed in macrophages including Cd14 and Cd11b after treatment with high levels of tamoxifen. Specific PU.1 occupancy within regulatory regions of the granulocyte specific genes including MMP9 was observed at intermediate levels of tamoxifen. Suprisingly, chromatin immunoprecipitation analysis revealed specific PU.1 occupancy within regulatory regions of the lymphocytic PU.1 target genes including Interleukin-7 receptor (Il-7r) and RAG1 at intermediate levels of tamoxifen even though expression of these genes was not detected. Accumulation of acetylated K9 and methylated K4 of histone H3 in gene loci of macrophage and granulocytic markers such as Cd14, Cd11b, and Mmp9 correlated with their mRNA expression. However, lymphocyte-specific regulatory regions including that of Il-7r gene were hypoacetylated in H3K9 despite a marked PU.1 recruitment suggesting additional factors may be required for PU.1 mediated transactivation. To identify these molecules we have tested PU.1-dependent transcription factors: Egr2, Nab2, Cebpa and Gfi-1 and found that upon increasing PU.1 levels, expression of Egr2/Nab2 and Gfi-1/Cebpa changed in a reciprocal manner and these changes preceded expression of the lineage specific markers. We are currently testing if PU.1 directly regulates expression of Egr2, Nab2, Cebpa and Gfi-1 during granulocytic/macrophage differentiation.

Beschreibung: CD26 (dipeptidyl peptidase IV, DPP IV) is a multifunctional type II cell surface glycoprotein that is widely expressed on T and natural killer cells, as well as on epithelial, endothelial and acinar cells of different tissues; its expression on B cells is very low but it is greatly upregulated following activation. We evaluated, by means of flow cytometry, the expression of CD26 in various types of B-cell lymphoid tumors: Follicular Lymphoma (Fo-Ly, 12 cases), Mantle cell Lymphoma (MCL, 12 cases) Multiple Myeloma (MM, 20 cases), Hairy cell Leukemia (HCL, 12 cases), B-cell Chronic Lymphocytic Leukemia (B-CLL, 112 cases), CD5 negative B-cell Chronic Lymphoproliferative Diseases (CD5neg-B-CLPD, 20 cases) and Diffuse Large cell Lymphoma (DLCL, 12 cases). CD26 expression was absent or low of Fo-Ly and MCL, high on MM and HCL, variable on B-CLL, CD5neg-B-CLPD and DLCL. Fluorescence intensity of positive cells was dim in B-CLL and CD5neg-B-CLPD, heterogeneous in DLCL, and bright in HCL and MM. Interestingly, in CLL patients, CD26 expression was significantly correlated with CD49d and CD38 expressions (p

Beschreibung: Due to the lack of large population-based studies, the incidence of polycythemia vera (PV) has not been well documented in the United States. In 2001, PV became reportable to the Surveillance, Epidemiology, and End Results (SEER) program, which consists of high-quality population-based cancer registries supported and sponsored by the National Cancer Institute. In the present analysis, we accessed the SEER data to estimate the incidence of PV in the United States during 2001 – 2003. Since the addition of PV to SEER reporting is fairly recent, and the diagnosis of PV is different from that of many other types of cancer, especially solid tumors, under-reporting is possible. Therefore, we also used health claims data from Medicare services to estimate the incidence of PV among individuals 65 years and older. SEER data suggest that the age-adjusted incidence rate of PV was 0.87 per 100,000 per year (95% confidence interval 0.83 – 0.91) during 2001 – 2003, which is lower than the estimates from other studies (Ania BJ et al. Trends in the incidence of polycythemia vera among Olmsted County, Minnesota residents, 1935–1989. Am J Hematol1994;47(2):89–93. Johansson P. Epidemiology of the myeloproliferative disorders polycythemia vera and essential thrombocythemia. Semin Thromb Hemost2006;32(3):171–3). The incidence of PV increased with age and was higher in men and white individuals than in women and other racial groups. The SEER-derived age-adjusted incidence rate of PV was 4.1 per 100,000 per year among individuals 65 years and older during 2003, which is considerably lower than the estimates made from nationwide Medicare claims data in the same age group during the same year, suggesting possible under-reporting to SEER. The addition of PV to SEER reporting represents a major step in generating population-based epidemiologic data for this understudied malignancy. However, it is critical to ensure the completeness in the ascertainment and reporting of PV so high-quality SEER data can be utilized to facilitate much needed research on the etiology and outcomes of PV, as well as other myeloproliferative disorders.

Beschreibung: Introduction: Filgrastim is widely used for mobilizing CD34+ cells into the peripheral blood that are easily collected by apheresis for allogeneic transplantation. With case reports documenting splenomegaly with life-threatening complications in normal donors, we prospectively evaluated spleen size using ultrasonography and clinical examination during PBPC mobilization and collection in a single-arm trial. Methods: Subjects ≥18 yrs eligible to be PBPC donors per institutional guidelines enrolled. Splenic assessments were done before, during, and after PBPC mobilization. Filgrastim dose and schedule and leukapheresis (LK) procedures were per institutional practice. The primary endpoint was fold change from baseline in splenic volume in post-baseline measurements during mobilization (measured by ultrasound [US]). Spleen size by US was measured in 3 dimensions similarly by all centers: longitudinal (craniocaudal), transverse, and diagonal (perpendicular to transverse in transverse image) diameters. Splenic volume was estimated by taking the cross-product of 3 dimensions and multiplying by 0.52, approximating the volume of an ellipse. Physical examination was performed on US days, assessing spleen palpability. US and palpation results were blinded from each other at assessment times. Timepoints included baseline (before first filgrastim dose), first LK (done before LK, typically day 4 or 5 of filgrastim), 2 and 4 days after first LK, and 7 days after last LK. Timepoints in the post-amendment cohort (n=219) were reduced to facilitate enrollment and were baseline and day of first LK (before LK). Results: 309 donors enrolled, median age 44yrs (range 18 to 74), 56% male. Mean daily filgrastim dose was 11.4mcg/kg (SD=3.0). Median number of LK was 1.5 (range 1 to 4). In all donors, the median increase in each measured dimension on first LK day was 1.4cm, 1.4cm, and 0.6cm (12.8%, 12.6%, and 15.0%), and the median fold volume increase from baseline to first LK was 1.47, resolving to near baseline 1 week after last LK. There was no apparent relationship between volume fold change and filgrastim dose, ANC, or CD34+ yield. Of 861 splenic palpation assessments reported in all donors, 98% were reported as nonpalpable (842 assessments), and 2% were palpable (19 assessments, 2 at baseline). Reporting of palpable spleens did not correlate with increased spleen size. Tenderness or guarding upon splenic palpation was reported in 2 donors with a spleen considered palpable and in 6 donors with nonpalpable spleens. No donor experienced a splenic rupture. Adverse events related to filgrastim were generally mild to moderate. Conclusion: During PBPC mobilization with filgrastim in normal donors, the spleen increased a median of approximately 50% from baseline to day of first LK and returned to near baseline 1 week after last LK. Size change was not associated with significant clinical sequelae. Timepoint Median fold change from baseline in splenic volume (Q1, Q3) *statistically significant (p

Beschreibung: Introduction: Fludarabine treatment has been shown to be beneficial for patients with Chronic Lymphocytic Leukemia (CLL), and fludarabine-based combinations may even further improve outcomes in patients with CLL. However, most CLL patients eventually become fludarabine refractory, a state which is associated with a relatively short survival. Treatment of fludarabine-refractory patients is challenging, with a median survival of about 10 months. Recently, 2 phase II clinical trials (Chanan-Khan et al. JCO 2006 and Ferrajoli et al. ASH 2006) demonstrated the clinical efficacy of lenalidomide, an immunomodulatory agent, in relapsed/refractory CLL patients. We conducted a subset analysis to examine the efficacy of lenalidomide in patients who are fludarabine refractory. Methods: All patients enrolled on the 2 phase II single agent lenalidomide clinical trials were evaluated and patients with fludarabine-refractory disease (progressed while on or within 6 months of fludarabine-based therapy) were assessed for clinical efficacy of lenalidomide. Lenalidomide was given orally either at 10 mg daily for 28 days followed by 5 mg increments every 28 days to a maximum dose of 25 mg or given at 25 mg on days 1–21 of each 28-day cycle. Response was assessed using the NCI-WG 1996 criteria. Results: A total of 80 patients were collectively enrolled in these clinical studies. Among these, 29 were identified to have fludarabine-refractory disease. Important clinical characteristics of these patients are reported in Table 1. The overall response rate in fludarabine-refractory patients was 34.5% (10/29). Complete remission was observed in 2 (6.8%) patients. Conclusion: Lenalidomide is a novel agent with immunomodulating properties demonstrating clinical efficacy in relapsed or refractory CLL patients. Interestingly, clinical responses to single agent lenalidomide were noted despite refractoriness to fludarabine (a subset of CLL patients with poor survival and limited therapeutic options). This observation of the clinical benefit of lenalidomide independent of responsiveness to prior fludarabine is encouraging and warrants further evaluation. Table 1 Ferrajoli et al. Chanan-Khan et al. Fludarabine-refractory (N=12) Fludarabine-refractory (N=17) ORR, overall response rate; PFS, progression-free survival; OS, overall survival. Median age, years (range) 62 (51–82) 68 (53–75) Sex, female/male 4/8 4/13 Median no. prior therapies (range) 4 (3–15) 4 (1–10) Median beta microglobulin (range) 5 (3–10) 5 (2–10) Advance Rai Stage (III/IV), n (%) 7 (58.3) 13 (76.4) ORR, n (%) 3 (25.0) 7 (41.2) Median PFS, months 12 14.9 Median OS, months All alive (range, 7–19) 23

Beschreibung: Dyskeratosis congenita (DC) is a congenital disorder characterized by very short telomeres. Clinical presentation includes a diagnostic triad (lacey reticular pigmentation, nail dystrophy, and leukoplakia), aplastic anemia (the main cause of premature death), myelodysplastic syndrome/leukemia and solid tumors. Allogeneic HCT is the only curative option for the hematologic complications of DC but has been associated with a high risk of peri-transplant morbidity and early death. Only about fifty HCT for DC have been performed to-date, and the five year survival after related donor HCT has been about 75%, but only approximately 35% when an unrelated donor was used. To improve survival in DC patients by decreasing transplant mortality, we introduced a reduced intensity regimen including cyclophosphamide (50 mg/kg), fludarabine (200 mg/kg), low dose total body irradiation (200 cGy), and (in patients 3 and 4) campath 1H (1 mg/kg). To decrease the risk of graft rejection, grafts were not T-cell depleted. We report outcomes in four consecutive patients, two adults and two children, all of whom engrafted with donor hematopoiesis: Age (years) Sex Graft and HLA match NC dose (×108/kg) CD34 dose (×108/kg) Follow-up (months) Donor chimerism 24 M URD dUCB 4/6, 4/6 0.55, 0.39 0.5, 0.43 1 (dead*) 83% 29 F REL PBSC 6/6 13.93 4.43 20 (alive) 100% 5 F URD BM 7/8 1.38 1.55 16 (alive) 100% 2 M URD BM 8/8 5.92 2.31 3 (alive) 100% Legend: M, male; F, female; NC, nucleated cell; URD, unrelated donor; REL, related donor; BM, bone marrow; dUCB, double umbilical cord blood; PBSC, peripheral blood stem cells; *Patient had autologous recovery after the first dUCB and died of sepsis 1 month after the second dUCB; HLA matching is reported for antigen level HLA-A, B and allele level DRB1 for cord blood, and allele level typing for HLA-A, B, C, DRB1 for PBSC or BM. The most recent donor chimerism is reported. To decrease the risk of graft rejection and prevent graft versus host disease (GvHD) patients received cyclosporine and mycophenolate mofetil. Patient 2 developed limited chronic GvHD and patient 4 developed grade III skin acute GvHD. Both were treated successfully with systemic and topical steroids. Our data suggest that this conditioning regimen results in a low rate of transplant related complications without compromising engraftment. Critically, early fatal pulmonary and vascular complications, common in post-transplant courses in DC patients, were not observed. This highlights the need to avoid drugs that are associated with pulmonary toxicity such as busulfan, and to limit radiation to the lung in patients with DC. This new less intensive conditioning regimen appears to result in a low rate of transplant related complications, and yet has adequate immunosuppressive activity to permit engraftment from alternative donors in DC patients.

Beschreibung: Extensive chronic graft-versus-host disease (ecGVHD) is characterized by fibrosis similar to that of patients with systemic sclerosis (scleroderma). Since stimulatory autoantibodies against the platelet-derived growth factor (PDGF) receptor (PDGFR) have been found in patients with scleroderma and are responsible for the activation of skin fibroblasts, we tested the hypothesis that these autoantibodies are also present in patients affected by ecGVHD. Serum from 39 patients subjected to allogeneic stem cell transplantation for hematologic malignancies (22 with ecGVHD and 17 without cGVHD) and 20 healthy controls was assayed for the presence of stimulatory autoantibodies to the PDGFR by incubating purified IgG with mouse-embryo fibroblasts lacking PDGFR α or β chains or with the same cells expressing PDGFR α. Stimulatory antibodies to the PDGFR were found selectively in all patients with ecGVHD but in none of the patients without cGVHD. Higher levels were detected in patients with generalized skin involvement and/or lung fibrosis. Antibodies recognized native PDGFR, induced tyrosine phosphorylation, accumulation of reactive oxygen species (ROS), and stimulated type 1 collagen gene expression through the Ha-Ras-ERK1/2-ROS signaling pathway. The biologic activity of these autoantibodies suggests a role in the development of fibrosis and argues for a common pathogenetic trait in ecGVDH and scleroderma phenotypes.

Beschreibung: Background: The initial genetic event in ∼85% of follicular lymphomas (FL), the most common B-cell lymphoma in North America, is the t(14;18)(q32;q21) resulting in over-expression of the anti-apoptotic protein Bcl-2. The secondary events associated with disease progression are not well understood. Alterations affecting the p arm of chromosome 1 are evident by standard karyotype analysis in ∼20% of FL. We have further examined the relationship between 1p deletion and FL using high resolution genomic analyses. Methods: The prevalence of 1p alterations was investigated in 139 cases of indolent and transformed FL using whole genome tiling path BAC array Comparative Genomic Hybridization (array CGH). Array-based single nucleotide polymorphism analysis was performed on a subset of cases using Affymetrix 500K SNP arrays. Results: Array CGH identified a minimum region of deletion spanning ∼0.5MB within 1p36.32 in 51 cases (37%). In 38 cases (27%) this loss was exhibited in the transformed sample but not the pre-transformation sample. The majority of cases displayed heterozygous deletion, while two cases showed homozygous deletion. The mechanisms of loss included simple deletions, unbalanced translocations with various partner chromosomes and eleven cases with an unbalanced t(1;1)(p36;q12). The Affymetrix 500 SNP array analyses showed copy neutral loss of heterozygosity or acquired uniparental disomy (aUPD) in three of ten cases that were negative for loss by aCGH. Contained within the 1p36.32 minimally deleted region are only a few candidate genes including tumor necrosis factor receptor superfamily 14 (TNFRS14), which has been implicated in growth inhibition of HT-29 human colon adenocarcinoma cells and induction of Fas-mediated apoptosis in non-Hodgkin’s lymphoma. Conclusions: Our data indicate that loss of heterozygosity at 1p36.32 through deletion or aUPD constitutes the most common secondary cytogenetic event in FL. LOH at 1p36 may represent an important step in the progression of indolent to transformed FL. Further studies have been initiated to investigate other possible gene inactivation events such as methylation and mutation.

Beschreibung: CD4+CD25+Foxp3+ regulatory T cells (CD25+ Treg cells) direct the maintenance of immunological self-tolerance by active suppression of autoaggressive T-cell populations. However, the molecules mediating the anergic state and regulatory function of CD25+ Treg cells are still elusive. Using differential proteomics, we identified galectin-10, a member of the lectin family, as constitutively expressed in human CD25+ Treg cells, while they are nearly absent in resting and activated CD4+ T cells. These data were confirmed on the mRNA and protein levels. Single-cell staining and flow cytometry showed a strictly intracellular expression of galectin-10 in CD25+ Treg cells. Specific inhibition of galectin-10 restored the proliferative capacity of CD25+ Treg cells and abrogated their suppressive function. Notably, first identified here as expressed in human T lymphocytes, galectin-10 is essential for the functional properties of CD25+ Treg cells.

Beschreibung: Background: Romidepsin is a bicyclic peptide that inhibits Class I and II HDACs. Piekarz et al noted responses to romidepsin in CTCL (ASCO, 2004). This pivotal phase II study sought to confirm the activity. Methods: This single arm, open label study enrolled CTCL (Stages 1B–1VA), including MF and Sézary syndrome (SS) patients (pts) from ∼40 sites in Europe, Russia, Ukraine, Georgia and the US. Pts with biopsy-proven CTCL (centrally reviewed) who failed ≥1 prior systemic therapy received romidepsin at 14 mg/m2 as a 4-hour IV infusion on Days 1, 8, and 15 q 28 days for up to 6 cycles but could continue if deriving benefit. Eligibility criteria included adequate organ function and ECOG PS ≤ 1. Exclusions included significant CV abnormality or treatment with QTc-prolonging or CYP3A4-inhibiting drugs. The primary endpoint is response rate measured by a combination of imaging, circulating malignant T-cell counts and a weighted scoring instrument to determine skin involvement (confirmed by photography). Target accrual of 64 evaluable pts (i.e. received 2 courses) has been reached and the study will close. Results: 92 pts were eligible with 68 evaluable for efficacy per protocol. Responses in evaluable pts are 1 CR, 3 CCRs, 20 PRs, 40 SD and 4 PD for an ORR of 35% (duration 2–21 months). Of pts who received ≥1 dose, the ORR is 26% (24/92) but includes 5 too early to be assessed. Median time to response is 8 weeks (range 4 – 20). Responses by stage at entry in evaluable pts, as available: Stage IB-IIA 7/23 (30%); Stage IIB-IVA 15/37 (41%). In pts with pruritus at baseline i.e. score of ≥ 30 mm on a 100 mm visual analogue scale (VAS), relief of ≥ 30 mm from baseline or a VAS score of 0 (no itching) for at least 2 cycles, was seen in 18/38 pts (47%). In those pts with severe pruritus (VAS score ≥70 mm), 14/24 (58%) experienced relief of itching. Adverse event (AE) data are available for 75 pts. AEs were reported in 54/75 (72%) of dosed pts but Grade (G) 3/4 events in only 12/75 (16%). Most frequent AEs are nausea/vomiting (G2) fatigue (G2/3), myelosuppression (G2/3), and asymptomatic ECG changes (transient mild QTc prolongation and nonspecific ST-T wave abnormalities). Thirteen pts withdrew due to AEs but there were no treatment-related deaths although 4 pts died of PD and 1 from right ventricular failure. Serious AEs considered possibly, probably or likely related to treatment were reported in 12 pts. Of these, 8 had ≥G3 events: tumor lysis, cardiac tamponade, sepsis, constipation, oral candidiasis, dermatitis, hyperglycemia/vomiting/nausea and bradyarrhythmia/atrial fibrillation. Conclusions: This study confirms the efficacy of romidepsin in treatment-refractory CTCL including relief of pruritus and an encouraging response rate with 4 CCR (1 pathology-confirmed). The low rate of discontinuation due to AEs and prolonged treatment duration of some patients illustrate that toxicity has been manageable.

Beschreibung: Waldenstrom’s Macroglobulinemia (WM) is an incurable B-cell malignancy characterized by bone marrow (BM) infiltration with a spectrum of clonally related cells, including small lymphocytes and lymphoplasmacytic cells (CD19+) as well as mature plasma cells (CD138+). The molecular pathogenesis of the disease remains to be defined. We therefore analyzed the gene expression profiles of CD19+ and CD138+ BM mononuclear cells from 30 untreated patients with WM and compared their gene expression profile to their normal counterparts from 10 healthy donors using Affymetrix microarrays (U133 plus 2.0). Since the microenvironment plays an important role in the pathogenesis of WM, we also profiled and compared gene expression profiling for CD19 and CD138 depleted BM mononuclear cells from the same patients and healthy donors. Gene expression analysis was performed using dChip software. Unsupervised hierarchical cluster analysis demonstrated distinct gene expression patterns between WM cells versus their normal counterparts. In supervised hierarchical cluster analysis selecting for genes with 〉 2 fold change in expression and a False discovery Rate (FDR) 〈 2%, a set of 1171, 582 and 360 genes were found to be differentially expressed between WM patient and healthy donor CD19+, CD138+, as well as CD19/CD138 depleted (microenvironmental) cells, respectively. Among the most significantly over-expressed genes in the CD19+ compartment in WM patients were: BCL2, TNFRSF13B, TNFRSF17, IGLL1, CCR2, CLLU1, whilst the AP1 family genes JUND and FOSB were among the most significantly down-regulated genes in both CD19+ and CD138+ cells in WM patients. Other interesting transcripts which were over-expressed in CD138+ cells from WM patients included those from genes involved in transcription regulation (ZKSCAN1, ZMYM1, ZNF189, ZNF19, and ZNF559) and interferon response (IFI16 and IFIH1). Of considerable interest was our observation that microenvironmental cells in WM patients demonstrated an overactive transcriptional profile composed of genes which are associated with immune and inflammatory responses including the Toll like receptors (TLR 1,5,7,8), interferon and cytokines (IFI16, IFNAR1, IL-10R, IL-8R), genes encoding extracellular matrix components (Fibronectin and Hepatocyte Growth Factor) as well as genes involved in apoptotic signaling (TNFSF10, TRAF4). These studies provide the first comprehensive molecular characterization of WM, dissecting the molecular features of the two immunophenotypically distinct populations of malignant cells, and providing for the first time evidence for a distinct molecular profile in BM microenviromental cells.

Beschreibung: Introduction: Event-free survival (EFS) for children with ALL is approximately 80%. Despite substantial success in achieving second and subsequent remissions, survival of patients with relapsed ALL (rALL) remains dismal. We propose that progress depends on identification of novel drug combinations with more activity in rALL than those commonly employed. The literature suggests a 40% CR rate for second and subsequent relapse (Br. J Haematol2005;131:579). However, CR rates depend on the number of prior therapeutic attempts and duration of any prior response, complicating identification of promising regimens. Limited patient numbers and a large number of potential candidate regimens discourage randomized trials. We surveyed local experience with rALL to establish a robust benchmark for evaluation of novel drug combinations. Methods: The TACL Consortium (www.tacl.us) was formed to develop novel drug combinations for patients with rALL. We initiated a review of all patients with rALL treated between 1995 and 2004 at eight TACL institutions. Detailed data on therapy, response, and duration of response were collected on all patients. Results: Of 313 rALL patients, 62% were males, 27% were older than 10 years at diagnosis, 26% had initial white blood counts (WBC) at diagnosis 〉=50,000/uL, and 46% were high-risk by NCI risk criteria. Re-induction attempts ranged between 1 and 9 and most commonly employed combinations of traditional ALL agents. Limiting analyses to patients with marrow involvement, we obtained 86% CR’s for 1st relapse (95% confidence interval 80%–90%), 44% for second relapse (35%–53%), and 30% for third relapse (19%–43%). CR rates declined with the number of prior treatment attempts (see Table, p

Beschreibung: Introduction: DIC is a complication that occurs during serious infection with Gram-negative bacteria. Endotoxin is a component of the bacterial cell wall that elicits a cytokine-mediated cascade of tissue factor-dependent hypercoagulable reactions. The resulting hypercoagulable state may be inhibited by potent anticoagulation. Rivaroxaban is an oral, direct Factor Xa (FXa) inhibitor in advanced clinical development for the prevention and treatment of thromboembolic disorders. The aim of this study was to examine the effects of rivaroxaban in a rat model of endotoxin-induced DIC. Methods: Rivaroxaban (0.1–10 mg/kg p.o.) or vehicle control (PEG400/H2O 60/40%, 5 mL/kg p.o.) were administered 30 minutes before endotoxin injection (lipopolysaccharide O55 B5 [LPS], 250 μg/kg i.v.) to conscious rats. Blood samples were withdrawn from anesthetized animals by heart puncture 4 hours after LPS injection and fibrinogen, platelet count, thrombin-antithrombin (TAT) complex levels and IL-6 were measured. Results: The induction of DIC was indicated in the placebo + LPS group vs vehicle control by decreased fibrinogen (2.12±0.08 vs 2.69±0.10 g/L) and platelet count (571±28 vs 904±30×109/L) an increase in TAT (75±9 vs 2±1 μg/L) and IL-6 (8.6±1.23 μg/L vs 0.028±0.020 μg/L). Pretreatment with rivaroxaban (0.1–10 mg/kg p.o.) dose-dependently ameliorated the effects of LPS on fibrinogen, platelets and TAT. Rivaroxaban 10 mg/kg p.o. normalized fibrinogen (2.58±0.07 g/L) and TAT (5.6±1.2 μg/L) and increased platelet count (703±19×109/L) (Table). Rivaroxaban also slightly reduced the plasma levels of the pro-inflammatory cytokine interleukin-6 (IL-6) (4.24±0.67 μg/L). Conclusions: These results show that direct, selective inhibition of FXa by rivaroxaban effectively normalized the hypercoagulable reactions of endotoxemia with a slight modulating effect on the generation of pro-inflammatory active cytokines, such as IL-6, in the rat DIC model. Further research into the use of rivaroxaban in the management of DIC is therefore warranted. Effects of rivaroxaban in an endotoxin-induced DIC model in rats 4 hours after LPS injection. Results show mean ± SEM. Vehicle control Placebo + LPS Rivaroxaban 1 mg/kg + LPS Rivaroxaban 10 mg/kg + LPS DIC, disseminated intravascular coagulation; IL-6, interleukin-6; LPS, lipopolysaccharide O55 B5; SEM, standard error of the mean; TAT, thrombin-antithrombin ## p

Beschreibung: Introduction: Radioimmunotherapy (RIT) is a new treatment for B non Hodgkin’s lymphoma (NHL) patients. 90Y ibritumomab tiuxetan (Zevalin®) consists of a murine monoclonal antibody to CD20, conjugated to a metal chelator tiuxetan for retention of the beta emitter 90Y. Thus Zevalin® delivers radiation to B-NHL, combining the tumor targeting attributes of a monoclonal antibody and the beta radiation of 90Y. Zevalin® is approved for the treatment of follicular lymphoma (FL) refractory to or relapsed after rituximab, on the bases of clinical trials where it achieved a response rate as high as 83%. Several ongoing registrational trials are evaluating the efficacy of Zevalin® in other NHL, as diffuse large B cell (DLCL) and mantle cell lymphoma (MCL). We are here evaluating the effect of Zevalin® as consolidation therapy in NHL patients that achieved a complete clinical response (CCR) with chemotherapy. Methods: In B cell NHL patients that achieved a CCR after 1st or multiple lines anthracyclines based chemotherapy +/− Rituximab, minimal residual disease was evaluated by PCR on bone marrow samples, for the following rearrangements: JH, Bcl-1, Bcl-2. Patients received Zevalin® 6-9 weeks post chemotherapy. Evaluation of molecular response was assessed after a follow up period at 12 weeks. The aim of the study was the role of Zevalin® in inducing a complete molecular response (CMR). Results: 23 B-NHL patients (13 FL, 6 MCL, 4 DLCL; male:female 13:10, median age 63, range 42–73. See table) in a CCR after chemotherapy (documented by TC scan and/or PET-scan negative for abnormal lesions or glucose captation) have been enrolled. 10 patients had a pathological rearrangement before RIT, while 13 were already in a CMR condition. Zevalin® was completed in all 23 patients and the post infusion evaluation was performed after 12 weeks. In the follow-up period thrombocitopenia was commonly documented, but it was not associated to bleeding or need of platelet transfusion, but in one singular case. After 12 weeks from RIT a new molecular evaluation was performed on bone marrow samples. All the 23 patients have completed the 12 weeks follow-up: 8 of 10 (80%) patients positive before RIT achieved a CMR with Zevalin® administration. The 13 PCR negative patients maintained the CMR. The 21 PCR negative patients are now under follow-up to evaluate the molecular disease free survival after Zevalin® RIT. Conclusion: Zevalin® is an efficient consolidation therapy in B cell NHL patients after chemotherapy. In this series of patients Zevalin® administration allowed to convert 8 of 10 CCR to CMR. In the remaining 13 patients Zevalin® maintained the CMR. Zevalin® addition to medication treatment is feasible and associated with manageable hematological toxicity. Pts disease sex age previous chemotherapy lines molecular response before RIT molecular response after RIT 1 FL M 68 1 POS NEG 2 FL F 53 1 NEG NEG 3 FL M 54 1 NEG NEG 4 FL M 51 4 NEG NEG 5 DLCL F 66 2 POS NEG 6 DLCL F 67 1 NEG NEG 7 FL F 42 1 POS POS 8 FL M 52 1 POS NEG 9 FL F 54 3 NEG NEG 10 FL M 57 2 POS NEG 11 FL F 62 2 POS NEG 12 FL M 58 2 POS NEG 13 FL F 69 2 NEG NEG 14 MCL M 62 1 POS NEG 15 MCL M 66 1 POS POS 16 MCL M 66 2 NEG NEG 17 MCL M 67 1 POS NEG 18 FL F 67 2 NEG NEG 19 DLCL F 67 3 NEG NEG 20 MCL M 70 2 NEG NEG 21 FL M 61 4 NEG NEG 22 DLCL M 43 2 NEG NEG 23 MCL F 73 2 NEG NEG

Beschreibung: INTRODUCTION: Janus kinase 2 gene (JAK2) encodes for a cytoplasmic tyrosine kinase involved in normal hematopoietic growth factor signaling. Point mutations of the JAK2 gene on chromosome 9, specifically V617F, a point mutation at amino acid 617, are associated with myeloproliferative disorders (MPD). The V617F JAK2 mutation has been found in 90% of patients with polycythemia vera, 50–60% of patients with essential thrombocythemia or idiopathic myelofibrosis and 1–5% of patients with other MPD. To our knowledge, previous studies involving the V617F JAK2 mutation were not performed on a control population of normal individuals. Therefore, the prevalence of this mutation has not been established. In this study, we tested volunteer blood donors from a hospital-based blood donation center for the presence of the V617F JAK2 mutation. METHODS: Citrated whole blood was obtained from volunteer blood donors, age 17 and older, who presented to donate whole blood at a hospital-based blood donation center. The donors met all qualifications to donate blood as defined by FDA regulations. DNA was extracted using the QIAagen and QIAamp DNA extraction columns, quantified and diluted to 100ng/ul. DNA was simultaneously amplified and detected using allele specific minor groove binder probes and primers for the V617F JAK2 mutation. The resultant amplification was recorded by real-time, quantitative PCR using an ABI 7500 (Applied Biosystems, Foster City, CA). A 1% limit of detection, determined from sensitivity and specificity studies using a known cell line control, was set as the technically reproducible threshold sensitivity of the test. Samples were defined as negative for the V617F JAK2 mutation if only the wild type allele was detected. Samples that had a mutant allele detected above the 1% limit of detection were defined as positive for the V617F JAK2 mutation. Samples that had a mutant allele detected below the 1% limit of detection were defined as negative for the V617F JAK2 mutation. RESULTS: A total of 181 DNA samples from volunteer blood donors were tested for the V617F JAK2 mutation. The test group consisted of 104 males (mean age 44, range 17–77) and 77 females (mean age 42, range 18–71). Of the 181 donors tested, 171 had only wild type allele detected and were considered negative. Ten donors had high background of the mutant allele detected below the 1% limit of detection and were considered negative. DISCUSSION: To our knowledge, this is the first report documenting the prevalence of the V617F JAK2 mutation in a healthy blood donor population. In this study of 181 volunteer blood donors none had the V617F JAK2 mutation. Although 10 of the 181 donors were found to have mutant allele detected, they were below the 1% technically reproducible sensitivity threshold of the test and were considered negative. We recommend that mutations detected below the technical threshold of 1% of our assay be considered false positives. The results of this study suggest that the V617F JAK2 mutation is not present in a healthy blood donor population and is significant when detected by our method.

Beschreibung: The capacity of CD4+CD25+Foxp3+ (Treg) cells to regulate adaptive and innate immune responses has led to studies investigating their application to regulate allogeneic T cell responses arising during hematopoietic stem cell transplants (HCT). With respect to HCT, a fundamental clinical concern is the reconstitution of the lymphoid compartment in recipients, particularly T cells since this process can be exceedingly delayed. We have previously found that host Treg cells can regulate resistance to engraftment following HCT, demonstrating that such cells survive and function at least transiently in recipients. The present studies investigated the residual host Treg compartment following HCT. Utilizing varying levels of total body irradiation (5.0 – 14Gy), we observed: recipient CD4+CD25+Foxp3+ cells can survive ablative as well as reduced intensity conditioning, the surviving, i.e. residual Treg cells undergo expansion as assessed by BrdU uptake and cell numbers, and these cells comprise the majority of the Treg compartment in recipients for several months post-HCT during which time donor derived Treg cells gradually arise and cede this compartment. Residual Tregs also dominated the compartment following allogeneic HCT of MHC-matched bone marrow depleted of T cells. To assess the functional capacity of the residual Treg cell compartment, the development of autoimmune disease following transplant of IL-2Rβ −/− (CD122−/−) bone marrow into syngeneic recipients with and without residual Tregs was examined. Autoimmune disease symptoms and T cell alterations were prevented in B6-wt but not T cell deficient recipients. Interestingly, the failure to transfer autoimmune disease following IL-2Rβ −/− HCT into lethally conditioned B6-CD4−/− recipients was associated with the presence of a peripheral CD8+FoxP3+ population not detected in B6-wt mice or the B6-wt mice transplanted with IL-2Rβ −/− BM. This finding indicates that in the genetic absence of CD4+ T cells, a CD8 regulatory population appears to emerge. In total, our observations support the notion that functioning host Tregs initially occupy a niche in the transplant recipient permitting lymphopenic expansion and an extended period of contribution to this compartment. Notably, this contribution reflected much greater levels than conventional T cell populations - even in aggressively conditioned recipients. Finally, these findings imply that the presence of host regulatory cells may be important to consider with respect to eliciting anti-tumor responses and vaccination in recipients during the early period post -hematopoietic cell transplantation.

Beschreibung: Imatinib mesylate has revolutionized treatment of Chronic Myeloid Leukemia. Yet for women of child bearing age problems may arise during pregnancy. While continuation of the drug is in the interest of maternal health, the safety of this approach for the fetus has been questioned. Therapy is therefore usually immediately interrupted on recognition of pregnancy. Only three successful pregnancies have been reported to date in which imatinib was continued. A 34-year-old woman of Chinese descent in complete cytogenetic and major molecular remission (i.e. 3-log reduction of BCR-ABL transcripts) of her CML in chronic phase on 400mg imatinib daily continued treatment during conception and throughout pregnancy. After an uneventful full term pregnancy she gave birth to an apparently healthy son. The mother retained her major molecular remission. Routine screening for congenital hypothyroidism of her child however repeatedly revealed an elevated TSH, rising from 11 mU/l at the age of 1 week to15 mU/l at 4 weeks, associated with a relatively low free T4 of 24.8 and 17.4 pmol/l respectively. Thyroglobulin and thyroxine binding globulin concentrations were normal for age. The mother was euthyroid and had no detectable anti-thyroid antibodies. A mild primary hypothyroidism was suspected. An ultrasound showed no developmental defect of his thyroid gland and a 123I scan with a perchlorate discharge test ruled out an intra-thyroidal iodide organification defect. There was no family history of congenital hypothyroidism. The child was put on levothyroxine and is currently doing well. Hypothyroidism has been noticed as a potential side effect of imatinib therapy in thyroidectomized patients receiving levothyroxine and has been ascribed to enhanced non-deiodination clearance of T4 (de Groot et al Clin Pharmacol Ther2005;78:433–8). An induction of uridine diphosphate-glucuronosyl transferases by imatinib was hypothesized as mechanism for an altered levothyroxine metabolism in these patients. Congenital hypothyroidism has to the best of our knowledge not been reported as a potential consequence of imatinib therapy.

Beschreibung: The relative importance of various human leukocyte antigen (HLA) loci and the resolution level at which they are matched has not been fully defined for unrelated donor transplantation. To address this question, National Marrow Donor Program data from 3857 transplantations performed from 1988 to 2003 in the United States were analyzed. Patient-donor pairs were fully typed for HLA-A, -B, -C, -DRB1, -DQB1, -DQA1, -DPB1, and -DPA1 alleles. High-resolution DNA matching for HLA-A, -B, -C, and -DRB1 (8/8 match) was the minimum level of matching associated with the highest survival. A single mismatch detected by low- or high-resolution DNA testing at HLA-A, -B, -C or -DRB1 (7/8 match) was associated with higher mortality (relative risk, 1.25; 95% CI, 1.13-1.38; P 〈 .001) and 1-year survival of 43% compared with 52% for 8/8 matched pairs. Single mismatches at HLA-B or HLA-C appear better tolerated than mismatches at HLA-A or HLA-DRB1. Mismatching at 2 or more loci compounded the risk. Mismatching at HLA-DP or -DQ loci and donor factors other than HLA type were not associated with survival. In multivariate modeling, patient age, race, disease stage, and cytomegalovirus status were as predictive of survival as donor HLA matching. High-resolution DNA matching for HLA-A, -B, -C, and -DRB1 alleles is associated with higher rates of survival.

Beschreibung: Risk factors for ONJ in MM pts include dental extraction, bisphosphonates (BP) use, older age and longer survival. There is also an increased risk of skeletal related events (SRE) in ONJ pts (Badros, JCO 2006). The current study provides long term follow-up data for ONJ pts with regard to ONJ recurrence, SRE and MM status. The study included 97 pts: 60 from Greece and 37 from the US. Pts’ characteristics are summarized in the table below. Median follow-up time has not been reached; lower limit of the 95%CI was 3.2 yrs. ONJ resolved in 60 of 97 pts (62%), resolved and recurred in 12 pts (12%), and did not heal over a 9 months period in 25 pts (26%). Dental extraction preceded ONJ in 46 of 97 pts (47%) and was more common in pts with a single episode of ONJ (35 of 60, 58%) than in the recurrent and non-healing pts (11 of 37, 30%) (p-value=0.007). The median number of ONJ episodes in the recurrent group was 3 (range, 2–6); recurrence of ONJ was precipitated by re-initiation of BP and by dental procedures in 5 and 4 pts of 12, respectively. There was a trend toward higher ONJ recurrence rate in the US (8 of 37, 22%) versus the Greek (4 out of 60, 7%) pts (p-value=0.053). Surgery was performed more often in the US than in Greece 17 of 37 (45%) versus 19 of 60 pts (32%). BP reinitiation was more frequent in US 16 of 37 (43%) than in Greece 3 of 60 (5%). Non-healing ONJ lesions were managed with antibiotics; 10 of 25 pts developed fistulas and needed surgery; in 9 pts the lesions remained asymptomatic. Twenty-one ONJ pts had SRE including fractures (ribs, vertebrae and long bones, n=13) and avascular necrosis of the femur (n=8). The rate of MM relapse was higher in pts with recurrent and non-healing ONJ (84%) compared to pts with a single episode (62%) (p-value=0.02). The median OS from diagnosis of MM was 10.8 yrs (95% CI; 9.3 yrs- not reached) and did not differ between pts with single, recurrent/non-healing ONJ (p= 0.2). In summary, pts in whom ONJ followed dental procedures were less likely to have recurrence or non-healing, both, although infrequent, were linked to BP re-challenge, mostly in the setting of relapsed MM. Non-healing ONJ lesions remained stable/asymptomatic without extensive intervention. BP should be discontinuation until ONJ lesions heal. The decision to restart BP should be individualized based on MM-SRE risk. ONJ Pts characteristics and outcome AA, African American; ttt, treatment; CR, complete remission; PR, partial remission; PD, progressive disease; Dex, dexamethasone, thal, thalidomide; Len, lenalidomide; Bort, bortezomib; A, pamidronate; Z zoledronic acid. The Fisher’s Exact test was used, all p-values reported are two-sided. ONJ, n= 97 one episode, n=60 recurrent, n=12 non-healing, n=25 age at MM; median (range) 60 (26–77) 61 (26–77) 55 (43–76) 61 (36–73) Sex; male/female 59/38 38/22 8/4 13/12 Caucasian/AA 87/10 54/6 10/2 23/2 Isotype; IgG, A, D, LCH 60/20/1/16 36/11/1/12 7/2/0/3 17/7/0/1 MM ttt at ONJ (n=93); none/dex/thal/len/bort 22/31/26/6/8 11/25/16/4/2 5/1/3/1/1 6/5/7/1/4 MM status at ONJ diagnosis; CR/PR/PD 7/54/33 4/37/17 3/8/1 0/9/15 BP use; AZ/Z 59/35 34/23 10/2 15/10 Dental extraction 46 35 5 5 Restarted BP 19 11 6 2 bone complciations 21 14 3 4 MM course after ONJ; continous remission/Relapse 29/68 23/37 2/10 4/21 MM status at last follow up; CR/PR/PD (died) 3/59/35(28) 3/35/22(20) 0/10/2(2) 0/14/11(6)

Beschreibung: Background: The phosphatidylinositol 3-kinase/mammalian target of rapamycin (mTOR) signal transduction pathway integrates signals from multiple receptor tyrosine kinases to control cell proliferation and survival. Everolimus (RAD001, Novartis Pharmaceuticals) is an oral investigational antineoplastic agent that targets mTOR. Objectives: To learn the anti-tumor activity and toxicity of single-agent RAD001 in pts with relapsed/refractory aggressive NHL. Patients and Methods: Patients were eligible if they had measurable disease, a platelet count 〉75,000, an absolute neutrophil count 〉1,000, and a creatinine and bilirubin

Beschreibung: Background: Mantle cell lymphoma (MCL) is a distinct type of non-Hodgkin’s Lymphoma characterized by being incurable with a low response rate and short progression free survival when treated with conventional chemotherapy agents. Lenalidomide (Revlimid®), an immunomodulatory drug, is approved for the treatment of relapsed/refractory multiple myeloma and myelodysplastic syndromes associated with a del(q5) cytogenetic abnormality. Lenalidomide has also shown activity in chronic lymphocytic leukemia and cutaneous T-cell lymphoma. Aim: To determine the activity and safety of lenalidomide in relapsed/refractory MCL. Methods: Patients with relapsed/refractory MCL and measurable disease ≥ 2 cm after at least 1 prior treatment regimen were eligible. Patients received 25 mg lenalidomide orally once daily on Days 1–21 every 28 days and continued therapy for 52 weeks as tolerated or until disease progression. Response and progression were evaluated using the IWLRC methodology. Results: Fifteen patients with MCL were enrolled. Median age was 67 (45–84) and 7 were female. Median time from diagnosis to lenalidomide was 5.1 (0.7–12.7) years and median number of prior treatment regimens was 4 (2–6). Eight patients (53%) exhibited an objective response (1 complete response (CR), 1 complete response unconfirmed (CRu) and 6 partial responses (PR)), 2 had stable disease (SD) and 5 had progressive disease (PD). All eight responses were in eleven patients having a tumor burden 〈 50 cm2 and a time since last rituximab therapy of ≥ 230 days. No responses were achieved in four patients having a tumor burden ≥ 50 cm2 or a time since last rituximab therapy of 〈 230 days. Four of 5 patients (80%) with a prior stem cell transplant responded. Progression free survival [PFS] is 5.7 months and ongoing. Seven patients (47%) required at least one dose reduction with a median time to first dose reduction of 2.3 months (1.2–4.9). Grade 4 adverse events were neutropenia (13%), thrombocytopenia (13%), and thromboembolism (13%). Most common Grade 3 adverse events were neutropenia (33%) and leukopenia (20%). Conclusion: Lenalidomide oral monotherapy produced a 53% response rate in relapsed/refractory MCL with manageable side effects.

Beschreibung: Methotrexate (MTX) is an antifolate widely used to treat childhood acute lymphoblastic leukemia (ALL). MTX is retained within cells as long-chain polyglutamates (MTX-PGs), after metabolism by the enzyme folylpoly-γ-glutamate synthetase (FPGS). Intracellular retention of MTX-PGs results in enhanced cytotoxicity due to prolonged inhibition of dihydrofolate reductase (DHFR), and the additional inhibition of thymidylate synthetase (TS). The FPGS gene was shown to be regulated by the transcription factors Sp1 and NFY. We performed DNaseI hypersensitive assays and identified a hypersensitive site mapping closely upstream of exon 1 suggesting that chromatin remodeling may contribute to FPGS gene regulation. Using co-immunoprecipitation and Western blotting we investigated the role of histone modifications and chromatin remodeling on the expression of FPGS and uncovered interactions between NFY, Sp1 and HDAC1. Our results demonstrate that HDAC1 complexes with NFY and Sp1 transcription factors in both B- and T-ALL cells. DNA affinity precipitation assay (DAPA) revealed that the HDAC1-NFY and HDAC1-Sp1 complex binds to the NFY and Sp1 binding sites in the FPGS promoter. These findings suggest that transcription of the FPGS gene may be regulated by acetylation of NFY and Sp1 factors and interaction with HDAC1, and/or chromatin remodeling. We then examined the effect of the histone deacetylase inhibitor (HDACi) sodium butyrate (NaBu) on the expression of FPGS and other folate-related genes. The level of FPGS, ATP-binding cassette subfamily C (ABCC1), ATP-binding cassette subfamily G (ABCG2), DHFR, γ-glutamyl hydrolase (GGH), solute carrier family 19/folate transporter (SLC19A1), and TS mRNA gene expression was determined by qRT-PCR in NALM6 (Bp-ALL), REH (Bp-ALL, t(12,21)/TEL-AML1), SupB15 (Bp-ALL, t(9,22)/BCR-ABL), and CCRF-CEM (T-ALL) cells treated with NaBu [2mM-5mM]. In all cell lines examined, treatment with NaBu induced 2- to 5-fold the level of FPGS and ABCC1 mRNA expression whereas the level of DHFR, SLC19A1, and TS mRNA expression was decreased. Expression of GGH and ABCG2 mRNAs was increased 2-fold in CCRF-CEM but remained unaltered in Bp-ALL NaBu treated cells. Promoters of butyrate-responsive genes have been shown to contain genetic elements such as Sp1/Sp3 binding sites which interact with HDAC1 to mediate the action of NaBu. On this basis, we hypothesized that pre-treatment of ALL cells with NaBu should lead to induction of FPGS expression and subsequently, higher synthesis of MTX-PG and enhanced MTX cytotoxicity in ALL cells. To test this hypothesis, CCRF-CEM, NALM6, REH, and SupB15 cells were treated sequentially with NaBu (24h) and MTX (4h), and assessed for cell viability. Treatment of NaBu and MTX increased cell death by ∼40% in NALM6, REH, and SupB15 Bp-cells, and ∼60% in CCRF-CCEM cells when compared to treatment with each drug alone. These data suggest that combination of HDACi and MTX may represent a novel therapeutic strategy for treatment of ALL. This strategy may be particularly useful to overcome MTX resistance in patients diagnosed with phenotypes that accumulate low levels of MTX-PGs and for patients after relapse.

Beschreibung: Context: Akt plays a pivotal role in the survival and proliferation of multiple myeloma (MM) cells and functions as a central link between upstream signaling pathways, such as growth factor receptors (e.g. IL-6/IL-6R, IGF/IGF-1R) and the kinase PI-3K with their downstream signaling effectors, such as the multifunctional mTOR-p70S6K cascade. The pivotal role of Akt in these proliferative/anti-apoptotic cascades for MM has provided the impetus for development of small molecule inhibitors against the kinase activity of Akt. We show that the dual Akt/p70S6K kinase inhibitor EXEL-6075 has potent anti-MM activity in preclinical models. Methods/Results: We tested a panel of 15 human MM cell lines for in vitro response to EXEL-6075 using MTT colorimetric survival assays, which showed that the majority of MM cells responded to EXEL-6075 with IC50 values 1-log differential activity against the non-neoplastic tissues tested in our study. This remains an interesting strategy for the treatment of MM. Future in vivo studies will be needed to confirm these interesting in vitro results.

Beschreibung: Recombinant nematode anticoagulant protein c2 (rNAPc2) is a specific inhibitor of tissue factor (TF)/factor VIIa complex with novel anti-metastatic, anti-angiogenic, and anti-thrombotic activities. TF is highly expressed in human colorectal tumors and the level of TF expression positively correlates with disease stage and inversely correlates with survival. To explore the therapeutic potential of rNAPc2 during tumor growth and metastasis, we tested rNAPc2 efficacy in experimental colorectal cancers in mice. Administration of rNAPc2 inhibited pulmonary metastasis in mice systemically disseminated with CT26 murine colon carcinoma cells in a dose-dependent fashion, as measured by either number of lung surface metastases or lung mass. While rNAPc2 treatment alone moderately reduced primary tumor growth, combining rNAPc2 with the cytotoxic agent 5-fluorouracil (5-FU) resulted in synergistic growth inhibition of HCT116 human colorectal tumor xenografts in nude mice. Likewise, rNAPc2 further reduced tumor growth in HCT116 human colorectal tumor xenograft-bearing mice receiving bevacizumab (humanized anti-vascular endothelial growth factor monoclonal antibody). Using CD31 and Ki67 immunohistochemisty, we found that rNAPc2 synergized with either 5-FU or bevacizumab in inhibiting microvessel density and tumor cell proliferation in HCT116 human colorectal tumor xenografts. Furthermore, rNAPc2 synergized with CPT-11 in inhibiting hepatic metastasis in nude mice with portal vein injection of HCT116 human colorectal tumor cells. Long-term administration of rNAPc2 also significantly suppressed formation of intestinal adenomas and adenocarcinomas in ApcMin/+ mice. The dosing regimens of rNAPc2 used in these studies were well tolerated up to a three-month period by recipient mice without major hemorrhage or other adverse effects. In conclusion, the synergistic tumor inhibitory activity of rNAPc2 in pre-clinical colorectal cancer models suggests that rNAPc2 may be an effective anti-tumor agent in human colorectal cancer patients to potentiate chemo- or anti-angiogenic therapies.

Beschreibung: Ras mutations occur in 40–60% of multiple myeloma (MM) patients and are implicated in progression to advanced MM (including plasma cell leukemia/extramedullary lesions). The small molecule PRLX (Prolexys Pharmaceuticals) was identified in the context of “synthetic lethal” chemical screening for genotype-selective cytotoxicity against cells transformed with forced expression of mutant Ras (but not against isogenic normal cell counterparts) and was tested for anti-MM activity. In MTT survival assays, 34 of 46 human MM cell lines (74%) responded to 48hr treatment with sub-μM PRLX concentrations (achievable in preclinical pharmacokinetic studies). (24 MM lines had IC50 values

Beschreibung: Salvage chemotherapy followed by high dose therapy (HDT) and autologous stem cell transplantation (ASCT) is the standard of treatment for chemosensitive relapses in diffuse large B cell lymphoma. Improvement of salvage chemotherapy was suggested with the association rituximab, Ifosfamide, etoposide, carboplatinum, R-ICE. What is the optimal chemotherapy regimen and can we reduce the post ASCT relapses rate? The ongoing CORAL trial was designed to answer these questions. DLBCL CD 20+ in first relapse or pts refractory after first line therapy were randomized between rituximab plus DHAP and R-ICE. Stratification was made on centers, prior rituximab exposure and refractory/12months, 108 refractory/early relapses; 97 pts with prior exposure to rituximab; Stage 3-4 107 pts; elevated LDH 88 pts; secondary IPI 0–1 112 pts; sIPI 2-3 63pts. The two arms were well balanced. In the prior rituximab cohort exposure more pts had refractory disease and adverse prognostic factors. However, at inclusion patients characteristics were not significantly different in the stratified subgroups. The overall response rate was 68%, with 41% complete remission rate. Toxicity was similar to what is expected with intensive therapy, 72 SAE were reported with 12 deaths during the initial salvage regimens. In univariate analysis factors significantly affecting response rate (p1 54% vs 77% and prior exposure to rituximab 54% vs 82%. In a logistic regression model only refractory/early relapse and secondary IPI remain significant for response rate. Intent to treat 2 yrs EFS and OS were 50% (CI 42–57%) and 69% (CI 61–75%) respectively. Only 107 pts in this prospective study received, per protocol ASCT. For patients transplanted, 2 yrs EFS was 75% (CI 63–84%) with OS 89%. Two yrs EFS was affected by: prior treatment with rituximab, 34% vs 66% (p=.0001); refractory/early relapse 36% vs 68% (p

Beschreibung: Vascular endothelial growth factor (VEGF)-mediated signaling has at least two potential roles in diffuse large B cell lymphoma: potentiation of angiogenesis, and potentiation of proliferation and/or survival induced by autocrine VEGF receptor-mediated signaling by lymphoma cells. We have recently shown that diffuse large B cell lymphomas expressing high levels of VEGF protein also express high levels of VEGF receptors 1 and 2. We have now assessed a larger multi-institutional cohort of patients with de novo diffuse large B cell lymphoma treated with anthracycline-based therapy to address whether either tumor vascularity as assessed by microvessel density counting, or expression of VEGF protein and its receptors as assessed by immunohistochemistry, contribute to patient outcomes. Increased tumor vascularity (Figure 1a, b, c -- increasing microvessel density) is associated with poor overall survival (p=0.047), and is independent of the international prognostic index (Figure 1d). In contrast, high expression of VEGFR1 by lymphoma cells is associated with improved overall survival (p=0.044) and a trend toward improved progression-free survival (p=0.054). High expression of VEGF by lymphoma cells is also associated with a trend toward improved overall survival (p=0.089). The concordant trend toward improved survival with increased lymphoma cell expression of VEGF and its receptor VEGFR1, together with their coordinate overexpression in a subset of diffuse large B cell lymphomas (p=0.00025, chi value 21.5), suggests the presence of an autocrine VEGF-VEGFR1-mediated feedback loop in these lymphomas. Indeed the combination of high VEGF and VEGFR1 protein expression identifies a subgroup of patients with improved overall (p=0.003) and progression-free (p=0.026) survival; these findings are independent of the international prognostic index (Figure 2). The presence of improved survival in a cohort of patients whose lymphomas potentially depend on autocrine signaling via VEGFR1 suggests that dependence on this pathway may render patients susceptible to the effects of anthracycline-based therapy; indeed, downregulation of VEGF expression at the mRNA level by anthracyclines and other chemotherapeutic agents has been demonstrated in a variety of non-hematolymphoid neoplasms. The relative importance of autocrine VEGF-mediated signaling versus vascularity should certainly be taken into account in clinical trials of anti-VEGF/VEGF receptor therapy in diffuse large B cell lymphoma. Figure Figure Figure Figure

Beschreibung: The importance of bone marrow examination in the evaluation of leukemia, multiple myeloma, anemia, pancytopenia, and other disorders is well established. The objective for this study was to evaluate the ability of a powered bone marrow aspiration device to penetrate the intraosseous medullary space of the iliac crest, and to aspirate bone marrow samples for the ultimate purpose of diagnosing disease and monitoring the course of disease and medical therapy. The device was used to obtain bone marrow samples in accordance with accepted practice guidelines and device’s directions for use. Among other data, insertion success, time to insertion, and complications were recorded. Patient pain levels were rated from 0 to 10 (10=extreme pain). Device operators rated the use of the device from 0 to 10 (10=outstanding). There were 55 patients in the study from three centers. Successful insertion and aspiration of bone marrow samples were achieved in 54 of the 55 patients (98.1%). Mean insertion time was 4.9±3.0 seconds; significantly faster than the 7.3 minutes reported by Kuball et al* (one-sample t-test, p

Beschreibung: B-cell chronic lymphocytic leukemia (CLL) is the most common type of adult leukemia in the Western world, however, infrequent in the Eastern. Although the median survival is around 10 years, CLL patients have a highly variable clinical course and prognosis. While some patients show an indolent disease and never require treatment, others suffer from a much more aggressive course requiring intensive treatment shortly or immediately after diagnosis. Identification of these subgroups and insight into the prognosis for each individual patient will be important to determine individualized treatment strategies. To explore the prognostic significance of CD38 expression in Chinese patients with CLL, multi-parameter flow cytometry was used to detect the expression of CD38 on CD5+CD19+ cells of 147 patients with CLL. CD38 expression and its association with some other prognostic factors such as Binet stage, lymphocyte count in peripheral blood, serum lactate dehydrogenase (LDH), β2-microglobulin (β2-MG), ZAP-70 expression and cytogenetic abnormalities were analyzed. The Kaplan-Meier method was used to construct survival curves, and results were compared using the log-rank test. Univariate and multivariate Cox regression analyses were used to assess associations between survival time and potential risk factors. Out of the 147 CLL patients, positive expression of CD38 was found in 45 (30.6%) cases. CD38-positivity identified a subgroup of CLL patients with aggressive disease of Binet stage at the time of the test (P=0.036). Furthermore, the presence of higher serum LDH and β2-MG levels at diagnosis was strongly correlated with CD38-positive (P=0.016 and P=0.025, respectively). Prognostically unfavorable cytogenetic abnormalities, including 17p13 deletions and 11q22 deletions were significantly more frequent in CD38-positive patients than in CD38-negative ones (P=0.047 and P=0.001, respectively). There was no significant difference between CD38-positive and CD38-negative subgroups in terms of age, sex, or lymphocyte count. In addition, in CD38-positive patients, the percentage of leukemic cells expressing ZAP-70 protein was not significantly higher than in CD38-negative ones (P=0.120). There was no significant difference between CD38-positive and CD38-negative groups in molecular cytogenetic aberrations of del(6q23), del(13q14), 14q32 translocation, or trisomy 12. CD38 expression was associated with poor outcome. Patients with positive expression of CD38 had significantly shorter overall survival (mean, 81 months) than patients without CD38 expression (mean, 179 months) (P=0.015). Univariate and multivariate analysis showed that serum levels of LDH and β2-MG, del(17p13) and CD38 expression were strongly associated with survival. It was showed that the patients with high level of CD38 expression had poorer outcome; CD38 was a good predictor of survival time in Chinese patients with CLL.

Beschreibung: Liposome-mediated gene transfer is known to exhibit a number of desirable features for gene delivery, such as decreased immunogenicity, lack of mutagenesis, efficient transgene expression, internalization of episomal DNA irrespective of size, reproducibility, compositional variability and ease of use. As we and others recently demonstrated, cationic Phosphonolipids (CPs) have pronounced gene delivery potency into human cells. Based on the well-established dependency of gene transfer efficiency upon lipoplex properties, the superior-performing CP EG.308 and a novel cationic lipophosphoramide vechicle (CL) KLN-5, were evaluated for delivery of the human A-gamma globin gene in K562 human erythroleukemia and 5637 human epithelial carcinoma cells. The plasmid vector used for Aγ-globin gene was pAγLuc (3.3 kb HindIII human Aγ-globin gene). Two other vectors were used as controls: pGL3bLuc+, which encodes for luciferase; and, pEGFP-CI, which encodes for the green fluorescent protein. Dynamic Light Scattering experiments underlined the cooperative effect of the liposome vesicle diameter, complex formation medium and charge ratio upon lipoplex size. At optimized lipoplex generation conditions, the initial lipid size was not found to be always predictive of lipofection potency, rather transfection efficiency is a function of the liposome vehicle to generate lipoplexes of enhanced size (300–3500nm). Dot-blot analysis and reporter gene-expression assays revealed that among a number of varying composition solvent-systems, optimal nuclear transgene incorporation and expression was attained in 300mM NaCl. Lipoplexes EG.308- and KLN-5/pAγLuc formed in 300mM NaCl mediated maximal transgene delivery and expression 1 and 3 days-post transfection, respectively (330pg luciferase/ng genomic DNA, 80000 RLU/μg protein, respectively). On day 14, sustained plasmid DNA quantity was still detected in the nucleus. Enhanced plasmid integrity was confirmed by Southern blotting analysis. Both linear and circular pAγLuc DNA delivery resulted in pronounced transgene entry (330pg/ng genomic DNA); however, by introducing the latter, transgene expression was almost 100% higher. As opposed to the effective KLN-5-mediated transfection of K562 cells (330pg luciferase/ng genomic DNA, 80000 RLU/μg protein), transgene targeting and expression in 5637 cells was low (73pg luciferase/ng genomic DNA, 13150 RLU/μg protein). Confocal fluorescence microscopy revealed similar lipoplex-cell membrane association events and overlapping endocytic activity in EG.308- and KLN-5- mediated lipofection of target cells. However, KLN-5/pDNA nuclear co-localization in a greater number of GFP+ transfected cells compared to EG.308/pDNA lipofected ones, support a lipid-specific complex behaviour. These results indicate that effective transgene nuclear targeting is feasible with the tested CP and CL. Human Aγ-globin transgene nuclear incorporation and expression in target cells by CPs lends credence to their use as a vehicle of therapeutic transgene delivery.

Beschreibung: Background: In this study we cloned a vector based on a self-inactivating lentiviral backbone containing MDR1 (multidrug resistance 1 gene) connected by an ECMV-IRES-element with MGMT P140K (O6-BG-resistant mutant of O6-methylguanine-DNA methyltransferase). The chemoprotective potential of this HR′SIN-MDR-IRES-MGMT combination vector was compared to single vectors (HR′SIN-MDR, HR′SIN-MGMT). Methods: HL60 and CD34+ cells were transduced with the various vectors. After chemotherapeutical treatment MTT assays were used to detect chemoresistance levels in HL60 cells, CD34+ cells were held in liquid culture under differentiation conditions and analysed by FACS for MDR1 expression. Results: HL60 cells transduced with the combination vector showed significant chemoresistance to O6-BG/ACNU (IC50 13x higher compared to untransduced control), the IC50 of cells transduced with HR′SIN-MGMT was 35x higher. The IC50 of paclitaxel (MDR1 substrate) was 24x higher in cells transduced with HR’SIN-MDR and 25x higher with HR’SIN-MDR-IRES-MGMT compared to untransduced control. Combined exposure of cells to O6-BG/ACNU and paclitaxel showed a survival advantage of cells transduced with the combination vector (IC50 6.25x higher), for the single vectors the IC50 was 1.63x higher (MDR1) and 2.08x higher (MGMT) compared to untransduced control. Treatment of CD34+ cells with increasing concentrations of doxorubicin (up to 0.8 μM) resulted in a higher fraction of MDR1-positive cells either with HR′SIN-MDR (26.6x) or with HR′SIN-MDR-IRES-MGMT (30.6x) compared to untreated cells. After combination treatment (20μM O6-BG/16μM BCNU and 0.4μM doxorubicin) the fraction of MDR1-positive cells was higher for HR′SIN-MDR-IRES-MGMT (14x) than HR′SIN-MDR (8x) transduced cells. Conclusion: The protective effect of the combination vector is comparable with the single vectors for monotherapy and superior for combined therapy. The combination vector presents simultaneous protective effects of two drug resistance genes, thus reducing the risk of insertional mutagenesis by one transduction process. Consequently our results might help to reduce myelotoxic side effects and increase the chemotherapeutic efficiency.

Beschreibung: Background: The discovery of JAK2V617F mutation has lead to the proposal of new algorithms to diagnose and classify MPD. Separation of PV from ET could become less clear, especially in JAK2V617F patients, if one considers that mutated PV and ET are similar conditions. However, the short-term risk of thrombosis, and long-term risk of evolution to leukemia have not yet been shown to be similar in JAK2V617F ET and PV. Current WHO criteria for PV require either RCM 〉125% of predicted value, or Hb level 〉18.5 g in men and 16.5 g in women. Some investigators recently proposed a threshold for Ht at 52% in men and 48% in women to diagnose PV in JAK2V617F patients. Diagnosis of ET requires excluding PV, and using Hb or Ht values for this purpose could avoid RCM measurement. Aims: To evaluate the prevalence and prognostic impact of erythrocytosis determined by RCM measurement in MPD patients classified as ET on peripheral blood counts. Methods: We reviewed all RCM measurements performed in patients suspected of MPD in 2 nuclear medicine laboratories during a 10-year period. Results: Among 566 patients referred for RCM measurement, 71 had isolated thrombocytosis (i.e. both Hb and Ht 〈 values used for PV diagnosis as defined above). RCM was normal in 38/71, but was 〉125% of predicted value, revealing inapparent erythrocytosis, in 33 cases. Thus, after RCM measurement, final diagnosis was ET in 53.5% (38/71), and PV in 46.5% (33/71) of the patients, respectively. Pts with elevated RCM had significantly higher Ht (p

Beschreibung: We previously reported that histone deacetylase (HDAC) activity is elevated, but is not correlated to the JAK-2 mutation status, in patients with myelofibrosis myeloid metaplasia (MMM) (Blood 107:319b 2005). Now we have studied more patients: totally, 17 with MMM, 19 with other myeloproliferative disorders (MPD), and 16 normal volunteers as controls. Significantly elevated HDAC levels again was shown in patients with MMM compared with other MPD patients and normal volunteer controls (p

Beschreibung: It has long been proposed that Myelodysplastic syndromes (MDS) arise into a context of genomic instability leading to accumulation of multiple mutations. However, the underlying mechanism remains elusive. Two different genetic instability pathways, chromosome instability (CIN) and microsatellite instability (MSI) can be study using repetitive polymorphic markers (STR). Mutations of CIN genes increase the rate of gross chromosomal changes and loss of heterozygosity (LOH), which is an important mechanism of tumor suppressor gene inactivation. MSI is manifested by alterations of the length of STR due to germline mutations or methylation-induced silencing of key DNA mismatch repair genes (MMR). To verify whether both mechanisms might be involved in MDS, microsatellite instability and loss of heterozygosity (LOH) were analyzed with 10 specific STR markers. Bone marrow samples from 21 de novo MDS patients (12 females/9 males) with a mean age of 70.7 years (range 38–93), including 13 RA, 4 RAEB, 1 RAEB-T, 2 RARS and 1 CMML, were analyzed. All patients gave informed consent and the study was approved by our Ethics Committee. Mononuclear cells were separated by density gradient centrifugation through Ficoll Hypaque and depleted of adherent cells by 1 h incubation at 37°C. Myelodysplastic DNA was obtained from non-adherent mononuclear cells, while normal DNA was extracted from polymorphonuclear cells. Ten STR were amplified at different annealing temperatures (55–58°C), depending on the average size of each primer. Four mono- or dinucleotide markers (BAT 25, BAT 26, D2S123 and D18S58) were chosen from the Bethesda reference panel for MSI studies. Six additional loci at CRTL (5q13-14), IRF (5q31), D5S209 (5q31-33), CSF1RT (5q33-35), D7S525 (7q21-31) and TP53 (17p13.1), were selected because of their location on specific genomic points involved in MDS and acute leukemia. PCR products were analyzed by medium size non-denaturing polyacrylamide (15%) gel electrophoresis and silver staining (0.1%). Allele variations between normal and myelodysplastic DNA were scored according international criteria. Six out of 21 cases (28.6%) presented an unstable phenotype with band shifts or losses. No STR alterations were observed with markers BAT 25, BAT 26, IRF and D2S123. Two cases (9.6%) presented LOH at TP53, D5S209 and D7S525. Bethesda panel revealed MSI only in one patient (4.8%) at D18S58. STR at hematological loci allowed the identification of 5 cases (23.8%) with amplifications or deletions of the repeat sequences at CSF1RT, CRTL and TP53. MSI was detected only at 1/9 or 2/9 STR per patient and therefore these cases were classified to exhibit low grade of MSI (MSI-L). The mean frequency of mutated STR/individual showed a significant difference between hematological markers (0.08±0.03) respect to Bethesda STR (0.01±0.03) (Student test, p=0.007), suggesting that colon markers are not useful to evaluate MSI/LOH in MDS. MSI-L was more frequent than LOH in patients with unstable phenotype, suggesting that putative tumor suppressor genes at the loci studied are not involved in MDS genomic instability. Our data suggest that a subset of patients with MSI-L, are not related to MMR mutations but are probably associated to other genetic alterations, critical in MDS development. Moreover, a STR panel specific for MDS should be defined in order to deep evaluate genomic instability in this disease.

Beschreibung: We performed a retrospective analysis of our Spanish database of patients with ET in order to assess the role of different response predictive potential factors to anagrelide treatment. 411 ET patients from 54 sites from February 2005 to August 2006 were included in a retrospective chart review. ET was diagnosed according to PVSG criteria (1997). All patients had started treatment with anagrelide before December 2004, either as a first line or as a second line therapy. The population was divided itself in three groups of risk at diagnosis: of high Risk: those patients older than 60 years or with previous history of thrombosis (40.6%); of low Risk: patients younger than 60 years with thrombocytosis lower than 1.5· 109/L, without cardiovascular risk factors and previous history of thrombosis (35.8%); intermediate risk: those patients who did not reunite the criteria of previously mentioned groups (23.6%). The response to the treatment was defined as complete remission (CR), when a reduction of platelets′ count were equal or less than 400×109/L, or a reduction higher than 50% with respect to the basal numbers; partial remission (PR), when the platelets′ count was between 400× 109/L and 600×109/L with respect to the basal numbers; and no response, those with a lower reduction than PR or increasing their platelets′count. CR was obtained in 219 patients (53.6%; 95%CI = 48.6–58.5) and PR in 113 (27.6%; 95%CI = 23.4–32.2), giving an overall response (OR) rate of 81.2% [95%CI = 77.0–84.9]. The influence of certain factors (such as age, gender, risk group, platelets levels before treatment with anagrelide, previous cytoreductive untreated patients and maximum anagrelide dose), in the objective response was explored. A chi-square test and a multivariate analysis (logistic regression) were performed. A worse response (p = 0.021) was associated with a higher dose (≥ 2.5 mg per day). A trend (p = 0.103) to better response in patients without previous treatment (anagrelide as a first line) was detected. Additionally a significant association (p = 0.02) between the previously treated with hydroxyurea and the presence of evolutive hematological transformations (myelofibrosis and acute myeloid leukemia/myelodysplastic syndrome) was observed. It is difficult to set a cause effect relation in all these findings due to the retrospective design of the analysis. As a conclusion, the response to anagrelide in ET patients was demonstrated to be independent of the age, gender, platelets′ count and level of previous risk.

Beschreibung: The acquired von Willebrand syndrome (AVWS) is a bleeding disorder, which is amongst others associated with myeloproliferative diseases. Its prevalence varies from 0,04–0,13% in literature. Typical laboratory findings such as a low VWF:RCo/VWF:Ag- Ratio, a prolonged aPTT, PFA and bleeding time, a decrease of VWF:RCo, FactorVIII:, VWF:Ag, typical VWF multimeric structure and the absence of a family history of bleeding are the basis for the diagnosis of AVWS. Bleeding episodes are typical for AVWS and are mostly of the mucocutaneous type (epistaxis, heavy menstrual bleeding, gingival and postoperative bleedings). Myeloproliferative diseases, particularily essential thrombocythemia, are with 15–18% the third most frequent observed comorbidity of patients with AVWS, but the prevalence of AVWS in patients with MPS is -at our knowledge- still unknown. We investigated 54 patients with the diagnosis of myeloproliferative disease (44 ET, 9 PV, 1 IMF), which was established according to the WHO-criteria over a period of 6 years (2000–2006). By analysing the typical laboratory parameters for AVWS, the VWF multimeric structure and the own and family history of bleeding we detected that 67% (36/45) of these patients had an AVWS: 27/44 ET (61%), 8/9 PV (89%), 1/1 IMF. The two most sensitive parameters in this context were VWF:RC/vWF:Ag- Ratio and VWF:RCo. It is well known that AVWS can disappear by treating the MPD. We could also show that treating the MPD with Hydroxycarbamid platelets, VWF multimeric structure, VWF:RCo/VWF:Ag-Ratio and VWF:RCo and the bleeding tendency were normalized. The JAK2-V617F mutation is frequently found in myeloproliferative disorders (MPD): Up to 97% patients with polycythemia vera (PV) and about 50% of patients with essential thrombocythemia (ET) carry this mutation. A further goal in this investigation was to find out a correlation between JAK2-V617F mutation, MPD and AVWS. JAK2-V617F diagnosis was performed in 27 of the 54 patients with MPD. 10 of these 27 patients carried a mutated JAK2-allele. However we observed no significant difference between mutated and unmutated patients in correlation to AVWS. In our investigation the high prevalence of 67% AVWS in patients with MPD confirm the hypothesis that until now AVWS is an underdiagnosed disease. Because of this high prevalence, the possibility of regression by treating MPD and the importance of early diagnosis for prevention of unexpected, sometimes letal bleeding complications, it would be benefical to introduce the AVWS- diagnosis-procedure as a routine step for patients with MPD.

Beschreibung: Background: Prevalent in the vast majority of patients at diagnosis, anemia associated with myelodysplastic syndromes (MDS) has historically been treated through red blood cell (RBC) transfusions. More recently, the erythropoiesis-stimulating agents (ESAs), epoetin alfa (EPO) and darbepoetin alfa (DARB), have also been employed for the treatment of MDS-associated anemia. To characterize real-world utilization of ESAs in adult patients with MDS, this retrospective analysis of medical claims examined EPO and DARB dosing patterns, ESA treatment costs, and RBC transfusion use. Methods: An analysis of de-identified medical claims from January 2004–March 2006 using the Integrated Health Care Information Services (IHCIS) national database, which encompasses over 35 health plans, was conducted. Patients included were ≥18 years old, had ≥1 claim for MDS (ICD-9 code: 238.7) prior to ESA therapy, were newly initiated on either EPO or DARB, and received ≥2 doses during the treatment period. Patients with cancer (ICD-9 codes: 140–209) within 180 days prior to ESA treatment initiation were excluded. The study period terminated with either the last ESA treatment dose, end of data availability, initial AML diagnosis, or initial stem cell transplant, whichever occurred first. Dosing frequency was classified into mutually exclusive categories: once weekly (QW; ≤9 days), once every two weeks (Q2W; 9.1 to 18 days), and once every three weeks or longer (≥Q3W; 〉18 days), based on the average dosing interval during treatment. Mean cumulative ESA dose was used to calculate drug cost (based on July 2007 WAC: $12.005/1,000 Units for EPO and $4.576/mcg for DARB) and dose ratio (Units EPO: mcg DARB). RBC transfusions were identified from medical procedure codes, and the proportion of patients transfused during treatment was compared between the two groups. Results: A total of 193 patients, 146 receiving EPO and 47 receiving DARB, formed the study population. Patients receiving EPO were older (69.9 vs. 66.2 years for DARB, p=0.018) with a similar proportion of women (56.2% vs. 51.1%, p=0.541). Extended dosing frequency (defined as every two weeks or greater, ≥Q2W) during treatment was observed in the majority of patients in both groups (EPO: QW 43%, Q2W 35%, ≥Q3W 22%; DARB: QW 15%, Q2W 60%, ≥Q3W 25%). Mean treatment duration was similar for both groups (EPO: 88.3 days; DARB: 87.1 days; p=0.944). The mean cumulative ESA dose administered was 406,685 Units for the EPO group and 1,509 mcg for the DARB group, corresponding to a dose ratio of 270:1 (Units EPO: mcg DARB). Use of concurrent white blood cell growth factors (G-CSF or GM-CSF) was similar (EPO 8.2%, DARB 6.4%, p =0.683). RBC transfusion was administered to 8.9% of EPO patients compared to 8.5% of DARB patients (p=0.934) Cumulative drug cost was lower in the EPO group by $2,022 (EPO $4,882; DARB $6,904; p=0.101) compared to DARB. Conclusion: Based on these data from actual clinical practice, the majority of MDS patients received extended dosing regimens (≥Q2W) of either EPO or DARB. A dose ratio of 270:1 (Units EPO: mcg DARB) between the two agents and 29% lower drug cost in the EPO group was observed. These findings are similar to those previously reported from published real-world ESA drug utilization studies in other therapeutic areas.

Beschreibung: Meningeal infiltration is extremely rare in CLL. Moreover, part of the cases found in the literature occurred in fact in patients with prolymphocytic leukemia. In the few CLL cases, this complication was reported at late stages of the disease, in elderly patients. A previously healthy 30-year-old man was referred to the emergency department for evaluation of blurred vision and headache that developed 2 weeks earlier. Bilateral papilledema was observed, with no other neurological abnormality. CT and MRI of the head and orbits disclosed thickening of both optic nerves. Hemoglobin and platelet count were within normal limits. The WBC count was 41800/μl with 83% small lymphocytes, which were SmIg+ λ(weak) CD19+, CD5+, CD23+, CD20+, CD79b+, CD11c+, bcl2+, CD38+, ZAP70+ and CD10 negative. Moderately enlarged cervical lymph nodes were present. A diagnosis of Binet stage A, Rai stage I B-CLL was made. Bone marrow aspiration and biopsy showed 95% lymphocytes, with a diffuse pattern of infiltration. No metaphases were obtained for karyotyping. FISH disclosed a rearranged IgH, and was negative for trisomy 12 and for deletions of 11q, 13q and 17q. Abdominal CT showed retroperitoneal lymphadenopathy. Serology for HIV and EBV was negative. The cerebrospinal fluid (CSF) revealed 〉1000 lymphocytes/μl which were immunophenotypically identical to the peripheral blood cells. Systemic corticosteroids and intrathecal chemotherapy (methotrexate and dexamethasone) followed by cranial irradiation led to resolution of headache, but blurred vision persisted. Clearance of leukemic cells from the CSF was slow, a normal CSF being achieved only after 6 alternate day intrathecal injections. Systemic chemotherapy and stem cell transplantation are planned. To our knowledge this is the first reported case of CLL presenting as meningeal infiltration. Two possible reasons for the apparent rarity of CLL meningitis can be put forward: meningeal invasion would frequently be clinically silent, a hypothesis that is strengthened by autopsy findings; and it would usually occur in very advanced stages of CLL (with misinterpretation of neurological symptoms in erderly patients) making underdiagnosis easy.

Beschreibung: Hypereosinophilic syndrome (HES) was a group of diseases associated with persistent eosinophilia without unknown causes, companied with tissue and organ impairments. In 2001, WHO classification of hematopoietic and lymphoid neoplasms classified chronic eosinophilic leukemia (CEL) / HES into the category of chronic myeloproliferative disease, and proposed that the principal basis for the identification of CEL and HES was whether to have the evidence of eosinophils clonal proliferation: CEL had the evidence of eosinophils clonal proliferation, while HES was lack of the evidence of eosinophils clonal proliferation. In this study, 20 cases of CEL/ HES patients were analyzed retrospectively, and nested RT-PCR was used to detect FIP1L1-PDGFRA (F/P) fusion gene; Allele-specific PCR (ASP) conjoint sequencing analysis was used to detect JAK2 V617F, and PCR-RFLP was adopted to detect the mutation status of JAK2 V617F; and PCR is applied to detect TCRγ rearrangement. The clinical and laboratory characteristics of CEL and HES were compared. The ratio of male and female in the 20 cases of patients was 19:1, and the median age was 33 (20–57). F/P detection was positive for 12 cases, and the sequencing confirmed that FIP1L1 break point was at intron 10–12, while PDGFRA break point was at exon 12. There was 1 case of patient found that had JAK2 V617F, and the mutation status analysis showed that it was the mutation on heterozygote. 6 cases were detected having TCRγ gene rearrangement, of which 4 cases were CEL patients, and other 2 cases were HES patients. Most of CEL patients had respiratory symptoms in the early stage, easily companied with circulatory systematic impairment and nervous systematic symptoms. The incidence of splenomegaly of CEL patients was obviously higher than that of HES patients (92.5% vs 42.5%, p=0.031), so did the incidence of anemia and myelofibrosis. There was no difference in EO, WBC, PLT and EO% in peripheral blood as well as bone-marrow eosinophils percentage and bone-marrow primitive cells’ percentage between two groups. Abnormal morphology of eosinophils was often found in CEL patients, with the main manifestation of eosinopenia, basopenia and plasma vacuoles. Our data showed that Eosinophilia is often caught by male, mainly by the young; CEL patients have the main manifestation of the circulatory systematic, respiratory and gastrointestinal symptoms, and have a high incidence of anemia and thrombocytopenia. The routine examination has a little significance for the identification o CEL and HES, while the bone marrow smears morphological examination has a certain help for the diagnosis of CEL; Some HES patients have JAK2 V617F mutation, and further studies on the effect of JAK2 V617F mutation on the pathogenesis of HES can contribute to the diagnosis of such patients in the future and the development of the new targeted drug therapy; Some CEL patients have TCRγ rearrangement, while the relationship of CEL and TCRγ needs a further study.

Beschreibung: Introduction: Although blast-rich specimens immunophenotype studies in myelodysplastc syndromes (MDS) could associate bone marrow (BM) blast expression of CD7 and/or CD117 antigens with poor outcome (Ogata et al., Blood 2002), the prognostic role of markers of myeloid cell immaturity and committment in not enriched BM samples is largely unexplored. Patients and Methods: The expression of CD33, CD34 and CD117 antigens in not enriched BM samples of 50 newly diagnosed MDS was compared with both BM blast WHO category and IPSS score. Immunophenotyping was carried out by using the panel of quadruple monoclonal antibodies CD34/CD117/CD45/CD33, conjugated with the fluorochromes FITC, PE, PerCP, APC, respectively. Acquisition of information on 1x105 stained cells corresponding to the whole BM cellularity was assessed on a dual-laser FACSCalibur flow cytometer using the CellQUEST software (Becton Dickinson, San José CA USA). Multiple group comparisons were made using non parametric ANOVA for BM blasts; general linear model with Wald’s test and Kruskal-Wallis (KW) test to confirm significance was used for IPSS. Results: According to IPSS, 5 (10%) low risk, 27 (54%) intermediate risk-1, 14 (28%) intermediate risk −2 and 4 (8%) high risk pts were identified, respectively. The expression of CD33, CD34 and CD117 significantly correlated with both blast WHO category and IPSS, as shown in the Table 1. Interestingly, by analyzing the subset of 30 pts with BM blasts

Beschreibung: Myelodysplastic syndrome (MDS) is a malignant disorder of hematopoietic progenitor cells and characterize by peripheral blood cytopenias with normo- to hyper-cellular bone marrow (BM) and morphologically dysplastic changes. Thrombocytopenia is observed in approximately 50% of MDS. The underlying pathophysiology is not fully understood. We analyzed the megakaryocytopoiesis and thrombocytopoiesis state by several parameters, including % reticulated platelet (%RP), which indicated platelet production state, glycoalicine index (GCI), which indicated platelet destruction state, and serum thromopoietin (TPO) levels in 47 refractory anemia in myelodysplastic syndrome (MDS-RCMD) patients with platelet counts less than 100 x 109/L. Furthermore, apoptosis frequency of megakaryocyte progenitors was analyzed in several patients. In all patients, dysmegakaryocytopoiesis findings such as hypolobulated micromegakaryocyte, non-lobulated nuclei in all sizes, and multiple, widely-separated nuclei were observed. The megakaryocytes in BM was normal to decreased in number in 32 patients (70%). Plasma TPO levels were significantly higher (718.7±746.0 pg/ml, n=50) in MDS-RCMD, while they were less than 205.0 pg/ml in normal volunteers (68.3 ±65.3 pg/ml, n=32)(p〈 0.01). The %RPs in MDS-RCMD and normal controls were similar (MDS-RCMD 1.7±0.9% vs control 1.2±0.6%), indicating that increased thrombocytopoiesis was not observed in MDS-RCMD, regardless of high TPO levels. GCI was similar to normal controls (MDS-RCMD, 1.5±1.3% vs controls, 1.6±0.3%), indicating no excess of platelet destruction. There was no correlation between %RP and GCI. These data strongly suggested that platelet life span be not shortend in MDS-RCMD. We have reported that excessive apoptosis of CD34(+) cells was observed in MDS patients in the previous ASH meetings. The three-color flow cytometric analysis of bone marrow mononuclear cells using PE labeled Annexin V, PerCP labeled anti-CD34 antibody and FITC labeled anti-CD41 antibody were carried out in 17 MDS-RCMD patients. Much higher frequency of apoptosis was observed in double positive cells for CD34 and CD41 (48.1%:13.4∼78.4%, median: range) in MDS-RCMD, compared to that in normal controls (7.7%: 4.4∼17.2%, median: range) (P

Beschreibung: Objective To investigate the quantitive and qualitive changes of peripheral blood CD4+CD25+ regulatory T cells and Th3 cells of the patients with myelodysplastic syndromes. Methods Whole peripheral blood samples of 40 MDS cases and 19 nromal control were assayed by flow cytometer for detecting the numbers of CD4+CD25+ regulatory T cell and CD3+CD4+TGFβ+ T cell(Th3). Expression of FOXP3 and TGF-β of peripheral blood mononuclear cell(PBMNC) were analysed by reverse transcriptase-polymerase chain reaction(RT-PCR). Commercial ELISA kits were used to measure plasma concentrations of TGF-β 1 according to the manufacturer’s instructions. Results. The percentage and number of CD4+CD25+ regulatory T cells in the CD4+T cell population were significantly higher in the peripheral blood of MDS patients than healthy controls[(21.5±2.2% vs 8.3±1.9%; P

Beschreibung: Background: Recently, a provisional entity called “refractory anemia with ringed sideroblasts with thrombocytosis” (RARS-T) has been characterized in the MDS/MPD overlap category of the WHO classification. Diagnostic criteria include RARS features in association with sustained platelet counts 〉 600 (500) x 109/l and exclusion of cytogenetic abnormalities such as 5q−, t(3,3) and inv(3). Reticulin fibrosis in the bone marrow has been significantly associated with RARS-T. Splenomegaly also seems more frequent than in other RARS subtypes. Among these patients, approximately two thirds present with a JAK2 V617F mutation. On the contrary, JAK2 V617F mutation appears extremely rare in patients with typical RARS (Szpurka H. Blood 2006, Cabello AI. Leuk Research 2005, Malcovati L. ASH 2006, Remacha AF. Haematologica 2006). Case report: In 2001, we observed a 67 years old patient with RARS (22 to 45% ringed sideroblasts). Conventional cytogenetics revealed a trisomy 8 and deletion of chromosome Y. A mild to moderate fibrosis was observed in the bone marrow. At diagnosis, the patient was anaemic but had normal platelet counts which remained strictly normal during the next 6 years. After failure of various treatments, the transfusion requirements increased due to progressive alloimmunization and probably a moderate splenomegaly which had appeared 3 years after the diagnosis (splenic volume evaluated by ultrasonography was completely normal until 2004). In July 2006, the haemoglobin level was 6.1 g/dl despite the transfusion of 11 RBC units in the previous month. In the aim to overcome the suspected hypersplenism, a splenectomy was performed. In the subsequent days following splenectomy, we observed a rapid and persistent increase in platelet count with a peak of 2550 x 109/l. A JAK2 V617F mutation was demonstrated. The normalization and maintenance of platelets was obtained with small doses of anagrelide (0.5mg four doses/week) with RBC transfusion requirements decreasing by 50%. Conclusion: This patient presented most of the main characteristics of RARS-T except thrombocytosis (note that trisomy 8 has been described in association with RARS-T). Our observation suggests that thrombocytosis could be absent in some patients presenting with a very similar pattern of features as described in RARS-T. We postulate that hypersplenism (and possibly other particular biological characteristics) could, in some cases, mask the complete picture of RARS-T.

Beschreibung: Myelodysplastic syndrome (MDS) represents a heterogenous group of myeloid neoplasms characterized by abnormal differentiation and maturation of myeloid cells, bone marrow failure and genetic instability. The recent clinical and laboratory investigations suggest that MDS is closely related to diseases in which the bone marrow failure is mediated at least in part by the immune system. The are few studies concerning the of efficacy of treatment of MDS pts with cyclosporine A (CSA) but they are limited to the group of pts with bone marrow hypoplasia. The aim of our study was to evaluate the efficacy of CSA treatment in MDS pts as first line or second line therapy. 48 pts with different forms of MDS were included in study. The group of first line CSA treatment included 30 pts, male-18/female-12, RA-3, RARS-1, RCMD-18, RAEB-7, RAEBt-1, 20-normal karyotype, 10-different abberations including: 5q−, 16q+, −7, 12q+, −Y, [11p+,7q−] and complex abb. Hypoplastic bone marrow was revealed in 15 pts, hyperplastic-8 pts, hypo/hyper-7 pts. Second line therapy group included 18 pts, male-9/female-9, RCMD-11, RARS-1, RAEB-5, RAEBt-1, normal karyotype-3, cytogenetic anomalies-13 (+8, 5q−,−7, +8 and complex abb.). Hypoplastic bone marrow was revealed in 12 pts, hyperplastic-4 pts, hypo/hyper-4 pts. The first line therapy consited of splenectomy in 8 pts, low doses of Ara-C-3, interferon-α-3, chemotherapy-2 and ATG-2 pts. CSA was applied at 5–10 mg/kg/day initially and then adjusted according to blood levels and toxicity. The maintenance dose was 1–3 mg/kg/day. Minimum time to response evaluation was one month. Complete response (CR) was defined as normal PB counts, BM aspirate; partial response-improvement of PB counts to 50% of normal and freedom from transfusions; stabilization-decrease of transfusion requirements and stabilization of PB counts for more then 1 month. Total response rate in first group was 60% (18 pts) with median follow up of 10 months (2–134). CR was estimated in 20% (6pts), median follow-up 72 months (44–134). 2 pts with CR are in clonal remission. 2 pts from response group developed acute leukemia (AL). 40% of pts showed no response.58% of pts without response developed RAEB or AL. 42% of pts were in stable disease and were treated with another modalities. The response rate in second group was 61% (11 pts) with median follow-up of 7 months (1–78). 22% (4 pts) achieved CR, median follow-up 60 months (43–78). 39% of pts showed no response. 71% from these pts (5) transformed to RAEB or AL. 2 pts remained in stable disease. In both groups response was registrated from 1 to 4 months from treatment initiation (median 3 months). CR was achieved in the majority of pts after 1 year of treatment. Response was achieved in 77% of pts with hypo/hyper and hypoplastic bone marrow and in 12% of pts with hyperplastic bone marrow. Overall survival was decreased in pts with more the 5% blasts in bone marrow (p=0,02 for 1st line group, p=0,075 for 2nd line group), and increased for pts with hypo and hypo/hyper bone marrow cellularity (p=0,002 for 1st line group). There was no impact of cytogenetics. We may conclude, that CSA demonstrates good efficacy in therapy of MDS pts, especially for pts with RA, RARS and RCMD with hypo and hypo/hypercellular bone marrow and reactive lymphoid nodules in bone marrow. It can be initiated as 1st or 2nd line therapy and should be continued at least for 3 months before evaluating of response.

Beschreibung: The occurrence of additional chromosomal abnormalities (ACAs) in Philadelphia chromosome (Ph’) positive cells, a phenomenon termed clonal evolution (CE), reflects an increased genetic instability of the leukemic population which progressively acquires a highly malignant phenotype. Few data are available regarding the therapeutic effects exerted by imatinib mesylate on Ph’ cells bearing ACAs. Herein, we report the activity of imatinib mesylate employed as single agent or in combination with recombinant interferon (rIFN)a2 in inducing cytogenetic and molecular responses in 14 CML patients (F:M=5:9, median age: 58 years) with CE already present at diagnosis (5) or occurring later during the course of the disease (9) as the sole sign of accelerated phase (AP) (12) or associated with other AP features (2). Overall, the analysis of ACA rate showed the presence of i(Ph’) in 4, +8 in 3, -Y in 3, variant translocation in 2 (t(9;19;22) and t(3;9;22)), -17 in 1, 17p- in 1, 3p- in 1, 22q- in 1, and additional translocations involving chromosomes other than 9 and 22 in 3 other instances. Of note, the two cases with variant translocation showed at diagnosis the classical t(9;22). The t(9;19;22) or t(3;9;22), which was documented later in the course of the disease was therefore a proven second event. Imatinib was given at 400mg po/daily in all, but 2 patients with signs of AP in addition to CE who received a daily dose of 600 mg. rIFNa2 was given at a daily dose of 1 to 3x106UI, according to patient’s tolerance. An overall cytogenetic remission rate of 64% (9/14) was documented in response to imatinib mesylate employed as single agent (6/10) or in combination with rIFNa2 (3/4). Suppression of the ACA population was documented within an interval of 3–14 months from the beginning of imatinib treatment and it was timely coincident with the occurrence of CCR in 6 patients, irrespective of the entity of the ACA population in the context of the entire Ph’ positive one. In 2 of them, a “biphasic” response to imatinib was observed. Initially, suppression of i(Ph’) and t(9;19;22) with re-emergence of the original t(9;22) hemopoiesis was documented 4 and 7 months after treatment initiation, respectively, and then CCR occurred after further 10 and 9 months of treatment, respectively. In the last patient, a mixture of non-clonal and clonal +8 Ph’ negative hemopoiesis was noted at the time when CCR was documented. All 9 CCR patients tested negative for BCR/ABL gene at FISH analysis. Six of the 9 CCR obtained a major or complete molecular remission 3–29 months from treatment initiation. In 3 additional patients, who never achieved a CCR, suppression of the secondary clones was documented. In 1 case, the clone bearing 17p- was no more detected from the 6th to the 42nd month of therapy, when a new clone showing the i(17q) was recorded. In the other 2 cases, the leukemic population bearing the 3q- and 22q- or -17 marker was suppressed 6 and 3 months after the beginning of imatinib mesylate treatment, respectively. After a median follow up of 70 months, 8 patients are still alive and in continuous CCR. Such a long follow up, along with the rate, quality and duration of the responses observed, suggest that CE does not compromise the therapeutic activity of imatinib alone or combined with rIFNa in CML.

Beschreibung: Background: Gene expression profiles have been associated with prognosis in myelodysplastic syndromes. WT1 is a tumor-suppressor gene coding for a transcription factor located on chromosome 11p13, which was originally identified for its involvement in the pathogenesis of the Wilms’ tumor. In normal bone marrow, WT1 expression is low or undetectable, whereas it is aberrantly expressed in hematological malignancies. Evidence indicates that WT1 is important in the lineage-specific differentiation of hematopoietic cells and leukemogenesis. In myelodysplastic syndromes (MDS), WT1 expression has prognostic significance: it is directly correlated with the type of MDS, with IPSS score and with disease progression. Recent data demonstrate that WT1 is a potent activator of the EPO gene under normoxia and it is suggested that WT1 may regulate paracrine EPO synthesis in a tissue-specific manner. Bmi-1 is a transcriptional repressor gene which may be expressed restrictedly in stem cells and progenitors and is required to regulate the adult self-renewing hematopoietic and leukemic stem cells. It appears that it also plays an important role in providing cells the potential for proliferation. A number of reports on Bmi-1 provide perspectives on the close association of its expression with the progression of hematopoietic malignancies. Furthermore, flow cytometry has shown that Bmi-1 positivity in CD34+ cells is positively correlated with IPSS score. Introduction: We have designed a study to evaluate changes in gene expression profiles of bone marrow mononuclear cells of primary low and intermediate-1 IPSS risk MDS patients receiving erythropoetic growth factors (darbepoetin or high-dose rHuEpo alpha). Associations with response, changes in Hb, in percentage of CD34+ and apoptotic cells are evaluated. We present preliminary results in 6 patients. Methods: Bone marrow samples were obtained before and after 12 weeks of treatment. Mononucleated cells were cryopreserved and later thawed for total RNA extraction and cDNA synthesis. Gene expression profiling and the expressions of WT1 and Bmi-1 by RT-PCR were evaluated. Results: Baseline median Hb was 9,5 g/dL (interquartile range 8,7 10,1). Baseline mean Bmi-1 was 14011 (± SD 3927). All patients had a major erythroid response to treatment. Preliminary results demonstrate a significant decrease in WT1 from baseline median 1635 (interquartile range 1251–2150) to 1192 (interquartile range 1005–1365, P=0.046). Fig. 1. Changes in WT1 expression during therapy. Fig. 1. Changes in WT1 expression during therapy. Discussion: Though few patients have yet been studied, it is suggested that WT1 decreases in patients responding to erythropoetic growth factors. Further evaluation with stratification for erythropoetic response in the extended study may furnish novel associations between gene expression, erythropoetic growth factors and prognosis in MDS patients.

Beschreibung: Background Because in the past Chronic Myeloproliferative Disorders (CMPD) were not considered to be malignant conditions, cancer registries rarely recorded data on these diseases. Thus, information on incidence and outcome of CMPD in the population is limited. The aim of the present study was to better define epidemiological data of CMPD by examining all cases identified by the Modena Cancer Registry (MCR). Materials and methods We considered all cases of CMPD diagnosed in the Province of Modena (population 633.993 at 2001 Census). Cases, except Chronic Myeloid Leukemia, diagnosed from 1997 to 2005, were identified using the MCR database and the archival files of the centralized hemolymphopathological laboratory at Modena Cancer Centre according to ICD-O-3 codes 9950, 9960–64. Death certificate, cytology and histology report, both local and national reports of hospital admission, ICD-9 code reported in medical records were used as sources for identifying new CMPD cases and their outcome. All cases were checked and validated by a hematologist (A.B.) and a pathologist (G.B.) by a review of the original pathology report. Uniform diagnostic criteria were adopted, because the large majority of bone marrow aspirate and biopsy were examined by the same pathologist (G.B.). Clinical and follow-up data were retrieved by active search of discharge letters, review of hospital records and interview of general practitioners. Information on vital status was achieved from official population registries. Age standardized rates (ASR) were calculated according to the World Standard population. The dates of diagnosis and death or the closing date of study (December 2006) were used to estimate survival. Observed survival and relative survival were calculated according to Kaplan-Meier method and the Hakulinen approach, respectively. Results According to the 2001 World Health Organization (WHO) classification, a total of 380 cases of CMPD were identified. There were 155 Essential Thrombocythemia (ET) (41% of all CMPD), 114 Policythemia Vera (PV) (30%), 75 Idiopathic Myelofibrosis (20%), 2 Hypereosinophilic Syndrome/Chronic Eosinophilic Leukaemia (0.5%), 1 Chronic Neutrophilic Leukemia (0.3%) and 31 CMPD not otherwise specified (8%). The ASR of CMPD was 3.2/100,000 varying slightly (from 2.5 to 4.1/100,000) during the study period (p = 0.11); the crude incidence rate was 6.6/100,000. Median age at diagnosis was 69 years. No statistically significant differences were observed between sex regarding incidence and age at diagnosis. Overall relative survival was 97%, 89% and 88% at 1, 3 and 5 years after diagnosis, respectively. Analyzing CMPD, we observed a better survival for ET and PV in comparison with other subtypes (p = 0.01). Conclusions To our knowledge, this study is the first in Italy providing information on the incidence and outcome of CMPD using population-based data. Our results confirm that the risk of developing CMPD increases with age. The incidence of CMPD was substantially stable during the study period. Overall survival patterns reflect the well known chronic course of these diseases. As expected, we observed important differences in overall survival by WHO subtypes. We believe that the availability of precise epidemiological data, in particular those regarding outcome could help clinicians in choosing the most appropriate cost-effective treatments.

Beschreibung: Background: Food can change the bioavailability of a drug, which can have clinically significant consequences. This study was conducted to investigate the effect of food on the oral bioavailability of dasatinib (SPRYCEL®) in healthy adult subjects. Methods: Fifty-four healthy adult subjects received a single dose of dasatinib 100 mg dose, as 2 x 50 mg film-coated tablets after an overnight fast and within 10 minutes after the ingestion of a low-fat meal (315 kcal [20% fat, 68% carbohydrates, and 12% protein]) and a high-fat meal (985 kcal [52% fat, 34% carbohydrates, and 14% protein]) in a randomly assigned sequence. Individual treatments were separated by at least a 7-day washout period. Serial blood samples were collected for 24 hours after each treatment to determine dasatinib plasma concentrations using a validated liquid chromatography/tandem mass spectrometric method. Dasatinib pharmacokinetic (PK) parameters were determined using a non-compartmental method. Safety was monitored throughout the study. Results: Of the 54 healthy adult subjects (85% male, 61% Caucasian, mean age 32 y, and weight 80 kg), 48 completed the study. There were no serious adverse events. Adverse events and laboratory abnormalities were, in general, typical of those seen with dasatinib administration. PK results are summarized in the table below. Conclusions: Compared to the fasted state, a low-fat meal decreased Cmax and AUC of dasatinib by 21%; a high-fat meal decreased Cmax by 24% and increased AUC by 14%. These results are not expected to be of clinical relevance and, therefore, dasatinib may be taken without regard to meals. The drug was generally safe and well-tolerated when administered in the fed or fasted state. Statistical Analysis of PK Parameters for Dasatinib Treatment PK Parameter Geometric Mean Ratios (95% Confidence Intervals) Fed versus Fasted Low-Fat Meal Cmax 1.216 (1.047, 1.413) AUC 1.212 (1.100, 1.336) High-Fat Meal Cmax 0.758 (0.651, 0.882) AUC 1.140 (1.034, 1.257)

Beschreibung: The immune system plays a role in the pathogenesis of myelodysplastic syndrome (MDS) but its precise contribution to disease development and control is not fully clarified. T cell activation could reflect undesired autoimmune reactions against normal hematopoietic precursor cells as well as effective immune-surveillance against dysplastic clones. We have investigated lymphocyte subsets and activation markers of 42 low risk and intermediate-1 risk MDS patients and compared them to those of intermediate-2 risk, high risk MDS patients, and healthy donors. In low and intermediate-1 risk MDS patients, we have found an activated state of lymphocytes, determined by increased percentages of effector T cells with cytotoxic profile, increased number of clonal expansions, increased frequencies of Wilms’ Tumor 1 (WT1) specific lymphocytes and decreased regulatory T cell activation, as compared to healthy donors. Moreover, we demonstrate autologous T cell mediated cytotoxicity against aberrant hematopoietic precursor cells. These findings provide evidence for the existence of immune-surveillance in the pathogenesis of low and intermediate-1 risk MDS patients. Our data are important for adequate evaluation of the role of immune-modulatory drugs in the future and justify the reconsideration of the role of immune-suppressing therapy for low and intermediate-1 risk MDS patients.

Beschreibung: Introduction. The myelodysplastic syndrome (MDS) is mostly observed in patients older than 65 years and therefore there are few reports refering to patients less than 50 years old. However, the interest for this group of MDS patients is increasing because: these patients are doing an active life and therefore the disease have higher socio-economic consequences, their comorbidities are much more rare, there are important ethnic differences in frequency between western and eastern countries, the age-related genetic instability could not be incriminated in the pathogenesis of their MDS, they are the main candidates for aggressive treatments (high doses chemotherapy, bone marrow transplantation). Patients and methods. Sixty six cases with age under 50 years were extracted from the data-base of the MDS Registry of the Clinic of Hematology, Fundeni Clinical Institute, Bucharest, Romania comprising 404 primary cases, collected between 1982 and 2004. The registration form, using the FAB classification, was kindly provided by MDS Foundation (USA)(Chairman Prof. J.M. Bennett). The parameters included in the analysis were: age at presentation, sex, place of residence, values of hemoglobin, neutrophil count, platelet count, percentage of bone marrow blasts, prognostic scores, duration until acute leukemia (AL) transformation. The frequency of different subtypes of MDS and the dynamics of the new cases during the analysed period were also determined. A comparison with the group of age above 60 years and with other similar reference studies was performed. Results. There were 66 (16,7%) cases from which 22 (62,6%) were under 40 and 19 (28.3%) under 30 years old, with a mean age of 34,7 years. A global predominance of the feminine gender and of the urban location with no geographic aggregation could be noticed. The mean values of hemoglobin, neutrophils and platelets were 7.6 g/dL, 2,500/μL and 142.000/μL, respectively. Patients with refractory anemia (RA) and refractory anemia with ringed sideroblasts (RARS) accounted for 46.7% of all cases (RA 34,8%, RARS 11,9%), refractory anemia with excess of blasts (RAEB) 20,8%, RAEB in transformation (RAEB-T) 13,4%, chronic myelomonocytic leukemia 4,4% and unclassified 13,4%. The annual number of new cases increased three times during the analysed period, the increase being not uniform, with a peak in 2000, and not homogeneous. The subtypes with the most important increase in time were RA and RAEB-T. The AL transformation could be registered in 25.7%, after a mean time of 5 months. Conclusions. This study indicates a higher proportion (16.7%) and a lower age (34.7) of young patients with MDS in Romania, considered almost in the middle among those reported for western and eastern countries. The tendancy of the annual frequency of the new cases indicated a three times increase. An urban predominant location, in contrast with that of the group above 60 years old, has been noticed. The female predominance appears as a characteristic feature of the patients with MDS from this group of age. The degree of anemia was obviously more severe than that reported in other studies.

Beschreibung: The optimal management of patients with chronic myeloid leukemia (CML) is increasingly reliant upon molecular studies. Loss of response to imatinib in CML is most commonly associated with selection for a limited number of BCR-ABL kinase domain mutations that impair the ability of imatinib to effectively bind to BCR-ABL Molecular understanding of imatinib resistance mechanisms has led to the development of effective “second generation” BCR-ABL kinase inhibitors, such as dasatinib and nilotinib, which have clinical activity against most, but not all, drug-resistant mutations. Analysis of the BCR-ABL kinase domain in patients who develop resistance to second-generation inhibitors has implicated further selection of drug-resistant BCR-ABL kinase domain mutants in nearly all cases reported to date. Encouragingly, the number of resistant mutations capable of conferring clinical resistance to the most clinically-advanced second-generation agents, dasatinib (approved by the US FDA and EMEA) and nilotinib (approved in Mexico and Switzerland), appears to be restricted to a relatively small number of amino acid substitutions. As clinical experience with dasatinib and nilotinib grows, an understanding of the relative sensitivities of dasatinib- and nilotinib-resistant BCR-ABL mutants to other kinase inhibitors, both approved and investigational, is critical to optimize clinical outcomes in patients with resistance to dasatinib or nilotinib. At the present time, kinase inhibitor therapy options for patients with resistance to one of these agents include the investigational options bosutinib and MK-0457 (VX-680), as well as dasatinib and nilotinib (for patients not yet exposed to one of these agents) and re-exposure imatinib. It is likely that the success of therapeutic intervention in these cases can be predicted based upon the preclinical sensitivity of the mutation(s) involved with the agent chosen. We have therefore conducted a thorough biochemical and biological cross-analysis of the activities of each of these clinically-useful kinase inhibitors against mutations that confer clinical resistance to dasatinib or nilotinib. These studies provide clinicians with a useful reference for choosing an appropriate kinase inhibitor based upon the identity of the resistant BCR-ABL kinase domain mutation(s) detected at the time of relapse when faced with a patient who has lost response to dasatinib or nilotinib. It is hoped that the application of such “personalized medicine” strategies to the clinical management of CML cases will further improve outcomes in patients treated with kinase inhibitor therapy.