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Relapse of hematological malignancy remains a major complication after allogeneic stem cell transplantation. This is especially true for patients receiving minimal or reduced-intensity conditioning therapy. Analysis of donor chimerism (DC) is an important diagnostic tool to assess the risk of relapse after allogeneic stem cell transplantation, especially in patients lacking a specific marker suitable for monitoring of minimal residual disease. The sensitivity of a standard PCR assay amplifying short-tandem-repeat sequences can be improved significantly by investigating sorted CD34+ peripheral blood cells. We prospectively compared the serial analysis of DC in selected CD34+ cells and unmanipulated whole blood (WB) within a randomised study in 131 patients with CD34+ hematological malignancies (AML, ALL and MDS) surviving more than 100 days after transplantation. The primary end-point was the association between decreasing CD34+ DC and haematological relapse. Whenever a decreasing CD34+ or WB DC was confirmed and no signs of active GvHD were present, a rapid taper of CsA or tracolimus (50% every 5–7 days) was suggested. If no GvHD occurred within 14 days after the stop of CsA or tacolimus, patients were scheduled to receive donor lymphocyte infusions (DLI) in incremental doses. The cumulative incidence of relapse was significantly increased in patients with decreasing or incomplete CD34+ DC (62% vs. 38%, p=0.01). This was associated with a lower probability of overall survival (20% vs. 39%, p=0.03). The interval between the decrease in CD34+ DC and hematological relapse was 35 days (range 0–567) in the study group compared to only 8 days (range 0–63) in the control group monitored by WB DC analysis (p=0.05). The median time between a decrease in CD34+ DC and WB DC was 14 days (range, 0 to 445). Patients receiving preemptive therapy triggered by decreasing CD34+ DC had a significantly higher probability of disease-free survival compared to cases monitored and treated according to WB DC (19% vs. 0%, p=0.009). Multivariate analysis revealed age, disease-risk and decreasing CD34+ DC as independent risk-factors for overall survival in the study group. In summary, we could demonstrate that the quantification of DC in CD34+ selected cells is a sensitive method to predict relapse and survival after allogeneic SCT. Although this technology opens a window for preemptive therapy, new treatment approaches have to be employed to improve the overall outcome of patients with recurrence of residual disease after allogeneic stem cell transplantation.

Graft versus host disease is a major cause of morbidity and mortality after allogeneic stem cell transplantation from unrelated donors. Strategies using intensified GvHD prophylaxis including T cell depletion did not result in better outcome due to increased risks of infection and relapse. The use of ATG in the conditioning regimen for in vivo-Tcell depletion for GVHD prophylaxis has been reported by several groups but not been tested in a large prospective randomized trial. Here we report on results from the first large prospective, randomized, multicenter, open-label, phase III trial comparing standard GvHD prophylaxis with cyclosporine A (CyA) and short course methotrexate (Mtx) days +1, +3, +6, +11 (15/10/10/10 mg/m2) with or without 3×20mg/kg ATG-Fresenius (ATG-F) after a median follow-up time of two years. Between 2003 and 2007, 201 patients, median age 40 (range 18–60) years with AML (n=101), MDS (n=10), ALL (n=70), CML (n=17), OMF (n=3) in early (1st CR or MDS-RA, n=107), or advanced status of disease (all other, n=94), were transplanted from HLA-A and -B (2 digit), DRB1, DQB1 (4 digit) identical unrelated donors after highdose myeloablative conditioning with marrow (n=37) or PBSC (n=164) grafts. Median follow up time was 732.5 (25%-quartile 604, 75%-quartile 1097) days. For treatment comparisons with regard to the occurrence of aGvHD grade III-IV or death within 100 days post Tx, logistic regression adjusted for status of disease, source of stem cells, and center was used. For treatment comparisons with regard to time-to-event variables, cumulative incidence rates considering relapse and death as competing events were estimated, and Cox regression modelling the event-specific hazard rates and adjusting for status of disease and source of stem cells was used. Engraftment with WBC > 1000/μl was achieved in 97% in the ATG-F group after median 26 days, and in 95% in the control group after median 19 days (p The addition of ATG-F to standard CyA/Mtx prophylaxis results in decreased incidence of acute and chronic GvHD without increase of relapse or TRM rates. This is the first randomized trial answering the long-standing question regarding the beneficial effect of additional ATG-F to a standard GvHD prophylaxis. A reduction of GvHD without compromising survival could be demonstrated.

Background : Peripheral blood progenitor cells (PBPCs) are routinely applied worldwide for allogeneic related and unrelated stem cell transplantation. Factors influencing the mobilisation of PBPC’s in healthy donors are poorly understood. The database of the Apheresis Centre at the University of Dresden was evaluated to identify predictors of PBPC mobilisation. Methods : 4050 PBPC collections (3928 first donations, 122 second donations) of healthy unrelated donors between 1/1996 and 1/2008 were carried out according to a standardized protocol and prospectively documented in a database. Peripheral blood CD 34 counts were performed before each leukapheresis. CD 34 yield of every PBPC product was calculated in absolute numbers and per kg body weight of the recipient. All parameters are presented as median and range. Mann-Whitney test was performed for discrete variables. Correlation analysis by Pearson was applied for continuous variables. Multivariate regression analysis was carried out to detect factors independently predicting mobilization efficacy. Results : The range of peripheral CD 34+ haematopoietic stem cells obtained on day 5 was 8.7–285/μl (median 67.5/μl) in male and 6–282/μl, (median 51/μl) in female donors. The median CD 34 yield from the first leukapheresis was 5.88 ×108. Inadequate CD34-yields (< 2 × 106/kg) were obtained only in 18 donors (0.45%). Peripheral CD 34 count at day 5 is significantly correlated with (positive linear regression): BMI, baseline platelet count, male sex, G-CSF application (split dose), baseline leukocyte count. Peripheral CD 34 count at day 5 correlates negatively with: female sex, single dose G-CSF application, regular alcohol consumption and regular smoking status. Linear regression analyses revealed no significant influence of donor age. Multivariate correlation analysis included sex, BMI, baseline platelets, G-CSF-application, baseline leukocytes, age and smoking. This model explained 21.2 % of the variabilityA strong correlation exists between peripheral CD 34-counts at day 5 and apheresis yield (70% of the variability explained). Conclusions : A dose of 7.5 μg/kg/d lenograstim proved to be safe and effective in a large cohort of unrelated donors for mobilizing sufficient haematopoietic progenitor cells for allogeneic transplantation. Our analysis of 4050 donations revealed significant, but very weak correlations of the peripheral CD 34-counts with donor characteristics and life style parameters. No single parameter derived from donor baseline investigation reliably predicts CD 34 yield. Further evaluation of healthy donors including comprehensive genomic linkage analysis is necessary to elucidate the variable efficiency of PBPC mobilization

Hypoxia increases IL-8 secretion of mesenchymal stroma cells affecting migratory capacity in an autocrine manner Manja Wobus, Katrin Müller, Gerhard Ehninger, Martin Bornhäuser Department of Hematology/Oncology, University Hospital Dresden, Fetscherstr. 74, 01307 Dresden, Germany Adult bone marrow-derived stem cells represent an important source of cells for regeneration and repair of a number of damaged tissues. Mesenchymal stromal cells (MSCs) give rise to the cellular components of the bone marrow microenvironment and support expansion and differentiation of hematopoeitic stem cells in the respective niche. Low oxygen tension is thought to be an integral component of the endosteal niche microenvironment. When used for therapeutic purposes, MSCs cultured in standard conditions must adapt from 21% oxygen to less than 1% oxygen in the ischemic tissue. Understanding the mechanisms by which the production of cytokines and growth factors by MSCs is regulated may represent an important way to optimize their beneficial paracrine and autocrine effects. Human primary MSCs were incubated under normoxic (20% O2) and hypoxic (0.5% O2) conditions over five days and the pattern of released cytokines in the cell culture supernatants was compared using a human cytokine array (R & D Systems). Amongst others, we found upregulated IL-8 levels under hypoxic conditions leading us to further investigation of IL-8 expression in MSCs and its role for in-vitro migration. As expected, IL-8 mRNA levels were significantly higher in hypoxic MSCs. The result of the cytokine array was confirmed by examination of secreted IL-8 in the cell culture supernatant by ELISA (PeproTech). The migration capacity was investigated in a 24-well transwell chamber assay with 8 μm pore size. Using recombinant IL-8 as a chemoattractant in the lower chamber, we detected an almost twofold enhanced MSC migration rate after 24 hours under hypoxic conditions. As a different approach to investigate the migratory capacity, we used an in-vitro scratch assay. A wound was applied to a MSC monolayer in absence or presence of IL-8 which clearly enhanced the migration of MSCs into the wound area after 24 hours. In summary, IL-8 secretion by human primary MSCs is clearly increased under hypoxic conditions. IL-8 in turn seems to be a chemotactic factor for MSCs and enhances their migratory capacity in an autocrine manner.

MicroRNAs (miRNAs) can regulate hematopoietic stem/progenitor cells (HSPC) by modulation of intrinsic cell components such as transcription factors and receptors. In addition, miRNAs could play a role in the microenvironment were HSPC host and consequently affect HSPC via extrinsic factors, which to our knowledge is an hypothesis that has not been tested. Since the chemokine stromal derived factor 1alpha (SDF-1alpha) is essential for both homing and retention of HSPC in the bone marrow, we tested if miRNAs expressed by human bone marrow-derived mesenchymal stem cells (MSC; a key source of SDF-1alpha), could potentially inhibit SDF-1alpha expression. In deed, using luciferase reporter-systems we show specific binding of miR-23a to the 3′UTR of SDF-1alpha. Consequently, transfection of MSCs with a precursor miR-23a (pre-miR-23a) leads to a 30% reduction of SDF-1alpha at both mRNA and protein levels. In contrast, inhibition of endogenous miR-23a with anti-sense oligonucleotides (anti-miR-23a), leads to a significant increase of SDF-1alpha also at both mRNA (30%) and protein (10%) levels as compared to controls (scramble pre-/anti-miR). As a result, migration of CD34+ HSPC in transwell assays is strongly affected upon overexpression or inhibition of miR-23a in MSCs (with pre-miR-23a 35% less migration; with anti-miR-23a 20% more migration, as compared to respective controls). Interestingly, transforming growth factor beta 1 (TGF-beta1) inhibits SDF-1alpha expression in MSCs in a concentration dependent manner, while miR-23a is increased under same experimental settings. Even more, silencing endogenous miR-23a significantly reduces the effect of TGF-beta1 on SDF-1alpha mRNA and protein levels, suggesting that at least in part TGF-beta1 inhibits SDF-1alpha expression via increasing miR-23a levels. This is to our knowledge the first established connection between miRNA biology and HSPC-niche related factors.

Objectives: Chronic myeloid leukemia (CML) is effectively treated by Imatinib (IM) via inhibition of the BCR-ABL tyrosine kinase. However, also related tyrosine kinases like abl, c-Kit, PDGF-R, and c-FMS are blocked by IM. As shown in adult humans and mice, abl-controlled protein folding as part of the endoplasmatic stress response in heart myoblasts as well as bone “remodeling” depending on PDGF-R and c-FMS is impaired under imatinib exposure (Dewar AL et al 2005, Kerkelä R et al 2006, Fitter S et al 2008). The influence of IM on the growing heart and skeleton of immature animals has not been studied so far. With respect to treatment of pediatric CML we report alterations in these organs of juvenile mice chronically exposed to IM during the growth period. Methods: From the age of 4–14 weeks (w) [development milestones of mice: weaning 3 w; puberty 7 w; epiphysial lines closure 18 w] C3H/Neu male and female wild-type mice were chronically exposed to IM via the drinking water at concentrations of 500 mg/l (group A), 750 mg/l (group B), and 1000mg/l (group C). Femur length and overall skeletal development was analysed by whole body X-ray analysis using a mammography device. Bone metabolic activity was assessed by total body Na18F PET and CT after 5w and 10w of exposure using dedicated small animal tomographs. Bone mineral density and microstructure of tibiae were analysed by pQCT and microCT (resolution 12.5μ m) while the number of osteoclasts and resorption lacunae in femora and vertebrae was assessed by histomorphometry. Plasma concentration of IM, osteocalcin, and activity of the tartrate resistant acid phosphatase (TRAP5b) was also determined. The heart was examined histologically and ultrastructurally by electron microscopy. Results: IM was tolerated well and mean uptake of 80 mg/kg/d 110 mg/kg/d and 150 mg/kg/d resulted in serum levels of 60–674 ng/ml, 36–242 ng/ml and 51–534 ng/ml, respectively. Body weight gain was delayed in groups B and C until the age of 8 w while no change in overall growth, development and behaviour was observed at 14 w. At higher doses of IM and at younger age there was a non-significant trend to a reduction in femur length. Heart morphological examination exhibited an increased number (p Conclusion: In juvenile mice, a chronic exposure of IM resulted in toxic damage of the cardiomyocytes at higher dose rates. However, these alterations do not necessarily imply also a functional impairment which can only be studied in vivo. In the skeleton, IM reduced the number of osteoclasts and resorption lacunae in long bones but not in vertebrae. IM showed an antiresorptive effect in cancellous bone and increased cortical thickness and trabecular number by inhibiting the expansion of the marrow cavity. The effects were more pronounced in male mice and at younger age.

In the German Multicenter ALL studies (GMALL) patients aged >55 years with high risk (B-lineage ALL with WBC at diagnosis >30000, late CR, t (4; 11), complex aberrant karyotype or prae-T or mature T-ALL) or very high risk (Ph+/BCR-ABL+) ALL are increasingly candidates for allogeneic stem cell transplantation (allogeneic SCT with a HLA identical sibling donor, MRD or a matched unrelated donor, MUD) or autologous SCT. Here, we report on 31 elderly patients transplanted within the GMALL studies 06/99 and 07/03. Median age of the patients was 61 years (56–65). 22 patients belonged to the very high risk group (VHR), 8 patients to the high risk group (HR) and 1 patient from the standard risk group (SR) was transplanted because of detection of minimal residual disease.17/31 patients were transplanted from a matched unrelated donor, 9/31 patients from a HLA identical sibling donor and 5/31 patients underwent autologous SCT. Conditioning regimens for MRD SCT were myeloablative (MAC) in 6 patients (TBI 12 Gy and chemotherapy n=2, radioimmunotherapy + chemotherapy n=2, chemotherapy only n=2) and 3 patients received reduced intensity conditioning (RIC).Conditioning regimens for MUD SCT changed over time with an increasing number of RIC in the study 07/03. In total, 7/17 patients received MAC (TBI 12 Gy and chemotherapy n=5, chemotherapy only n=2) and 10/17 patients received RIC. Conditioning regimens in autologous SCT were myeloablative (MAC) in 5/5 patients. Results: After allogeneic MRD SCT 4/9 patients (44%) are alive in CCR (d+ 24, d+ 611, d+ 1721, d+ 2321), 3/9 patients died due to leukemia, 2/9 due to transplant related mortality (TRM). After allogeneic MUD SCT 8/17 patients (46%) are alive in CCR (from d+ 165 to d+ 2176). 1 further patient is alive after re- SCT for treatment of relapse. 7/17 patients died due to TRM and 1 patient died due to relapse. After autologous SCT 2/5 patients are alive in CCR (d+ 1703, d+ 1731), 3/5 died due to relapse. Risk factors for TRM: In allo SCT and MAC 8/13 patients died due to TRM in contrast to 1/13 patients with RIC. In auto SCT none of the patients died due to TRM. Risk factors for relapse: In allogeneic MRD SCT 3/9 patients died due to relapse and 2/17 patients relapsed after MUD SCT. Due to the small number of patients, no difference between MAC and RIC could be found. In autologous SCT 3/5 patients died due to relapse. In conclusion: The study shows that allo MRD but also MUD SCT is very effective in a selected population of elderly ALL patients. Since the survival of elderly patients with chemotherapy only is about 25%, more patients should be encouraged to have a MRD or MUD SCT.

FLT3-mutations are among the most common abnormalities in adult acute myeloid leukemia. These mutations induce constitutive activation of the receptor and downstream signaling pathways, including RAS/ERK, PI3K and STAT5-signalling. The most frequent aberration is an internal tandem duplication (ITD) of the juxtamembrane domain coding sequence, which is present in up to 27% of the patients with AML and has been associated with inferior prognosis. We have recently demonstrated by microarray analysis that leukemic samples of patients with FLT3-ITD mutations have significantly upregulated expression levels of Autotaxin (ATX). The ATX protein acts as a secreted lysophospholipase D (lysoPLD) through generating lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC). LPA has several important functions in cell migration and proliferation and acts via G-protein coupled receptors. It has been shown that ATX represents an aberrantly expressed motility and growth factor in a variety of cancer cells. However data on ATX in myeloid leukemias and especially in AML are missing. To study more deeply the role of ATX in leukemogenesis, we screened a series of human leukemic model cell lines for ATX expression. Using retroviral transduction, two alternatively spliced transcripts of ATX were expressed in several human leukemic cell lines without detectable levels of endogenous ATX. To investigate the ATX function and expression in normal haematopoiesis we used CD34+ human progenitor cells. The expression of ATX transcripts was confirmed at both mRNA and protein levels by RT-PCR and Western blotting, respectively. Transwell migration assays in the presence of LPC or LPA were performed to study effects of ATX on cell motility. Proliferation and clonogenic potential were investigated using MTT and colony forming assays. Moreover, we examined the effect of the FLT3 inhibitor N-benzoylstaurosporine (PKC412) on ATX expression and ATX mediated migration in MV4-11 cells. High ATX expression was found primarily in malignant cells. Western blot analysis showed that detectable levels of ATX are secreted into medium within 2 hours. In normal cells, highest ATX mRNA expression was found in purified CD34 cells, but could also be found at lower levels in T-cells. Stable overexpression of FLT3-ITD in OCI-AML3 cells induced an increased ATX expression (up to 6 fold). Vice versa, inhibition of FLT3-ITD by sublethal doses of PKC412 in MV4-11 cells resulted in a significant reduction of ATX expression down to 10% of the initial expression level. Furthermore, PKC412 treatment resulted in a complete loss of LPC or LPA induced specific migratory capacity in MV4-11 cells. The transduction of ATX increased the colony-forming capacity by 75% and significantly increased the short term proliferation. LPA increased chemotaxis in human leukemic cell lines and human CD34+ progenitors in a dose dependent manner and induced significantly higher migratory rates by at least 50%. LPC induced chemotaxis by 80–200% only in cells with high expression of endogenous or exogenous ATX, demonstrating the autocrine activity of ATX. Ongoing studies on the mechanism of ATX expression showed that ionomycin, an activator of the Ca2+–calcineurin–NFAT signalling pathway, upregulated ATX mRNA in several leukemic cell lines as well as in human primary progenitors up to 5-fold, which could be completely blocked by cyclosporine A treatment, indicating an involvement of NFAT in the regulation of ATX. Our data suggest that the production of bioactive LPA through ATX is involved in controlling proliferation and migration of hematopoietic stem cells and its deregulation may contribute to the pathogenesis of AML, especially in patients with FLT3-ITD mutations.

Introduction: Haploidentical hematopoietic cell transplantation (HHCT) after high dose conditioning with a megadose of CD34-selected stem cells has been complicated by regimen related toxicities, slow engraftment and delayed immune reconstitution leading to a high treatment related mortality (TRM). A new regimen using graft CD3/CD19 depletion and reduced intensity conditioning (RIC) may allow HHCT with lower toxicity and faster engraftment. CD3/CD19 depleted grafts not only contain CD34+ stem cells but also CD34 negative progenitors, dendritic-, natural killer- and other graft-facilitating cells which may allow stable engraftment even without the use of a megadose of CD34+ cells. Methods: A multicenter phase I/II study of HHCT using RIC with fludarabine (150–200 mg/m2), thiotepa (10 mg/kg), melphalan (120 mg/m2), OKT-3 (5 mg/day, day −5 to +14) and graft CD3/CD19 depletion with anti-CD3 and anti-CD19 coated microbeads on a CliniMACS device has been initiated. No post grafting immunosuppression was applied if the graft contained Results: To date, 36 patients (median age=46 [range, 21–65] years) have been enrolled in this study. Diagnosis were AML (n=20), ALL (n=7), NHL (n=3), MM (n=2), CML (n=2) and MCL (n=2). Patients were high risk with refractory disease or relapse after preceding HCT (auto=6, allo=12). The CD3/CD19 depleted haploidentical grafts contained a median of 7.5 × 106 (range, 3.4–17×106) CD34+cells/kg, 2.8×104(range, 0.4–44×104) CD3+T-cells/kg and 3.8×107 (range, 0.02–37.3 x107) CD56+cells/kg. Donor-recipient KIR-ligand-mismatch was found in 20 of 36 patients. The regimen was well tolerated with maximum acute toxicity being grade 2–3 mucositis. Five cases of reversible peripheral neuropathy and 3 cases of progressive multifocal leukencephalopathy (PML) occurred posttransplant in heavily pretreated patients. Engraftment was rapid with median time to >500 granulocytes/μL of 12 (range, 9–21) days, >20000 platelets/μL of 11 (range, 7–30) days and full donor chimerism after 2–4 weeks in all but one patient. Incidence of grade II-IV GVHD was 36% with grade II=9, III=2 and IV=2. TRM in the first 100 days was 10/36 (27%). Overall survival is 16/36 patients (44%) with deaths due to relapse (n=9), infection (n=9), GVHD (n=1) and PML (n=1) with a median follow-up of 194 days (range, 14–1271), resulting in a Kaplan-Meier 1 year survival estimate of 41%. We did not observe a statistical significant survival advantage for patient transplanted from a KIR-mismatched donor. Conclusion: This regimen is promising in high risk patients lacking a suitable donor, and a prospective phase I/II study is ongoing.

Notch, expressed on hematopoietic progenitors plays a crucial role in hematopoiesis. Mesenchymal stem cells (MSC) express both, Notch and its ligand Jagged and are known to support self renewal of hematopoietic progenitors via cell-cell contact and cytokine secretion. The Jagged/Notch signaling pathway has been implicated in the differentiation process of MSC, however it is not completely understood and current observations are contradictory. In order to analyze the effect of Notch signaling on human MSC differentiation we constructed lentiviral vectors that contained either the GFP-marker gene, hJagged1 IRES GFP, hNotch1 intracellular domain (NICD) IRES GFP or a gene fusion between dominant negative Mastermind1 (MAML1dn - inhibitor of Notch signaling) and the Cherry reporter gene. Primary hMSC that were obtained from bone marrow of 3 different donors were transduced with respective lentivirus vectors to greater than 98%. After exposure to adipogenic and osteogenic differentiation stimuli hMSC differentiation was quantified by Alizarin red or oil red staining, alkaline phosphatase (AP) activity and expression levels of adipogenic or osteogenic markers by Real-time PCR. Jagged1 transduced hMSC demonstrated enhanced calcium phosphate deposits and enhanced AP activity and expression levels in osteogenic differentiation medium, while adipogenic differentiation was strongly inhibited as quantified by oil red staining and low mRNA expression of genes upregulated during adipogenic differentiation (pprY, Fabp4). Similarly, overexpression of NICD induced strong and rapid osteogenic differentiation while inhibiting adipogenic differentiation and reducing cell viability. Moreover, NICD overexpression upregulates the expression of endogenous Jagged1 up to 5-fold. Inhibition of Notch signaling via overexpression of MAML1dn partially blocked the effect of hJagged1 and NICD in co-transduction experiments. In another approach MSC samples obtained from 20 donors with various osteogenic differentiation potential as measured by AP activity were analyzed for Notch1 and Jagged1 expression. While there was no correlation between AP activity and Notch1 levels we observed a significant positive correlation for AP activity and Jagged1 expression. In summary, our data strongly suggest that increased Jagged/Notch signaling enhances the osteogenic differentiation of hMSC while inhibiting their adipogenic fate.

NFAT (Nuclear Factor of Activated T cells) is a family of calcium-induced, calcineurin-dependent transcription factors, well characterized as central regulators of inducible gene expression in T lymphocytes but now known to function also in several other cell types in various adaptation and differentiation processes. Activation of NFAT by the phosphatase calcineurin is counteracted by several inhibitory kinases and can be completely blocked by the immunosuppressant Cyclosporin A. The Down syndrome critical region 1 (DSCR1; also termed CSP1, MCIP1 or RCAN1) gene belongs to the calcipressin family of endogenous calcineurin inhibitors and is expressed in several isoforms, one of which (isoform C, coded by exons 4–7) has been described to be a transcriptional target for NFAT in striated muscle, endothelial, and neural cells. The DSCR1 gene is located within the Down syndrome critical region of human chromosome 21 and is, together with 200–300 other genes, overexpressed about 1.5-fold in patients with Down syndrome (DS). Previously, dysregulation of NFAT signaling by overexpression of DSCR1 has been implicated in causing various of the pathophysiological features observed in DS patients. Children with DS also suffer from an about 500-fold increased incidence of acute megakaryocytic leukemia; the respective roles of NFAT or DSCR1 in megakaryocytes of either normal individuals or those with DS, however, has not yet been established. Here we show that DSCR1 is upregulated during megakaryocytic differentiation in a lineage-specific manner, and in mature megakaryocytes is further strongly induced by calcineurin stimulation. DSCR1 expression in megakaryocytes is regulated by NFAT, since overexpression of NFATc2 enhances, while overexpression of the specific inhibitor of NFAT activation, VIVIT, suppresses expression of the gene. We further demonstrate that DSCR1 does not only represent an NFAT target in megakaryocytes, but itself acts an inhibitor of NFAT signaling in these cells. Overexpression of DSCR1 in CMK cells as well as in primary megakaryocytes by retroviral transduction profoundly suppressed ionomycin-induced dephosphorylation and nuclear translocation of NFATc2, as well as transactivation of an NFAT-dependent promoter construct. Finally, overexpression of DSCR1 in megakaryocytes markedly downregulated both the constitutive and induced expression of Fas Ligand, a pro-apoptotic gene recently established as a NFAT target in megakaryocytes. Together, these results suggest that DSCR1 acts as an NFAT-induced NFAT inhibitor in megakaryocytes and, when overexpressed, interferes with the expression of NFAT-dependent megakaryocytic genes.

Allogeneic hematopoietic cell transplantation (HCT) using reduced intensity conditioning (RIC) regimens offers a potential curative therapy to patients with advanced NHL. RIC HCT induces potent graft-versus-lymphoma effects with best results in patients with low tumor burden at time of HCT. Combined use of radioimmunotherapy (RIT) with RIC may increase anti-lymphoma activity of RIC while HCT provides rescue from hematologic toxicity of RIT. This may allow dose escalation of RIT. A multicenter phase I/II study of allogeneic HCT combining RIT using yttrium-90-ibritumomab tiuxetan (Y90-CD20) with two RIC regimens for treatment of patients with NHL has been initiated. Patients with indolent NHL (Arm A) receive RIT with Y90-CD20 (0.4 mCi/kg) on day −14 combined with RIC using fludarabine (30 mg/m^2 day −4 to−2) and 2 Gy TBI (day 0). Patients with aggressive NHL (Arm B) receive an escalated dose of Y90-CD20 (0,6–0,8 mCi/kg) on day −14 combined with RIC using fludarabine (30 mg/m^2 day −8 to−4), melphalan (140 mg/m^2 day −3) and campath (20–30 mg day −3 to−2). For postgrafting immunosuppression either CSA/MMF (Arm A) or CSA alone (Arm B) is used. To date, 31 patients have been enrolled (Arm A=23, Arm B=8). Diagnoses in Arm A were FL (n=12), MCL (n=6), CLL (n=4) and Immunocytoma (n=1). Diagnoses in Arm B were DLBCL (n=6), blastoid MCL (n=1) and transformed CLL (n=1). Median age was 55 (range, 34–67) years. PBSC grafts were either from matched related (n=10) or matched unrelated donors (n=21). All patients were high risk with refractory disease or relapse after preceding HCT. Disease status after salvage therapy at time of HCT was in Arm A: CR=1, PR=18, SD=4 and in Arm B: PR=8. No additional toxicity due to RIT was observed. Engraftment was rapid and sustained with no graft rejections. In Arm A median time to >500 granulocytes/μL was 13 (range, 0–69) days and to >20000 platelets/μL 3 (range, 0–69) days (in 11 patients platelets never dropped 500 granulocytes/μL was 17 (range, 9–23) days and to >20000 platelets/μL 11 (range, 8–29) days. TRM in the first 100 days was 3%, overall 19%. Incidence of grade II-IV GVHD in Arm A was 35% (II=3, III=4, IV=1) and in Arm B 25% (II=2). Best disease response observed was in Arm A: CR=18, PR=5 and in Arm B: CR=3, PR=5. To date, 16/23 patients in Arm A and 6/8 patients in Arm B are alive with a median follow-up of 271 (range, 20–390) days, resulting in a Kaplan-Meier 1 year survival estimate of 65% in Arm A and 62% in Arm B. Causes of death were infection=5, GVHD=1, relapse=1 in Arm A and relapse=2 in Arm B. A combination of RIT with RIC is feasible with no additional toxicity due to RIT and stable engraftment in all patients. Preliminary response data suggest that this strategy may improve early post-transplant disease control, but long-term disease-free survival remains to be determined.

BACKGROUND: The toxicity of commonly used myeloablative regimens is the main factor limiting applicability and restricting upper age limit for allogeneic hematopoietic stem cell transplantation (alloHSCT). However, reduced intensity conditioning (RIC) protocols are associated with high risk of relapse. Previous dose-escalation trials revealed high-dose treosulfan-based conditioning an alternative treatment with myeloablative as well as antileukemic potential, accompanied by low non-hematological toxicity. The goal of this multicenter phase II trial was to evaluate safety and efficacy of treosulfan/fludarabine conditioning in AML patients. PATIENTS AND METHODS: Seventy-five patients (median age 45 years, range 19–59) with AML in 1st (80%), 2nd (17%), or 3rd (3%) complete remission were treated with alloHSCT from either matched related (MRD, 40%) or unrelated donors (MUD, 60%). Preparative regimen consisted of treosulfan 14 g/m2/day on days −6 to −4, fludarabine 30 mg/m2/day on days −6 to −2, and in case of MUD-HSCT additionally ATG (Fresenius) 10 mg/kg/day on days −4 to −2. Graft-vs.-host disease (GvHD) prophylaxis consisted of cyclosporine and short-course methotrexate. 25% of the patients had an increased risk of toxicity for standard myeloablative conditioning regimens because of higher age or co-morbidities. RESULTS: All patients engrafted after a period of absolute neutropenia with the median time to ANC >0.5 G/L and PLT >50 G/L of 21 (13–39) days and 19 (11–48) days, respectively. The cumulative incidence of complete donor chimerism was 72% on day +28 and 92% on day +100. Most frequent CTC grade 3 or 4 complications until day 28 after transplantation were febrile neutropenia (28%), infection in neutropenia (16%), and mucositis (5%). None of the patients experienced severe hepatic toxicity, including VOD. After a median follow up of 457 days (98–988), the probabilities of overall (OS) and disease-free survival (DFS) at 2 years equaled 67% resp. 58% (MRD-HSCT) and 58% resp. 45% (MUD-HSCT, p=NS). The 2-year cumulative incidence of relapse was 34% for MRD-HSCT and 37% for MUD-HSCT, whereas non-relapse mortality was 8% and 17%, respectively. Outcome was not significantly influenced by disease status (CR1 vs. ≥CR2) or age. CONCLUSIONS: Conditioning therapy based on treosulfan at a dose of 3x14 g/m2 in combination with fludarabine is very well tolerated, resulting in a low NRM. The regimen demonstrated reliable efficacy in AML patients with confirmed first or subsequent remission. Further randomized studies are warranted to verify advantageous survival results.

The major limitation of allogeneic stem cell transplantatiopn (SCT) in patients (pts) with myelofibrosis is the high treatment related mortality. We performed a prospective multicenter trial of a dose-reduced conditioning regimen, consisting of busulfan (10 mg/kg orally or 8mg/kg i.v), fludarabine (180 mg/m2) and anti-thymocyte globulin (ATG Fresenius: 30–60 mg/kg) followed by allogeneic SCT in pts with myelofibrosis. From 2002 to 2006, 104 pts with a median age of 55 years (range: 32–68) andlow risk with constitutional symptoms (18%) or intermediate risk (n= 58%) and high risk (n=19%) were included. Cytogenetic abnormalities and JAK2 mutation were noted in 22% and 48%, respectively. Bone marrow histology showed advanced fibrosis (MF 2 and 3) in all pts. All but 3 pts received peripheral blood stem cells as graft source either from related (n=33) or unrelated donor (n=71). All but one (1%) pts showed leukocyte and platelet engraftment after a median of 18 and 21 days, respectively. The median duration of leukocyte aplasia was 9 days (range: 3–21). Acute GvHD grade II to IV occurred in 19% and severe aGvHD III/IV in 7%, while chronic GvHD was seen in 32% of the pts. Non-relapse mortality at 1 year was 19% (95% CI: 11–27%) and significantly lower for pts younger than 50 years of age (0% vs 27%, p=0.004) and for pts with low risk vs intermediate/high risk disease (0% vs 27%, p= 0.02). The cumulative incidence of relapse at 3 years was 29% (95%CI: 15–43%). The 3 year overall (OS) and event-free survival (EFS) was 70% (95% CI 60–80%) and 55% (95% CI 42–68%). Significant factors for improved 3 year OS and EFS were age less than 50 years (92% vs 62%, p=0.003 and 79% vs 46%, p= 0,004) and low vs intermediate/high risk (100% vs 62%, p=0.01 and 72% vs 48%, p= 0.02), while no impact on survival was seen for cytogenetic abnormalities, JAK2 mutation status and donor (related vs unrelated). These prospective multicenter study show excellent outcome of an busulafan/fludarabine based reduced conditioning regimen followed by allogeneic SCT in pts with myelofibrosis.

Infections with herpesvirus and adenovirus is a major cause of morbidity and mortality in patients undergoing stem cell transplantation (SCT). In those patients, the frequency of herpesvirus and adenovirus co-infections as well as their contribution to the infectious burden remain so far unclear. In this study, we have postulated that co-infections with herpesviruses and adenoviruses are frequent in the 100 days following SCT, contributing significantly to the infectious burden. To address this issue, 615 blood samples collected from patients undergoing SCT (n=35) were retrospectively tested by quantitative real-time PCR for the presence of HSV 1/2, VZV, EBV, CMV, HHV6 A/B, and adenovirus. Infections with a single virus were detected in 46.1% (283/615) of the samples, whereas co-infections with two or three viruses were detected in 9.9% (61/615) and 2.1% (13/615) of the samples, respectively. Interestingly, about 50% of the patients (17/35) displayed a co-infection with two or more viruses, with [CMV+HHV6 A/B], and [EBV + HHV6 A/B +adenovirus] being most frequently detected, respectively. Comparison of viral loads indicated that the infectious burden increases proportionally to the number of viruses detected. Moreover, a DNA-microarray allowing simultaneous detection of HSV 1/2, VZV, EBV, CMV, HHV6 A/B, and adenovirus was developed. The assay displayed a high diagnostic accuracy, being an attractive device for routine assessment of the infectious burden in immunocompromised patients after SCT.

In a up-front randomized study, 939 adult patients up to the age of 60 years received a double induction therapy. One course of MAV (mitoxantrone 10 mg/m2 days 4–8, cytarabine 100 mg/m2 continuous infusion days 1–8, etoposide 100 m g/m2 days 4–8) was followed by one cycle of MAMAC (cytarabine 1 g/m2, every 12h days 1–5; amsacrine 100 mg/m2 days 1–5). Patients with intermediate risk cytogenetic (IRCG) and a HLA matched sibling received an allogeneic transplantation, those with poor risk cytogenetic (PRCG) were intended to be transplanted from a sibling or unrelated donor. All AML patients without an available donor received the randomly assigned first postremission therapy (PRT) mitoxantrone combined with intermediate-dose cytarabine (I-MAC; total dose 12 g/m2) or high-dose cytarabine (H-MAC; total dose 36 g/m2). As second PRT, patients with t(8;21) received an additional cycle of chemotherapy. An autologous transplantation was scheduled for IRCG and PRCG without an allogeneic donor. The CR rate was 88% for patients with t(8;21), with IRCG 71%, and 50% with PRCG. The 5-year-survival was 21% (95% CI: 16–27%) in the PRCG, 40% (95% CI: 36–45%) in the IRCG and 74% (95% CI: 60–88%) in the t(8;21) group. No difference was observed between the I-MAC and the H-MAC group. In a multivariate analysis, a significant (p50*10^9/L, and IRCG compared to PRCG. The relapse incidence was higher in patients without an allogeneic donor, a Flt3 mutant/wildtype ratio > 0.8 or PRCG. A risk score build out of the sum of the individual hazard ratios (SHR) was able to discriminate two groups for the IRCG with a marked difference in the 5-year-survival (low SHR: 55% [95% CI: 48–62%]; high SHR: 33% [95% CI: 28–38%]) was well as for the PRCG group (low SHR: 44% [95% CI: 32–56%]; high SHR: 13% [95% CI: 7–18%]). The risk score identified in this large patient cohort may allow individual tailoring of therapeutic interventions in future AML trials.

The majority of patients with acute myeloid leukemia (AML) are older than 60 years at diagnosis. However, treatment results for these elderly patients are still unsatisfactory. This is thought to be due to a more aggressive disease, preexisting co-morbidities or a decreased tolerance for intensive treatment approaches. As for younger patients there is growing evidence that elderly AML patients may be divided into prognostic subgroups. So far data on prognostic factors in this group of patients are still sketchy. Between February 1996 and March 2005 a total of 827 elderly AML patients with a median age of 67 (61–87) years were treated within the prospective AML96 trial of the German Study Initiative Leukemia (DSIL). 643 patients had de novo and 184 patients secondary disease. All patients were scheduled to receive a double induction therapy with Daunorubicin and Ara-C (DA3+7). The consolidation therapy consisted of one course of m-Amsacrine and intermediate-dose (10g/m2) Ara-C. 265 (32%) patients reached CR criteria after double induction therapy. Forty-two patients (5%) had only a PR, 307(37%) displayed refractory disease, 126(15%) died during induction therapy and 77(10%) received only one course of induction therapy due to severe toxicity. Out of the 265 patients in CR 120 (45%) patients received the consolidation course. The strongest independent prognostic factors for achieving a CR were less than 10% blasts in the day 15 bone marrow, the presence of a NPM mutation or a low-risk karyotype (p

Animal models of acute graft versus host disease (GVHD) suggested the critical role of host antigen-presenting cells (APC) in the pathogenesis of acute GVHD (Shlomchik et al Science285:412, 1999). Depletion of host B cells reduced the incidence of acute GVHD (Schultz et al BMT16:289, 1995). We hypothesized that B-cell depletion by prior rituximab (RTX) therapy in lymphoma pts might result in reduced incidence of acute GVHD (Ratanatharathorn et al Blood96:391a, 2000). We now analyzed the outcomes of allogeneic PBSCT in 435 B-cell lymphoma pts reported to CIBMTR from 1999 to 2004. Pts who had prior therapy with anti-CD52 or anti-T-cell antibody or recipients of conditioning regimens containing any of these antibodies were excluded. All patients received unmodified PBSC grafts. Pts were considered to have prior RTX therapy if they received RTX within 6 months of transplantation. There were 256 pts who did not receive RTX within 6 months of transplantation (No-RTX group) and 179 pts received RTX within 6 months of transplantation (RTX group). In RTX group, most pts (67%) received the last dose of RTX within 3 months of transplantation and 54% received at least two doses of RTX. Baseline characteristics of the two groups were similar except for significantly more unrelated donor (32% vs 19%, P=0.002)), tacrolimus use (47% vs 26%, P Clinical outcomes Relative risk (95% CI) P value Grade II-IV acute GVHD 0.72 (0.53–0.97) 0.03 Grade III-IV acute GVHD 0.55 (0.34–0.91) 0.02 Chronic GVHD 0.89 (0.67–1.18) 0.41 TRM 0.68 (0.46–1.0) 0.05 Progression or relapse 0.7 (0.42–1.19) 0.19 Progression-free survival 0.68 (0.50–0.92) 0.01 Overall survival 0.63 (0.46–0.86) 0.004

Activating mutations of the FLT3-receptor tyrosine kinase such as the short internal tandem duplication (ITD) represent the single most common genetic aberration in patients with acute myeloid leukemia (AML). FLT3-ITD mutations are present in about 25% of AML patients and are associated with high blast counts and a higher rate of relapse after conventional chemotherapy. CXCR4 and its ligand SDF-1 are of central importance for stem cell homing to the bone marrow. In FLT3-ITD positive AML, CXCR4 expression on stem cells is negatively correlated to overall survival. Using retroviral vector transduction we achieved overexpression of the human FLT-ITD transgene, the FLT3-wt or the GFP control gene in greater than 70% of cord blood hematopoietic progenitors (HP). In contrast to previously published data in murine cell lines, we found that FLT3-ITD positive human cells display strongly reduced migration towards the CXCR4 ligand SDF-1. Analysis of SOCS3 (suppressor of cytokine signaling 3) expression revealed strongly increased mRNA expression in FLT3-ITD overexpressing CD34+HP and FLT3-ITD positive AML blasts. Since SOCS3 is a known antagonist of CXCR4 signaling, increased SOCS3 levels might cause the observed inhibition of SDF-1 dependent migration. For individually tested CD34+HP from 6 cord blood donors, co-cultivation of FLT3-ITD overexpressing HP on the irradiated mouse stromal cell line M210-B4 or on primary mesenchymal stromal cells resulted in an increased formation of early cobble stones and a dramatic proliferation advantage for suspended cells compared to naïve, FLT3-wt or GFP-transduced HP. Addition of the CXCR4 chemokine inhibitor AMD3100 to the co-culture decreased the cobble stone formation and growth advantage of FLT3-ITD transduced cord blood HP. Similar results were obtained using FLT3-ITD positive AML cells from 3 patients. Using CFSE to track cell proliferation and PI/Annexin staining to analyze apoptosis, we demonstrate that the addition of AMD3100 had no effect on apoptosis but decreased the proliferation rate of FLT3-ITD positive blast cells. Using primary AML blasts and FLT3-ITD transgene overexpressing cord blood HP, we demonstrate that the robust FLT3-ITD mediated proliferation advantage can be partly inhibited by the CXCR4 antagonist AMD3100.

We analyzed the prognostic impact of the most frequent genetic abnormalities detected by fluorescence-in situ-hybridization combined with immunofluorescent cytoplasm immunoglobulin staining (cIg-FISH) in 101 patients with multiple myeloma, who underwent allogeneic stem cell transplantation after reduced melphalan/fludarabine-based conditioning from related (n=34) and unrelated (n=67) donors. The abnormalities were del(13q14) [62%], t(11;14)(q13;q32) [11%], t(4;14)(p16.3;q32) [16%], CMYC-gains (8q24) [17%], del(17p13.1) [16%], t(14;16)(q32;q23) [4%], whereas none of the patients had t(6;14)(p25;q32). Translocation t(4;14), CMYC-gains and del 17p were frequently associated with del(13q14): 64%, 80% and 92%, respectively. The complete remission (CR) rate was 45% for all patients. Patients with del(17p13) achieved fewer complete remission than others (7% vs. 56%; p=0.001), while no difference was seen for t(4;14) and other abnormalities. Univariate analysis revealed higher relapse rates for age > 50 years (p=0.002), del(13q14) (p=0.006) and del(17p13) (p=0.003). Patients with translocation t (4;14) had a similar four year event-free survival than others (50 vs 45%). In a multivariate analysis, only del(13q14) [HR: 2.34, p=0.03] and del(17p13) [HR: 2.24; p=0.04] influenced the risk of relapse, while for event-free survival, only age [HR 2.8; p=0.01] and del(17p13) [HR: 2.05; p=0.03] retained the prognostic value. These data seem to indicate that some adverse cytogenetic risk factors such as t(4;14) can be overcome by allogeneic stem cell transplantation, probably due to the graft versus myeloma effect. Del(17p13.1) is a significant factor for a lower chance of complete remissions and shorter event free survival following allogeneic stem cell transplantation. The presented data will have implications for risk-adapted strategies in the future.

Megakaryocytes, as precursor cells of platelets, comprise a cell population crucial for the maintenance of adult hematopoiesis. Since platelets are anuclear cellular fragments, the platelet transcriptome as well as the proteome are largely determined by the megakaryocyte. Calcium signaling in response to agonists such as collagen or thrombin has a central function in platelet activation and therefore likely represents an important signaling pathway in megakaryocytes as well. Indeed, megakaryocytes show considerable elevation of intracellular calcium levels in response to selected platelet agonists, but the downstream effector molecules of calcium signaling in these cells or the transcriptional responses induced are unknown. We here establish calcineurin and the NFAT (Nuclear Factor of Activated T cells) family of transcription factors as components of a calcium-induced signaling cascade in megakaryocytes. In resting megakaryocytes, NFAT is cytoplasmic and inactive, but can be activated by fibrillar collagen type 1, a physiological agonist of platelets and megakaryocytes known to induce a sustained increase in intracellular calcium levels in these cells. In contrast, treatment with SDF-1a, thrombopoietin, or VEGF remained without noticeable effect, presumably because of the short duration of the calcium transients induced by these agents. Collagen-induced NFAT activation in megakaryocytes requires dephosphorylation by calcineurin and is completely sensitive to the calcineurin inhibitor cyclosporin A. Activation of NFAT by collagen was paralleled by the induction of the expression of Down Syndrome Critical Region I (DSCR1) and Fas Ligand (FasL), two genes recently identified as NFAT targets in megakaryocytes. Collagen-induced expression of DSCR1 and FasL occurred in a calcineurin- and NFAT-dependent manner, as it was blocked by both cyclosporin A as well as the specific peptide inhibitor of NFAT, VIVIT. These experiments show that the calcineurin pathway is a target for selected physiological ligands capable of inducing sustained calcium mobilization in megakaryocytes and regulates megakaryocyte gene expression in a cyclosporin A- and NFAT-dependent manner.

Gemtuzumab ozogamicin (GO) has been associated with an increased risk of liver sinusoidal obstruction syndrome (SOS) when applied within three months before or after allogeneic stem cell transplantation. In addition, excess of liver toxicity has been observed when GO was used directly before conventional intensive preparative regimens. We hypothesized that GO might be safe and effective as part of a fludarabine-based reduced-intensity conditioning regimen. 30 patients relapsing after conventional induction chemotherapy (n=17) or after previous transplantation (autologous n=3; allogeneic n=10) have been included in a prospective phase I/II trial. Per protocol the preparative regimen contained 6 mg/sqm and 3 mg/sqm GO on day-21 and day-14 followed by fludarabine 120 mg/sqm, 200 or 800 cGy total-body irradiation (TBI; depending on age and pretreatment) and stem cell infusion during GO-induced aplasia. Five patients who had previously undergone TBI received melphalan 140 mg/sqm. GvHD prophylaxis was performed with tacrolimus (starting day-1) and mycophenolate mofetil (1.5g BID from day 0). Four patients with progressive disease after GO did not proceed to conditioning therapy and went off study. A reduction of marrow blast counts after GO monotherapy was achieved in 15/30 patients (50%). In 11 cases without response to GO, an additional salvage regimen containing anthracyclines and high-dose cytarabine was administered before conditioning therapy and transplantation. 26 patients received G-CSF mobilized peripheral blood progenitor cells from matched-sibling (n=6) or unrelated donors (n=20). Primary engraftment was observed in all cases. With a median follow-up of 20 months (range 6–55) only one patient experienced sinusoidal obstruction syndrome which was reversible after treatment with defibrotide. Grade II-IV acute GvHD occurred in 15 patients (54%). Nine patients are alive in complete remission. Reasons for death in the other patients were relapse/progression (n=10) and GvHD/Infection (n=7). The probability of overall and disease-free survival at two years for patients having received a transplant is 38% and 32%, respectively. Blast reduction after GO was associated with a superior disease-free survival (48%, p=0.1). These data suggest that GO can be integrated into reduced-intensity conditioning therapy before allogeneic stem cell transplantation in patients with relapsed AML. Refractory patients with no response to GO monotherapy have a low chance of cure and should probably receive other combination therapies.

No standard treatment is available for relapse of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) after allogeneic stem cell transplantation (alloSCT). Efficacy of donor lymphocyte transfusion (DLT) was limited, at least in part, by the rapid pace of the disease, overwhelming any allogeneic immune response. However, induction chemotherapy before DLT was complicated by severe toxicity. Based on a pilot trial conducted in Munich, a prospective, multicenter phase II trial for relapsed AML and MDS after alloSCT was initiated in 2000 by the German transplant cooperative group. The study evaluated the sequence of low-dose(ld)AraC for leukemic control (intensive chemotherapy for progressive disease only), transfusion of donor PBSC without immunosuppression as adoptive immunotherapy, and systemic application of GM-CSF. GM-CSF was included, since in combination with other cytokines, it has been able to improve the antigene presentation capacity of myeloid blasts in vitro. Between 2000 and 2006, 41 patients with hematological relapse of AML or MDS >3 months after alloSCT were included. Median age was 47y, 50% had an unrelated donor. Median remission after SCT was 223d (93–1614), median percentage of BM blasts at relapse was 40%. Median follow up was 28 months. Control of leukemic proliferation by ldAraC was achieved in 61%, allowing outpatient care in the majority of these patients. 39% required intensive chemotherapy. Three patients died from infections before donor cell transfusion (DCT), one patient was not transfused due to progressive leukemia. Median time from relapse to DCT was 52 days. 25/34 evaluable patients were found to be free of blasts in BM at d35 after PBSC and were considered initial responders. In an intent-to-treat analysis, overall survival (OS) of the entire cohort at 1 and 2 years from relapse was 41% and 32%. Among initial responders, 1y- and 2y-OS was 68% and 49%. A remission >6months after alloSCT, and control of leukemia by ldAraC prior to DCT were associated with better outcome. At last follow up, 8 patiens were in continous CR, 2 were alive with second relapse, 18 had died from leukemia, and 13 had died in remission or aplasia. In conclusion, ldAraC seems to be effective for initial control of leukemic proliferation in relapsed AML and MDS after alloSCT. A longer duration of post-transplant remission and response to ldAraC may identify patients who will benefit from adoptive immunotherapy. On an intend-to-treat-basis, the overall results of our trial compare favorably to other published strategies; nevertheless, outcome is still unsatisfying and warrants further investigation.

Between 1996 and 2003, 994 patients aged under 60 years with de novo AML received a double induction chemotherapy consisting of MAV (mitoxantrone 10 mg/m2 day 4–8, Ara-C 100 mg/m2 continuous infusion day 1–8, etoposide 100 mg/m2 day 4–8) and MAMAC after an interval of at least 14 days (Ara-C 1 g/m2 q. 12 hrs day 1–5, amsacrine 100 mg/m2 day 1–5). A cytogenetic high risk profile (complex aberrations, −5/del(5q), −7/del(7q), hypodiploid karyotypes other than −5, −7, −X, −Y, abnl3q, abnl1q, abnl12p, t(6;9), t(9;22), t(9;11), +11, +13, +21, +22) was present in 252 patients (de novo AML [N=160], prior MDS [N=41], and secondary AML [N=51]). 119 out of 252 (47%) were in complete remission after 2 induction cycles. 35 (14%) additional patients showed complete remission after a further cycle. Patients with a HLA matched sibling donor (N=37) or an unrelated donor (N=34) received an allogeneic transplantation as consolidation therapy. The conditioning protocol consisted of 12 Gy TBI and cyclophosphamide. Cyclosporine and methotrexate were used as GvH prophylaxis. The actuarial survival at 5 years is 42% (CI: 26%–58%) in the related donor group and 53% (CI: 36%–70%) in the unrelated donor group. The disease free survival at 5 years is 40% (CI: 24%–56%) in the related group and 43% (CI: 26%–60%) in the unrelated group (for both, p=ns). In conclusion, the results after unrelated donor transplants are comparable with those after related donor transplants in patients with high risk AML. These data of the AML’96 study will be compared with the experience of our current upfront concept using allogeneic transplantation in aplasia after the 1st or 2nd induction cycle in high risk AML.

Haploidentical hematopoietic cell transplantation (HHCT) after high dose conditioning with CD34-selected stem cells has been complicated by high regimen related toxicities, slow engraftment and delayed immune reconstitution leading to increased treatment related mortality (TRM). A new regimen using graft CD3/CD19 depletion with anti-CD3 and anti-CD19 coated microbeads on a CliniMACS device and reduced intensity conditioning may allow HHCT with lower toxicity and faster engraftment. CD3/CD19 depleted grafts not only contain CD34+ stem cells but also CD34 negative progenitors, natural killer-, graft-facilitating- and dendritic-cells. Reduced intensity conditioning was performed with fludarabine (150–200 mg/m²), thiotepa (10 mg/kg), melphalan (120 mg/m²) and OKT-3 (5 mg/day, day −5 to +14) and no posttransplant immunosuppression. Twenty two consecutive patients (median age=43 (range, 21–59) years) have been transplanted with this regimen. Diagnosis were AML (n=12), ALL (n=5), NHL (n=2), MM (n=2) and CML (n=1). Patients were “high risk” with refractory disease or relapse after preceding HCT. The CD3/CD19 depleted haploidentical grafts contained a median of 7.6 x 106 (range, 3.4–17x106) CD34+cells/kg, 6.7x107 (range, 0.02–18.2 x107) CD56+cells/kg and 5.5x104(range, 0.006–44x104) CD3+T-cells/kg. Donor-recipient KIR-ligand-mismatch was found in 14 of 22 patients. The regimen was well tolerated with maximum acute toxicity being grade 2–3 mucositis. Because of severe neurotoxicity in 4 patients treated with 200 mg/m² fludarabine, the dose was reduced to 150 mg/m² with no further cases of neuropathy thereafter. Engraftment was rapid with median time to >500 granulocytes/μL of 12 (range, 10–21) days, >20000 platelets/μL of 11 (range, 7–28) days and full donor chimerism after 2–4 weeks in all patients. Incidence of grade II–IV GVHD was 59% with grade II=9, III=2 and IV=2. One patient, who received the highest T cell dose, developed lethal grade IV GVHD. TRM in the first 100 days was 6/22 (27%) with deaths due to idiopathic pneumonia syndrome (n=1), mucormycosis (n=1), pneumonia (n=3) or GVHD (n=1). Six patients died after day 100, five due to relapse and one with systemic adenoviral infection. Overall survival is 10/22 patients (45%) with a median follow-up of 185 days (range, 17–988). In conclusion, this regimen is promising in high risk patients lacking a suitable donor, and a prospective phase I/II study is ongoing.

Deletions of chromosome bands 13q14 and 17p13 (P53) as well as t(4;14)(p16;q32) or overrepresentation of CMYC are known to be adverse prognostic factors for time to progression (TTP) and overall survival (OS) in myeloma patients receiving conventional or high-dose chemotherapy followed by autologous stem cell transplantation. We investigated the impact of these chromosomal abnormalities as detected by fluorescence in situ hybridization combined with immunofluorescent cytoplasm immunoglobulin staining (cIg-FISH) on outcome after dose-reduced melphalan/fludarabin based allogeneic stem cell transplantation (SCT) in 64 patients (pts) with multiple myeloma (MM). Median age was 52 years (34 – 67 ys.), 38 patients were male, 26 female. 45 pts received stem cell graft from an unrelated donor and 19 pts from HLA-identical sibling. Deletion of 13q14 was found in 35 out of 64 pts (55%), P53 deletion in 7 out of 64 pts (11%), t(4;14)(p16;q32) in 11 out of 56 pts (20%) and trisomy of CMYC in 12 out of 55 pts (22%). The following strong correlations among the genetic changes have been seen: six out of seven pts with P53 deletion also showed deletion in chromosome band 13q14 (86%), which was also found in nine out of eleven pts with t(4;14) (82% del 13) and nine out of twelve pts with CMYC trisomy (75% del 13). The estimated event-free (EFS) and overall survival (OS) at four years for the entire study population was 48% and 57%, respectively. No significant difference of the estimated event-free survival at 3 years was seen for pts with del 13 (42% vs 54%, p=0,4), with P53 deletion (43% vs 49%, p=0,27) and for pts with t(4;14) (46% vs 62%, p=0,69) or CMYC overrepresentation (37% vs 50%, p=0,35). Comparison of pts showing both del 13 and P53 deletion, del 13 and t(4;14), del 13 and trisomy of CMYC or t(4;14) and CMYC trisomy with the rest of the study population did not show a significantly reduced EFS either. Even the comparison of pts with any chromosomal abnormalities and pts without any genetic changes detected by FISH did not show a significant difference in EFS (48% vs 50%, p=0,8). These preliminary data suggest that the negative prognostic impact of chromosomal abnormalities such as deletion of 13q14, P53 deletion, t(4;14) and overrepresentation of CMYC may be overcome by allogeneic SCT probably due to the graft versus myeloma effect.

Purpose: To analyse hematopoietic engraftment after allogeneic stem cell transplantation (SCT) in patients with chronic lymphocytic leukemia (CLL) who had received Campath prior to SCT. Patients and Methods: 40 patients with relapsed CLL were included into the analysis. All patients received Busulphan 8 mg/kg and Fludarabine 150 mg/m2 as conditioning regimen. The first 20 patients (Bu/Flu/Cam) received a cumulative dose of 75 mg Campath on days -9 to -5 and Cyclosporine (CSA) as GVHD-prophylaxis. The second 20 patients (preCam/Bu/Flu) received a conventional treatment (3 × 30 mg weekly) with Campath prior to SCT. Transplantation was scheduled 14 days from the last dose of Campath and GVHD-prophylaxis consisted of CSA and Methotrexate. The two groups did not differ in age, median number of prior chemotherapy regimens, prior fludarabine treatment or donor type. The observation of late secondary graft failure during the first phase of the study led to the analysis of lineage-specific engraftment (CD4+T-cells, CD8+T-cells, NK-cells and Neutrophils) and of Campath levels with an ELISA at the day of transplant. Complete donor chimerism was defined as more than 95% donor-DNA in a specimen. Results: Campath levels are available for 22 patients. The median antibody level was 307 ng/mL (range, 41 to 1820 ng/mL) after Bu/Flu/Cam and 62 ng/mL (detection limit to 490 ng/mL) after preCam/Bu/Flu. All patients reached neutrophil engraftment (>0.5/nl) after a median of 18 days (range, 14 to 27). The cumulative incidence of complete leukocyte chimerism at day +100 was 75% after Bu/Flu/Cam and 78% after preCam/Bu/Flu (p=0.858). The cumulative incidences of CD4+ and CD8+ T-cell donor chimerism at day +100 were 45% after Bu/Flu/Cam and 70% after preCam/Bu/Flu for both T-cell subsets (CD4+subset, p=0.481; CD8+subset, p=0.398). Of note, the Campath-level at the day of transplantation discriminated better between patients with prompt versus impaired T-cell engraftment. The incidences of complete CD4+ and CD8+ T-cell chimerism at day +100 were 14% and 29% in patients with Campath levels >250 ng/mL compared to 82% and 82% in patients with antibody levels 250 ng/mL compared to 0 out of 17 patients with Campath levels Conclusions: T-cell chimerism should be analysed in patients who have received Campath recent to SCT. The level of Campath at the day of transplant may be an important factor for delayed T-cell engraftment. Figure Figure

Recent data show that the tyrosine kinase inhibitor Imatinib mesylate (IM) also affects normal hematopoietic stem cells (HSC), T lymphocyte activation and dendritic cell function not relying on the specific inhibition of bcr-abl activity. Mesenchymal stem cells (MSC) have been identified in the bone marrow (BM) as multipotent non-hematopoietic progenitor cells that differentiate into osteoblasts, adipocytes, chondrocytes, tenocytes, skeletal myocytes, and cells of visceral mesoderm. MSC interact with HSC, influencing their homing and differentiation through cell-cell contact and the production of factors including chemokines We evaluated possible effects of IM in vitro on human bone marrow-derived MSC. Screening the activity of fourty-two receptor tyrosine kinases by a phospho-receptor tyrosine kinase (RTK)-array revealed an exclusive inhibition of platelet-derived growth factor receptor (PDGFRβ) by IM which consequently affects downstream targets of PDGFRβ as Akt and Erk1/2 signalling pathways in a concentration and time dependent manner. Furthermore, perinuclear multivesicular bodies harbouring PDGFRβ were found within 18–20 hours culture of MSC in the presence of 5 μM IM. Cell proliferation and clonogenicity (evaluated as the capability to form colony forming units - fibroblasts (CFU-F)) of MSC were significantly inhibited by IM in a concentration dependent fashion. IM inhibits significantly the differentiation process of MSC into osteoblasts as evaluated by decreased alkaline phosphatase activity and reduced calcium phosphate precipitates. In contrary, differentiation of MSC into adipocytes was strongly favoured in presence of IM. All these functional deficits described, probably contribute to an observed 50% reduction in the support of clonogenic hematopoietic stem cells, as evaluated by a long term culture-initiating cells (LTC-IC)-based assay. In summary our experiments show that IM inhibits the capacity of human MSC to proliferate and to differentiate into the osteogenic lineage, favouring adipogenesis. This effect is mainly mediated by an inhibition of PDGFRβ autophosphorylation leading to a more pronounced inhibition of PI3K/Akt compared to Erk1/2 signalling. This work confirms the role of PDGFRβ recently described for the proliferation and differentiation potential of MSC and provides a first possible explanation for the altered bone metabolism found in certain patients treated with IM.

Seventy-one patients with acute myeloid leukemia (AML), most of them (63/71) considered ineligible for conventional allogeneic hematopoietic stem cell transplantation (HSCT), were enrolled into a phase II study on reduced intensity myeloablative conditioning with fractionated 8 Gy total body irradiation (TBI) and fludarabine (120 mg/m2) (Blood. 2005 Nov 1;106(9):3314–21). Patients received mobilized peripheral blood stem cells (n=68) or bone marrow (n=3) from siblings (n=39) or unrelated donors (n=32). HLA-typing was performed for HLA-A, -B, -Cw (serological matching or intermediate resolution DNA typing), DRB1 and DQB1 (high resolution DNA typing). Three patients had unrelated donors with an allele mismatch in HLA DRB1 (2 with an additional mismatch in HLA Cw) and 7 patients were transplanted from unrelated donors with an antigen mismatch in HLA Cw. Thirty-six patients were transplanted in complete remission (CR) and 35 with untreated or refractory disease (non-CR). Median patient age was 51 years (range, 20–66). Sustained engraftment was attained in all evaluable patients. With a median follow-up of now 41.3 months (range, 20.4–70.4) in surviving patients, probabilities of overall survival for patients transplanted in CR and non-CR were 80% (95% CI, 66 to 94%) and 17% (95% CI, 5 – 29%) at 4 years, respectively. Relapse-free survival rates were 57% (95% CI, 39 – 75%) and 14% (95% CI, 2 – 26%). Of the 35 evaluable patients transplanted in CR, 10 patients suffered a relapse between days 68 and 868 after transplantation (cumulative incidence 29%). Five patients with late relapse (>1 year after transplantation) achieved a subsequent CR after conventional chemotherapy, blood stem cell boost and treatment with granulocyte-macrophage colony-stimulating factor, lasting 2000+, 1841+, 909+, 847+ and 480 days, respectively. Depending on donor type, relapse-free survival was similar in patients transplanted from unrelated or sibling donors. Overall survival in patients transplanted in complete remission from unrelated vs. sibling donors was 84% (95% CI, 73 – 95%) vs. 77% (95% CI, 68 – 86%). The cumulative incidence of non-relapse mortality (NRM) in CR patients was 11% at 4 years and beyond (3 patients deceased before day 100 and 1 patient 25 months after transplantation), but amounted to 37% at 4 years in non-CR patients. Nine of the 33 surviving patients (27%) have actually active chronic GvHD (5 limited and 4 extensive disease). This update confirms that allogeneic HSCT from related or unrelated donors with 8 Gy TBI/fludarabine conditioning is feasible with low NRM and preserved long-term antileukemic activity in AML patients in first or later CR.

Only recently, a class of gene regulatory molecules (miRNA’s) has been described that have the potential for posttranscriptional and translational gene regulation. MiRNA’s can influence critical cellular properties like proliferation and differentiation. Since these properties vary substantially in genetically defined AML subsets we have chosen to analyze AML patients with inv(16) (n=12); standard risk cytogenetic classification (mainly comprising the normal karyotype) (SR) (n=21) and with monosomy 7 or deletion7q (n=17) using DNA oligonucleotide arrays which are employing the mirVANA miRNA probe set. Principal Component Analysis (PCA) of AML samples from patients with inv(16) showed only minor differences to normal bone marrow (BM). In contrast AML patients with SR could be clearly divided in two subgroups - one subgroup that showed miRNA expression pattern close to BM and a distinctly different subgroup. Comparison of SR patient samples with BM using ANOVA tests revealed 9 differentially expressed miRNA’s (mir-223; mir-15a; mir-16; mir-203; mir-103; mir-23b; mir-107, mir-17–5p and mir23a). AML patients with aberrations of chromosome 7 showed highly distinct PCA pattern as compared to BM samples. 64 miRNA’s were found to be differentially expressed in those patients as compared to BM. All miRNA’s located on chromosome7 that were included in the array were found downregulated in AML patients with aberrant chromosome 7 (e.g.mir-335). To further substantiate the microarray data we analyzed a total of 108 AML patients with inv(16), SR cytogenetics and aberrations of chromosome 7 for mir-23a, mir-223, mir-16 and mir-335 expression by qRT-PCR. Supporting the microarray data it could be shown that the median expression levels of mir-23a, mir-223 and mir-16 were lowest in patients with SR cytogenetics, whereas AML patients with chromosome 7 aberrations showed lowest median expression for mir-335 transcripts. This was in clear contrast to the expression of mir-150, a miRNA which could be previously shown to associate with immature T-cells and which had the highest expression in AML samples with chromosome 7 aberrations. Expression of mir-150 was correlated with a high CD34 percentage in the investigated AML samples (p In conclusion, the presented data demonstrate specific expression profiles of miRNA’s in AML patients with different cytogenetic risk profiles. As it could be shown for patients with SR cytogenetics the observed expression profiles are likely to contribute to a deregulated differentiation and may influence therapeutic outcome.

Activation of type III receptor tyrosine kinase FLT3 by its ligand controls cell proliferation and differentiation in normal hematopoiesis. Constitutively activating mutations of FLT3 are common abnormalities in acute myeloid leukemia. Internal tandem duplication (ITD) mutations of the FLT3 receptor are found in approximately one third of patients with AML and normal karyotype and have been shown to be associated with inferior response towards chemotherapy. One possible way how mutant FLT3 receptors may be mediating its pathological effects is by altering the normal localization of the receptor protein. To determine the intracellular localization of wild type and mutant FLT3 receptors, confocal laser scanning microscopy was performed in cultured leukemic cell lines (THP-1, EOL-1, MV4-11) and primary hematopoietic stem cells (HSC) from healthy donors. HSC (CD34+) cells were prepared from human bone marrow and leukapheresis products of healthy donors by MACS technique using CD34 and CD135 (FLT3) coupled microbeads. Additionally, different leukemic cell lines were transduced with retroviral vectors encoding FLT3wt or FLT3-ITD receptors. In normal HSC, only 4–13% are expressing FLT3, and unexpectedly FLT3 staining was observed both in the plasma membrane and evenly distributed in the cytoplasm. The intracellular FLT3wt was not localized to the endoplasmic reticulum (ER), but part of it co-localized with the Trans-Golgi-network (TGN) and early endosomes. In leukemic cell lines endogenously expressing either wt or mutant FLT3, wtFLT3 localized to the plasma membrane and uniformly to the cytoplasm whereas mutant FLT3 strongly accumulated in a perinuclear region but did not localize to the plasma membrane. Stable transfection of leukemic cell lines with wt or mutant FLT3 confirmed this localization pattern. In leukemic cell lines neither FLT3wt nor FLT3-ITD accumulated in the ER or in the Golgi apparatus, but instead FLT3wt and FLT3-ITD seem to reside in the TGN and in early endosomes as shown by immunofluorescence costaining with the organelle markers. These data indicate that although perinuclear accumulation of mutant FLT3 is observed in all cells analyzed, wtFLT3 is also found intracellularly. FLT3-ITD might reside in a different state concerning glycosylation and/or phosphorylation (Schmidt-Arras et al., Mol Cell Biol, 2005) and therefore it may activate alternative or additional signaling cascades by having access to different or additional substrates. Further studies will clarify, whether intracellularly located wtFLT3 in healthy HSC and FLT3wt-expressing leukemic cells is also involved in FLT3 signaling.

NFAT (Nuclear Factor of Activated T cells) transcription factors are a family of five proteins that are primarily known for their central role in the regulation of inducible gene expression in activated T cells. It is now clear that NFAT proteins are also expressed in various non-immune cell types, where they regulate the expression of genes involved in such diverse cellular processes as proliferation, apoptosis and differentiation. We have previously shown that NFATc2 is strongly expressed in human CD34+ cells and megakaryocytes, but not in purified peripheral blood neutrophil granulocytes and monocytes. Furthermore, granulocytic differentiation of CD34+ cells in vitro was paralleled by the rapid and profound suppression of NFATc2 mRNA and protein. The function of NFATc2 in CD34+ cells, however, is unknown, and no information exists on the expression or regulation of other NFAT family members in CD34+ cells or during heamtopoietic differentiation. To provide a systematic basis for further functional analysis, we established in the present study a comprehensive expression profile of all five NFAT family members in CD34+ cells and during their in vitro differentiation into neutrophil, eosinophil, erythroid and megakaryocytic lineages. CD34+ cells were purified from umbilical cord blood and cultured in the presence of cytokines or cytokine combinations inducing differentiation of the respective lineages. At several time-points during the culture, the efficacy and specificity of the differentiation was monitored by morphological examination of cytospin preparations as well as by analysis of lineage-specific cell surface markers. By quantitative RT-PCR, NFATc3 and NFAT5 were the NFAT family member found to be most prominently expressed in CD34+ cells of both peripheral blood and umbilical cord blood, as well as in the immature CD34+CD38− subpopulation of cells. NFAT expression during the differentiation of umbilical cord blood CD34+ cells into the diverse hematopoietic lineages followed a family member- and lineage-specific pattern. Neutrophil differentiation was accompanied by a rapid suppression of transcript level for all NFAT family members. In contrast, eosinophil, erythrocyte and megakaryocyte differentiation was paralleled by an upregulation of NFATc3, NFATc1/NFATc3 and NFATc1 mRNA, respectively. The most obvious lineage-specific pattern was observed for NFATc4, where transcript levels were low in CD34+ cells and either not or only transiently increased in neutrophil, eosinophil and erythrocyte differentiation; in contrast, they were specifically upregulated about 10-fold in the megakaryocytic lineage. The expression profile of NFAT family members in developing hematopoietic cells of diverse lineages presented here will allow predicting and directly assessing the role of individual NFAT family members in hematopoietic differentiation.

Myeloproliferative disorders such as polycythemia vera (PV), essential thrombocytosis (ET) and chronic idiopathic myelofibrosis (CIMF) are clonal hematopoietic diseases with clinical similarities including the risk of transformation into acute myelogeneous leukemia. By definition, these diseases have been separated from Philadelphia chromosome positive (Ph+) CML requiring negativity for the BCR-ABL transcript in PCR studies of bone marrow or peripheral blood. Several groups independently discovered a gain of function mutation of the Janus kinase 2 (JAK2) gene in Ph-negative myeloproliferative diseases. This mutation has been associated with the proliferation of clonogenic progenitors independently of exogenous cytokine stimulation. A sixty-six year old male patient presented with moderate splenomegaly (3 cm under the costal marigin), mild anemia (11.3 g/dl), elevated lactate deyhdrogenase, an increased count of circulating CD34+ cells and a dry bone marrow aspirate. Marrow histology confirmed a prefibrotic stage of chronic idiopathic myelofibrosis (CIMF). Metaphase cytogenetics as well as BCR-ABL FISH were performed on samples from bone marrow, blood and sorted CD34+, CD3+, CD19+ and CD14+ cells from a steady-state back-up leukapheresis. The JAK2(V617F) mutation was confirmed by an allele-specific PCR assay. A screen for BCR-ABL was performed by FISH and PCR in sorted cells as well as in individual colonies (CFU-GM and CFU-E). Four Philadelphia-chromosome positive metaphases could be detected out of 86 derived from the autologous leukapheresis product harvested and cryopreserved as back-up shortly after diagnosis. The BCR-ABL translocation could be detected by fluorescence in-situ hybridisation (FISH) in 2/16 (12.5%) isolated granulocyte/macrophage colonies only whereas all erythroid colonies were negative. The JAK2 mutation was detectable in all clones and was enriched in CD34+ selected cells. The patient experienced progressive splenomegaly despite the achievement of a molecular response measured by quantitative BCR-ABL PCR after treatment with imatinib mesylate. Our in-vitro investigations suggest that the secondary BCR-ABL translocation within the myeloid compartment was of minor pathophysiological relevance in this patient with CIMF harbouring a heterozygous JAK2 mutation.

Recent studies suggest that one of the most important treatment resistance mechanisms of cells with the BCR-ABL translocation could be the interaction with the hematopoietic niche. During the contact with the respective microenvironment, leukemic stem and progenitor cells acquire both a state of cell adhesion mediated drug resistance (CAM-DR) and may change their proliferation and differentiation potential. Studies on the effect of the BCR-ABL oncogene on cell adhesion and migration have yielded conflicting results due to different cell type models (primary cells vs. transduced cell lines), non-standardized quantification methods and diverse substrates (fibronectin vs. bone marrow stromal cells) studied. In the present work, we investigated the adhesion properties of 32D cells which were stably transformed with the BCR-ABL fusion transcript in comparison to an empty vector transformed control. The commonly used panning-style adhesion assay determining the percentage of hematopoietic cells adhering to a stroma cell line (M2-10B4) clearly demonstrated higher adhesion capacity of BCR-ABL transformed cells. Accordingly, an atomic force microscopy (AFM)-based method to measure adhesion forces of a single leukemic cell over stroma cells, showed an increased ‘stickyness’ of BCR-ABL transformed cells. AFM experiments demonstrated highly different forces that were needed to retract attached 32D-vector cells as compared to 32D/BCR-ABL cells (1000 pN vs. 3500 pN; p To further gain insight in the mechanisms of adhesion we used a cDNA microarray (Affymetrix-Gene chip 430A 2.0) for the identification of BCR-ABL related gene expression with special focus on adhesion associated genes. P-Selectin upregulation (40 folds) in cells harbouring BCR-ABL was seen as the most dramatic change in adhesion related molecules. Consequently, a monoclonal antibody against P-selectin inhibited adhesion capacity of BCR-ABL cells on M2-10B4. In conclusion, we describe an increased adhesion capacity of 32/BCR-ABL transformed cells which is associated with an up-regulation of P-selectin expression. Moreover, whilst 32D/BCR-ABL cells show a high potential to primarily attach to the stroma layer, physiological functions like cobblestone formation is impaired by a reduced migration and consecutively impaired homing potential of the cells into the supporting niches formed by the feeder cells.

Herpesviruses and adenovirus are an important cause of end-organ disease (EOD) and are associated with graft versus host disease (GVHD) in patients undergoing allogeneic stem cell transplantation (SCT). Detection and monitoring of those viruses is routinely performed by simplex quantitative real-time PCR. Since more than one herpes- or adenovirus are reported to be present shortly after transplantation, detection of all possible (sub-)clinical viral infections is still time-consuming and cost-intense. We therefore designed a DNA-microarray for the simultaneous detection of herpes-simplex-virus-1 and -2 (HSV-1/2), cytomegalovirus (CMV), varicella-zoster-virus (VZV), Epstein-Barr-virus (EBV), human herpesvirus-6 (HHV-6) and adenovirus. The DNA-microarray was validated in comparison to simplex quantitative PCR routinely used in our diagnostic laboratory. We retrospectively screened 608 samples of 35 patients undergoing allogeneic SCT on a regular basis until day 100 post-transplantation. The detection limit of the DNA-microarray is 10 genome equivalents (GE)/ml. We detected one or more viruses in 367 samples (60,2%). Of the positive samples, CMV was detected in 272 (74,1%), EBV in 103 (28,1%), HHV-6 in 81 (22,1%), adenovirus in 35 (9,5%), HSV-1/2 in 21 (5,7%) and VZV in 14 samples (5,2%), respectively. Of the samples containing two viruses (n=62), we detected CMV/EBV in 19 (30,6%), CMV/HHV-6 in 27 (43,6%) and HHV-6/EBV in 7 (11,3%) samples, respectively. CMV/EBV/HHV-6 was detected in 4 (36,4%) of the samples containing 3 viruses (n=11). Results of the DNA-microarray showed good overall agreement in comparison to the simplex quantitative PCR. We conclude that the DNA-microarray is a highly sensitive and specific method for the simultaneous detection of up to six viruses in a single sample. By screening and monitoring SCT-patients for viruses known to be associated with EOD or GVHD we herewith introduce an innovative molecular technique that can simultaneously detect multiple viral infections on a large scale. The assay is currently used to determine the impact of multi-virus detection on clinical end-points like GVHD and infectious complications in patients undergoing allogeneic stem cell transplantation.

New immunosuppressive compounds are currently investigated to reduce the rate of lethal Graft-versus-Host disease (GvHD) after allogeneic hematopoietic cell transplantation without adding relevant toxicity. Although positive reports on the efficacy of Mycophenolate Mofetil (MMF) in patients with acute and chronic GvHD are available, there is only limited data on the optimal prophylactic dosing schedule in patients at high risk for GvHD. Since very low trough-levels of MPA are measured in recipients of allogeneic stem cell transplants, we performed a prospective phase I/II trial targeting daily MMF doses according to MPA AUC levels determined at several time-points after transplantation. Twenty-nine patients (18 male, 11 female) with a median age of 53 years were included. The indication for allogeneic transplantation from matched sibling (n=7) and unrelated donors (9/10 alleles matched as minimum requirement; n=22) was high-risk AML/MDS (n=19) or relapsed lymphoma/multiple myeloma (n=10). Conditioning therapy included 30 mg/m2 Fludarabine on days −9 to −6 (4 x 20 mg/m2) combined with intravenous Busulfan 3.45 mg/kg from day −5 to day −2 (4 x 3.45 mg/kg i.v.). Tacrolimus was given orally starting on day −1 at 0.03 mg/kg in order to achieve trough blood levels of 5–10 ng/ml. MMF was started on day 1 at 1500 mg intravenously every 12 hours. AUC measurements of mycophenolic acid (MPA) and its metabolite MPAG by HPLC were scheduled on day 3, 8, 14, 21 and 28 after transplantation. The MMF dose was modified in order to achieve an AUC of 35–60 μg/m*h. MMF was tapered from day 56 on, if possible The dose of MMF had to be increased in 15/29 (52%) patients to 1750–2500 mg every 12 hours on day 4. No patient required a reduction of dosing on day 4. With the respective dose adjustments 52% and 80% of patients reached the AUC target on day 8 and 14, respectively. There was no direct association between dose level and extramedullary toxicity. Early grade 3–4 gastrointestinal toxicity occurred in 4 patients and lead to a reduction of MMF back to 1000 mg every 12 hours. Trilineage engraftment and complete donor chimerism was observed in all patients included. Only one out seven patients with a matched related donor experienced acute GvHD > grade II. The respective proportion of grade III–IV acute GvHD in the unrelated setting was 7/20 (33%). The rate of viral (CMV) and fungal infections was not increased compared to historical controls using standard antimicrobial prophylaxis. With a median follow-up of 18 months 15 (52%) patients are alive and 14 are in complete remission. Reasons for death were relapse (n=5; 17%), pneumonia/sepsis (n=2; 7%), GvHD (n=5; 17%) and organ failure (n=2; 7%). So far, 8 out of 24 evaluable patients (33%) suffer from limited (n=3) or extensive (n=5) chronic GvHD. A retrospective analysis revealed a significant correlation between Cmax levels and the AUC for MPA. The respective target Cmax was shown to be 16 μg/ml. Given the high-risk patient population and the high proportion of unrelated donors (70%) the clinical results observed with the combination of tacrolimus and MMF as prophylactic regimen are encouraging. The regimen has to be optimised for recipients of unrelated transplants. MMF doses of up to 2500 mg every 12 hours can be infused early after stem cell transplantation without an increased risk of toxicity. A simplified MMF targeting strategy based on MPA Cmax levels seems to be warranted in future trials.

Haploidentical hematopoietic cell transplantation (HHCT) has been made feasible with the use of megadoses of CD34+ peripheral blood stem cells. Despite this progress, HHCT is still complicated by high treatment related toxicity, slow engraftment and immune reconstitution. A new regimen using graft CD3/CD19 depletion with anti-CD3- and anti-CD19-coated microbeads on a CliniMACS device and dose reduced conditioning may allow HHCT with lower toxicity and faster engraftment. CD3/CD19 depleted grafts not only contain CD34+ stem cells but also CD34 negative progenitors, natural killer-, dendritic- and graft-facilitating-cells. As dose reduced conditioning fludarabine (150–200 mg/m2), thiotepa (10 mg/kg), melphalan (120 mg/m2) and OKT-3 (5 mg/day, day -5 to +14) without posttransplant immunosuppression was used. So far 9 consecutive adult patients have been transplanted with this regimen. Median age of the patients was 37 (range, 27–58) years and the diagnoses were AML (n=3), ALL (n=3), NHL (n=2) and multiple myeloma (n=1). All patients were high risk with relapse after preceding hematopoietic cell transplantation. The CD3/CD19 depleted haploidentical grafts contained a median of 8 x 10E6 (range, 5.2–17x10E6) CD34+ cells/kg, 6x10E7 (range, 0.02–8.6 x10E7) CD56+ cells/kg and 2x10E4 (range, 0.006–8.2x10E4) CD3+ T-cells/kg. 8 of 9 patients had a donor-recipient KIR-mismatch. The conditioning regimen was well tolerated with maximum acute toxicity being grade 2–3 mucositis and nausea. In 4/9 patients reversible peripheral neuropathy grade 2–3 occurred, combined with multifocal leucencephalopathy in one patient. Subsequently the dose of fludarabine was reduced to 150 mg/m2. Engraftment was rapid with median time to neutrophils >500/μL of 13 (range, 10–17) days, platelets >20000/μL of 11 (range, 8–14) days and full donor chimerism after two weeks in all patients. All evaluable patients had fast immune reconstitution with T-cells >100/μL by day +100. Five cases of grade 2 skin GVHD rapidly responded to steroids. Three patients with controlled fungal pneumonia at time of transplant had resolution of infiltrates with neutrophil reconstitution and antifungal therapy. Treatment related mortality in the first 100 days was low with one death due to idiopathic pneumonia syndrome. Three patients died after day 100, one each due to relapse, systemic adenoviral infection and CMV infection. Overall survival is 5/9 patients with a median follow-up of 406 days (range, 44–661). In conclusion, this regimen seems to be promising in adult high risk patients lacking a suitable donor and a prospective phase I/II study is ongoing.

Understanding the mechanisms underlying the reconstitution of the hematopoietic system after blood stem cell transplantation is of crucial importance for improving therapeutic modalities. In order to address these issues, we have recently established a novel co-culture system consisting of immuno-isolated human CD34+ hematopoietic stem and progenitor cells (HSPCs) growing on primary human mesenchymal stem cells (MSCs) as feeder layer in the presence of relevant cytokines. Here we have investigated i) the morphological modification of HSPCs induced by their interaction with MSCs; ii) the sub-cellular localization of the stem cell markers CD34 and CD133 (prominin-1) as well as other cell surface molecules potentially involved in such intercellular interaction, and iii) the intracellular pathway(s) responsible for the migration as well as the interaction of HSPCs with MSCs. The scanning electron microscopy analysis revealed that the adherent HSPCs display various morphologies; they are either round with, in some cases, the appearance of a microvillar pole or exhibit several distinct types of plasma membrane protrusions such as lamellipodium, filopodium and magnupodium. We could also observe very long and thin plasma membrane processes between adjacent HSPCs suggesting the formation of nanotubes, which have been implicated in intercellular communication. As previously reported (Giebel et al., 2004, Blood, 104:2332), the highly motile HSPCs acquire a polarized cell shape with the formation of a uropod at the rear pole and a leading edge at the front pole. The immunofluorescence analyses revealed that CD34 is randomly distributed over the entire surface of HSPCs, irrespective of their morphological shape, whereas CD133 shows various specific sub-cellular localizations, which are depending on the state of the cell. In migrating cells, CD133 is concentrated in the uropod at the rear pole, which contrasts with the enriched localization of the adhesion receptor β2 integrin in the leading edge. In HSPCs harboring other types of plasma membrane protrusions, which are in close contact with the feeder cell layer, CD133 is concentrated therein. In round adhesive cells, CD133 is located in one pole of the cell suggesting that the HSPCs are highly polarized even in the absence of plasma membrane outgrowths. Interestingly, the formation of uropod and microvillus-like structures is highly reduced in presence of Y-27632 inhibitor suggesting that the Rho GTPase/ROCK pathway is somehow involved in the polarization of HSPCs. Time lapse videomicroscopy revealed that the retraction of filopodia is also affected in Y-27632-treated HSPCs as well as their migration. Taken together, these data provide new insights regarding the molecular cell biology of HSPCs, and increase our understanding of cellular events associated with mobilization and engraftment in the context of blood stem cell transplantation.

Deletion of chromosome band 17p13 (P53) is known to be an adverse prognostic factor for time to progression (TTP) and overall survival (OS) in myeloma patients receiving conventional or high-dose chemotherapy. In this retrospective multicenter study, we investigated the impact of P53 deletion as detected by fluorescence in situ hybridization combined with immunofluorescent cytoplasm immunoglobulin staining (cIg-FISH) on outcome after allogeneic blood stem cell transplantation (SCT) in 50 patients (pts) with advanced or relapsed multiple myeloma (MM). Median age was 51 years (34 – 67 ys.), 32 patients were male, 18 female. Thirty pts received a stem cell graft from an unrelated donor, and 20 pts from a HLA-identical sibling. P53 gene deletion was found in 5 out of 49 pts (10,2 %). Deletion 13 detected by cIg-FISH was found in 20 out of 47 pts (42%). There was a strong correlation between P53 deletion and deletion 13: four out of the five patients with P53 deletion also showed deletion in chromosome band 13q14. The estimated event-free (EFS) and overall survival (OS) at three years for the entire study population was 43% and 65%, respectively. For patients with del 13 only a trend for a worse 3 year EFS was seen (38% vs 42%, p=0.2), while pts with P53 deletion had a significant reduced EFS (0% vs 46%, p=0.0001). Three out of five pts with P53 deletion relapsed very early after allogeneic SCT (day 81, 108 and 287 respectively). These data suggest that P53 is a risk factor for patients with MM treated with allogeneic SCT which can not be overcome by this treatment strategy.

It is known that the crosstalk between adhesion molecules, bone marrow microenvironment, and cytokines facilitates the multi step process of stem cell mobilization from bone marrow to peripheral blood. A combination of G-CSF plus AMD3100 - a CXCR-4 antagonist - has been shown to be safe and efficient in stem cell mobilization of healthy donors and cancer patients. Nevertheless, data predicting the efficacy of this approach are still missing. The present study investigated the correlation of the expression of CXCR-4 (CD184) and adhesion molecules with the kinetics and efficacy of stem cell mobilization in nine patients with Multiple Myeloma (MM) or NHL, respectively. Steady-state mobilization was performed using a combination of G-CSF (Filgrastim, 10μg/kg/d, 8 am) for 4 days followed by AMD3100 (240μg/kg) on day 4 at 10pm. Autologous aphereses were started on day 5. Bone marrow and peripheral blood (PB) before AMD3100 application (day 4) and PB on day 5 were investigated with a 4-color flow cytometric procedure. Bone marrow aspirates of healthy donors (n=20) served as control. The qualitative (%) and quantitative (mean fluorescence intensity, [MFI]) antigen expression of CXCR-4 in relation to CD34 was assessed as well as the expression of certain adhesion molecules including LFA-1, PECAM-1, VLA-1, L-selectin and CD44. First, the median percentage of CXCR-4 surface expression in healthy bone marrow was significantly higher (92%; range: 52 – 99%) than in patients bone marrow (70%; 30 – 88%; p=0.002), PB before AMD3100 (87%; 35 – 97%; p=0.050) and on day 5 (17%; 2 – 74%; p

Respiratory failure is a serious early complication confounding the clinical effect of allogeneic stem cell transplantation and early detection may be crucial for efficient treatment. We introduced a system of intensive oxigenation monitoring in patients transplanted in our center between November 2001 and December 2003. All allogeneic transplanted patients were followed by a twice daily measurement of oxi-index (paO2/FiO2) during the transplantation period (untill day + 25). Patients that fulfilled criteria of acute lung injury (oxi-index

The use of anti-B cell antibodies before allogeneic hematopoietic stem cell transplantation (HSCT) induces a depletion of recipient B cells. This leads to a reduction of the number of potential antigen presenting cells and thereby might hamper the induction phase of graft-versus-host disease (GvHD). The humanized anti-CD20 antibody Rituximab has been used with increasing frequency to treat patients with B cell Non-Hodgkin’s Lymphoma. Only recently, several investigators have reported on the efficacy of Rituximab for the treatment of chronic GvHD. We therefore hypothesized, that patients who received Rituximab before being scheduled for allogeneic HSCT may have a decreased risk of GvHD. A retrospective analysis in 72 patients after allogeneic hematopoietic stem cell transplantation for indolent (n=18), aggressive (n=45) B cell lymphoma or mantle cell lymphoma (n=9) was performed at two centres. Whereas 28 patients had received chemotherapy only before allogeneic HSCT, 44 patients had received 4–8 (median 6) cycles of 375 mg/m2 Rituximab before the start of conditioning therapy. Patient characteristics’ were comparable except for a higher proportion of unrelated donors (73% vs. 54%) and a higher median age (48 vs. 42 years) in the cohort with Rituximab pre-treatment. In addition the proportion of patients having received a previous autologous transplantation was higher (63% vs. 46%) and therefore more recipients received dose reduced conditioning before allogeneic tranplantation (70% vs. 50%) in the Rituximab group. With a median follow-up of 24 months for all patients, no significant differences in overall survival, progression-free survival and treatment-related mortality could be detected between both groups. Although the proportion of transplants from unrelated donors was lower in the No-Rituximab group, the rate of acute GvHD grade I–IV was higher (88% vs. 63%, p=0.03). The incidence of grade II–IV and grade III–IV GvHD was not significantly different between both cohorts. Chronic GvHD occurred more often in patients who had not received Rituximab before allogeneic transplantation (52% vs. 23%, p=0.02). Although the retrospective nature of our study does not allow to draw definitive conclusions, the rate of chronic GvHD seems to be lower when anti-CD20 treatment is performed before allogeneic HSCT. Prospective studies incorporating Rituximab for the prophylaxis and therapy of GvHD are warranted.

Allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative treatment option for most high-risk patients with acute myeloid leukemia (AML). However, many high-risk patients regenerate with blasts or relapse early after induction therapy. Thus, consolidation with allogeneic HSCT in first CR is often not possible. Performing allogeneic HSCT within induction therapy can circumvent these problems and may furthermore reduce cumulative toxicity in high risk patients. Therefore, the prospective randomized treatment trial AML2003 for patients Within the first 8 months 107 AML patients with a median age of 48 (17–60) years were included in the study. Fast search diagnostics was complete within a median of 15 (range 5–31) days after arrival of the bone marrow samples for all patients. 57/107 patients were randomized into the intensified treatment arms. Out of these 25 (44%) patients with high risk characteristics have been identified. A suitable related or unrelated donor was found for 22 (88%) of those high-risk patients. Nine of those high risk patients with a donor (41%) received early allogeneic stem cell transplantation in aplasia after the first (n=4) or the second (n=5) induction therapy course on an intend to treat basis within the protocol. Three were transplanted with stem cells of related and six of unrelated donors. The preparative regimen consisted of melphalan 150mg/m2 and fludarabine 150mg/m2. So far no treatment associated death had to be recognized. These encouraging preliminary results show that fast risk-profiling and early donor-search is feasible in a large multi-center study. This leads to a significant proportion of early allogeneic stem cell transplants in aplasia after induction therapy within the group of high risk AML patients, which may improve the disastrous prognosis of this group of patients in the future.

Expansion of hematopoietic stem cells from neonatal cord blood is an important issue for clinical uses since the number of CD34+ cells in individual cord blood samples is limited and often not sufficient for a successful engraftment in adult individuals. In vivo, hematopoietic stem cells reside in the bone marrow in close vicinity to stromal cells and extracellular matrix molecules. We have established a culture system for the ex vivo expansion of CD34+ cord blood cells utilizing fibrillar collagen 1 as a bioartificial matrix to enable cellular adhesion during cell culture. CD34+ hematopoietic stem cells were isolated via immunomagnetic separation from umbilical cord blood after informed consent and cultivated in presence of recombinant cytokines and reconstituted collagen 1 fibrils as matrix. After seven days of cultivation, expansion of cells, expression of surface molecules cells and expansion of colony forming units were assessed. Additionally gene expression profiling was performed with Affymetrix HG U133A chips interrogating 22,253 probe sets. As control, CD34+ cells were expanded in liquid culture without fibrillar collagen. The overall expansion of CD34+ cells was 4.2 fold + 1.7 compared to 11.1 fold + 2.9 for the control sample. The number of colony forming units (CFU) was increased in the collagen 1 containing samples was elevated (65.1 + 10.3 compared to 26.1 + 7.6 in the control). Gene expression analysis with chip technology showed up regulation of several cytokines (e.g. interleukin 8, interleukin 1a) and also of transcription factors with antiproliferative features like BTG2. The chip data have been verified with quantitative PCR using the Taqman technology. Our data support the idea that direct contact of CD34+ cells with fibrillar collagen 1 results in a delay in cell cycle progression which prevents a subsequent differentiation into more committed progenitors. Therefore fibrillar collagen 1 may serve as supportive matrix for the ex vivo expansion of cord blood derived CD34+ cells.

Both number and origin of dendritic cells (DC) in the blood have been associated with acute graft-versus-host disease (aGvHD), graft-verus-leukemia effect, relapse and graft failure after human allogeneic haematopoietic cell transplantation (aHCT). Aim of this study was the systematic simultaneous investigation of skin and blood DC subtypes, the analysis of their host/recipient origin after HCT and the correlation of DC kinetics with treatment, outcome and incidence of aGVD and its treatment in 31 recipients of allogeneic HCT. Epidermal shave biopsies and peripheral blood samples were collected from patients pre and at defined time points following aHCT, DC from skin and blood isolated and examined by FACS and quantitative PCR for Short Tandem Repeat markers. A significant reduction in number of skin and blood DC subtypes was observed already before start of conditioning chemotherapy in patients compared to healthy volunteers. A majority of donor derived epidermal Langerhans Cells (median 95%, range 53–100%) and also dermal DC (median 94%, range 39–100%) was found in all examined patients even early after transplant independent from chemotherapy regimen, graft, occurrence of skin GvHD or time point after transplantation. Numbers of both skin DC subsets remained low for months post HCT. Reconstitution kinetics of CD 11c+ preDC1 and CD 123+ preDC2 blood DC were similar to each other and pre transplant numbers were reached again around day +112 post transplant. Recipients of grafts containing higher T cell numbers had lower preDC1 counts on day +28. PreDC2 reconstitution was negatively affected by steroid treatment. Patients with aGvHD tended to have more preDC1 on day +28 and lower numbers of both preDC subsets on day 56 post transplant. Residual host blood DC were rare at all time points (median

Dendritic cells (DCs) are characterized by their unique capacity for primary T cell activation, providing the opportunity of DC-based cancer vaccination protocols. Recently, we defined a novel major subset of human blood DCs by using the monoclonal antibody M-DC8 which recognizes a carbohydrate modification of P selectin glycoprotein ligand-1 (PSGL-1) selectively expressed on these cells (Immunity2002;17:289-301). In addition to a marked capacity to activate tumor-reactive cytotoxic T cells M-DC8+ DCs efficiently mediate antibody-dependent cytotoxicity (Blood;2002;100:1502-1504). In the present study, we analyzed the capacity of M-DC8+ DCs to kill tumor cells in the absence of antibodies and to enhance the tumor-directed cytotoxicity of NK cells. To determine whether M-DC8+ DCs exhibit tumoricidal activity, DCs were isolated at high purity (>93%) from the blood of healthy donors by immunomagnetic separation. These cells were cultured for 6 h in the presence or absence of 200 U/ml interferon (IFN)-gamma. Subsequently, DCs were coincubated with four chromium-labeled tumor cell lines and two normal cell lines for 18 h. Whereas unstimulated DCs demonstrated only moderate tumor-directed cytotoxicity (specific lysis: 7–13%), IFN-gamma-stimulated M-DC8+ DCs displayed potent killing of each of these tumor cell lines (specific lysis: 27–35%). Only a marginal cytotoxic effect was seen when normal human cells such as lung fibroblasts or endothelial cells were used as targets. When evaluating the cytotoxic effector mechanisms FACS analysis and ELISA assays revealed that IFN-gamma-stimulated M-DC8+ DCs secreted a high amount of tumor necrosis factor (TNF)-alpha induced by direct cell-to-cell contact with the different tumor cell lines. This effect was already observed after 3 h of cocultivation. Interestingly, no significant induction of TNF-alpha was detected during contact of M-DC8+ DCs with the normal human cell lines. These results suggest that tumor-associated surface molecules are important for the observed increase of TNF-alpha production in M-DC8+ DCs. Inhibition experiments with neutralizing antibodies clearly demonstrated that tumor cell-induced TNF-alpha play an important role in tumor-directed cytotoxicity mediated by M-DC8+ DCs. To investigate whether M-DC8+ DCs enhance the tumoricidal activity of NK cells freshly isolated DCs were cultured for 6 h in the presence or absence of IFN-gamma. Thereafter, DCs were coincubated with highly enriched (>90%) NK cells. The cytotoxic potential of the stimulated NK cells was tested towards various tumor cell lines in a 4 h chromium release assay. We observed a two- to threefold increase of NK cell-mediated cytotoxicity towards all analyzed tumor cell lines by IFN-gamma-stimulated M-DC8+ DCs. In addition, transwell experiments demonstrated that this triggering effect was mainly dependent on cell-to-cell contact. In conclusion, our data provide evidence that a major subpopulation of circulating human blood DCs exhibits efficient tumoricidal activity and clearly enhances NK cell-mediated tumor-directed cytotoxicity. The capacity of DCs to induce tumor-specific T cell responses and to kill tumor cells either directly or by activating NK cells points to the pivotal role of DCs in triggering the innate and adaptive immune response against tumors.

Recently we demonstrated that RIC with busulfan, fludarabine and ATG followed by allogeneic hematopoietic stem cell transplantation (HSCT) induced molecular remissions in patients (pts) with advanced CLL. However, this approach was hampered by severe GVHD. In an attempt to lower the rate of severe GVHD we replaced ATG by campath in a new study protocol. Patients and Methods: 20 pts with a median age of 54 years (range, 43 to 64) and advanced CLL were included. A median of 3 prior chemotherapy regimens had been given before HSCT, including fludarabine-containing regimens in all but two pts with autoimmune hemolysis. High risk cytogenetic features (17p−, 11q−, +12) were present in 9 pts. After conditioning with busulfan (8 mg/kg), fludarabine (150 mg/m2) and campath (75 mg) on days −9 to −5 peripheral blood stem cells from matched related (n=4) or unrelated donors (n=16) were transplanted. GVHD prophylaxis consisted of CSA monotherapy. Campath levels were analysed in frozen serum samples by BioAnaLab, Oxford, UK. Results: Two pts had no detectable campath level at the day of HSCT, while four pts had levels between 0.5 to 1.8 microgram/mL. Regeneration of neutrophils (>0.5/nl) and platelets (>20/nl) required a median of 17 (range, 14–25) and 10 (range, 0–27) days, respectively. Incomplete T-cell chimerism ( Differences in the extent of in vivo T-cell depletion might therefore explain the individually varying T-cell engraftment pattern. In addition, the high incidence of severe viral infections reflects impaired immunoreconstitution. Including pts after DLI we observed a substantial rate of severe GVHD. Based on these data we decided to skip the strategy of in vivo T-cell depletion with campath in patients with CLL.

Objectives: In patients with acute myeloid leukemia and high-risk cytogenetic aberrations or minor response to the first cycle of induction chemotherapy (IC) the probability of achieving a sustained complete remission is low. Thus early treatment intensification may be warranted in order to achieve long-term disease control. We performed a prospective trial to evaluate whether reduced-intensity conditioning followed by allogeneic hematopoietic stem cell transplantation (HSCT) from related or unrelated donors can be performed during the aplastic phase of IC in patients with poor-risk AML. Methods: Seventeen patients (n=17) aged between 17 and 63 years (median 45) with acute myeloid leukemia and high-risk cytogenetic aberrations (n=14, complex, inv3 or t(3;3), t(3;5), −7 or del 7q, +8) or more than 10 % marrow blasts on day 15 after the first cycle of IC (n=3) were included so far. During aplasia a median of thirteen days (range 7–35) after the first (n=8) or second (n=9) cycle of IC patients received 5 x 30 mg/m2 fludarabine i.v. combined with either 8 mg/kg busulfan p.o. (n=4) or 150 mg/m2 melphalan iv. (n=13) followed by allogeneic G-CSF mobilized peripheral blood stem cells (PBSC, n=16) or bone marrow (n=1) from related (n=7) or unrelated (n=10) donors. Nine out of seventeen patients were not in complete remission before conditioning therapy was started. Patients with unrelated grafts received antithymocyte globulin (4 x 10 mg/kg ATG Fresenius). GvHD prophylaxis was performed with cyclosporine A (CSP). Results: All patients engrafted (ANC > 0.5 Gpt/l on day 11, range 8–19, platelets > 50 Gpt/l day 15, range 11–32) and went into remission. Acute GvHD grade II-IV occurred in 8 patients and extensive chronic GvHD was documented in 5 patients with a follow-up of > 100 days. Two patients died while being in remission from infectious complications associated with acute (n=1) or chronic (n=1) GvHD and two patients died during relapse eight and twelve months after PBSC. With a median follow-up of 15 months (range 1–65) thirteen out of seventeen patients (76 %) are alive and in remission. Conclusion: Early allogeneic HSCT as part of primary induction therapy seems to be an effective strategy in AML patients with either poor risk karyotype or minor response to the first induction cycle.

Stem cell mobilization is achieved by short term administration of G-CSF in healthy donors and G-CSF +/− chemotherapy in patients with malignant diseases. It is known that the crosstalk between adhesion molecules, bone marrow microenvironment, and cytokines facilitates the multi step process of stem cell mobilization from bone marrow to peripheral blood. But still, the biological basis of the large variability in mobilization efficacy remains unclear. The aim of the present study was to evaluate if poor and good mobilizers - healthy donors as well as patients with hematologic malignancies - show differences in the expression of adhesion molecules (VLA-4, L-selectin, LFA-1, PECAM-1, CD44), the chemokine receptor CXCR4 and the G-CSF receptor measured by flow cytometry on CD34 positive cells. Therefore, we investigated 200 aphereses from 132 healthy PBSC donors and 68 patients with a 4-color staining: CD34/CD45 combined with two different adhesion molecules in each sample. The quantitative (mean fluorescence intensity) and qualitative (%) antigen expression was assessed. Donors/patients were divided into three groups according to the peripheral CD34-positive cell count at the day of apheresis: ≤ 30/μl (poor mobilizer; 36 donors, 30 pts.), 31–100/μl (standard mobilizer; 56 donors, 23 pts.), > 100/μl (good mobilizer; 40 donors, 15 pts.). The quantitative antigen expression was significantly higher for LFA-1 in poor vs. good mobilizing donors (GeoMean: 22 vs. 17; p=0.007) and patients (26 vs. 18; p