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Platinum-based chemotherapeutics exhibit excellent antitumor properties. However, these drugs cause severe side effects including toxicity, drug resistance, and lack of tumor selectivity. Tumor-targeted drug delivery has demonstrated great potential to overcome these drawbacks. Herein, we aimed to design radioactive bisphosphonate-functionalized platinum (195mPt-BP) complexes to confirm preferential accumulation of these Pt-based drugs in metabolically active bone. In vitro NMR studies revealed that release of Pt from Pt BP complexes increased with decreasing pH. Upon systemic administration to mice, Pt-BP exhibited a 4.5-fold higher affinity to bone compared to platinum complexes lacking the bone-seeking bisphosphonate moiety. These Pt-BP complexes formed less Pt-DNA adducts compared to bisphosphonate-free platinum complexes, indicating that in vivo release of Pt from Pt-BP complexes proceeded relatively slow. Subsequently, radioactive 195mPt-BP complexes were synthesized using 195mPt(NO3)2(en) as precursor and injected intravenously into mice. Specific accumulation of 195mPt-BP was observed at skeletal sites with high metabolic activity using micro-SPECT/CT imaging. Furthermore, laser ablation-ICP-MS imaging of proximal tibia sections confirmed that 195mPt BP co-localized with calcium in the trabeculae of mice tibia.

Introduction: The most common cause of treatment failure after allogeneic hematopoetic stem cell transplantation (aHSCT) is relapse. The combination of venetoclax and the hypomethylating agents (HMA) azacitidine (AZA) or decitabine (DAC) have shown promising efficacy in elderly patients with AML. We here present clinical data on 32 patients, who were treated with an HMA/venetoclax combination therapy (HMAClax) for relapse of a myeloid malignancy after aHSCT, collected retrospectively from 11 German centers. Results: Sixteen patients (50%) were male, median age was 54 years (30.8-71.5). Diagnoses at aHSCT were 25 AML (17 primary, 8 emerging from MDS, CMML or OMF), 5 MDS, 1 CMML and one atypical CML. Twenty six patients were treated for relapse after their 1st and 6 after their 2nd aHSCT. Only 9 patients were in CR at aHSCT. The majority received a graft of a matched unrelated donor (21), 4 from an HLA-identical sibling and 7 from a haploidentical relative. Conditioning was myeloablative in 15 and RIC in 17patients. Median time from aHSCT to last relapse was 5.7ms (1.1-67.8). Five patients had molecular (MR) and 23 had hematologic relapses (HR), 4 patients had extramedullary manifestations 3 with concurrent HR and 1 with MR. Twenty-one patients were treated for 1st and 5 for 2nd relapse after 1st aHSCT. Four patients were treated for 1st and 2 for 2nd relapse after 2nd aHSCT. HMAClax was first line therapy for relapse in 8, 2nd line in 22, 3rd line 1 and 4th line in 1 patient. In 21 patients relapse had been refractory to HMA (+/- DLI, +/- lenalidomide). Median time from relapse to HMAClax was 1.8 ms (0.3-42.9). Twelve patients received AZA and 19 DAC with venetoclax. One patient was switched from AZAClax to DAClax because of rising MRD after 6 cycles and back to AZAClax after another 7 cycles. Six patients received DLI. Median number of cycles was 2 (1-15). Six patients are still on therapy. In total 75 cycles were given. Three patients had non-fatal tumor lysis syndrome. All but one patient had grade 3/4 neutropenia and 25 patients (78%) had grade 3/4 thrombocytopenia. Hospital admission for grade 3/4 infections was necessary in 23 patients (72%), 5 of these infections (22%) were fatal. Overall response rate was 43% (12/28, 2 CR MRD-, 4 CR, 2 CRi, 3 PR, 1 MLFS). Two patients died of infection before first response evaluation and in another 2 response has not been evaluated yet. ORR for patients who received first line HMAClax was 80% (4/5) and 35% (8/23) for salvage treatment. Three of 5 patients with MR reached CR, 2 received HMAClax first line. Time to best response was 1.2ms (0.7-3.8). Six patients lost best response after 1ms (0.4-3.3.) 2 underwent second transplant in remission, 4 have ongoing responses (0.4, 0.7, 3.1 and 8.8ms at last follow up). On July 25th 2019, median follow up was 3.3 ms (0.9-17.3), 20 patients (63%) had died and 12 were alive. Six were continuing HMAClax. One patient developed cGvHD and 4 underwent second aHSCT (2 in remission). Estimated median overall survival was 3.7ms (CI 2.9-4.7). Four responders are continuing treatment with HMAClax. Patients, who responded had an estimated OS of 11.1ms (2 underwent second aHSCT in remission). Median survival of patients with HMAClax first line therapy was 5.8ms and of patients with HMAClax salvage therapy 3.7ms. Conclusion: For patients relapsing after aHSCT, venetoclax plus AZA or DAC seems to be an effective, but also highly hematotoxic therapy. Responses occurred fast and were more frequently seen during 1st line treatment for relapse. Duration of response was short, especially in patients receiving HMAClax as 2nd, 3rd or 4th line therapy. Therefore HMAClax should be explored as 1st line therapy for relapse after aHSCT in combination with DLI or as a bridge to 2nd transplant. Disclosures Schuler: Celgene: Other: travel grants; Novartis: Honoraria, Other: travel grants; Alexion: Other: travel grants. Bug:Gilead Sciences: Membership on an entity's Board of Directors or advisory committees, Other: Travel grants; Hexal: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel grants; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Sanofi: Other: travel grants; Celgene Neovii: Other: travel grant. Crysandt:Incyte: Membership on an entity's Board of Directors or advisory committees; Amgem: Other: travel grant; celgene: Other: travel grant; Pfizer: Other: travel grant; Gilead: Other: travel grant. Jost:Jazz Pharmaceuticals: Honoraria; Sanofi: Honoraria; Gilead: Other: travel grants; Daiichi: Honoraria. Kaivers:Jazz Pharmaceuticals: Other: Travel Support. Mueller:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; CTI Life Sciences: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Financing of Scientific Research; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria; Gentium: Honoraria; Gilead: Honoraria; Janssen: Honoraria; Jazz: Honoraria; Pharmaceuticals: Honoraria; Neovii: Honoraria; Novartis: Honoraria; Sanofi: Honoraria. Rautenberg:Jazz Pharmaceuticals: Other: Travel Support; Celgene: Honoraria, Other: Travel Support. Wolff:Takeda: Honoraria; Mallinckrodt: Honoraria; Novartis: Honoraria; Neovi: Honoraria. Schroeder:Celgene Corporation: Consultancy, Honoraria, Research Funding. Off Label Use: Venetoclax was used in combination with azacitidine or decitabine. The combination is not approved in the EU so far.. Kobbe:Pfizer: Honoraria, Other: Travel support; Abbvie: Honoraria, Other: Travel support; Biotest: Honoraria, Other: Travel support; Roche: Honoraria, Other: Travel support; Jazz: Honoraria, Other: Travel support; MSD: Honoraria, Other: Travel support; Celgene: Honoraria, Other: Travel support, Research Funding; Takeda: Honoraria, Other: Travel support; Amgen: Honoraria, Other: Travel support, Research Funding; Neovii: Honoraria, Other: Travel support; Medac: Honoraria, Other: Travel support; Novartis: Honoraria, Other: Travel support. OffLabel Disclosure: Venetoclax was used in combination with azacitidine or decitabine. The combination is not approved in the EU so far.

Introduction The only curative treatment approach for patients with myelodysplastic syndromes (MDS) is allogeneic hematopoietic stem cell transplantation (HSCT), but disease relapse after transplantation is a major concern. Predictors for disease outcome after HSCT are limited. However, unfavorable cytogenetic abnormalities have been shown to serve as predictors for MDS-relapse after transplantation. Similar to the data available in MDS-patients not undergoing HSCT (Schanz et al. J Clin Oncol 2012), there is evidence that the novel 5-group cytogenetic classification has a better predictive value for outcome after HSCT than standard IPSS cytogenetics (Deeg et al. Blood 2012). The aim of this large multicentric, international study was to retrospectively determine the impact of the new 5-group cytogenetic MDS classification on outcome after HSCT. Patients and Methods Patients were selected from the EBMT database who had received HSCT for the treatment of MDS between 1982 and 2010 and for whom sufficient cytogenetic information was available. In total, 903 patients were included into the study. At time of HSCT, 97 (10.7%) patients had untreated MDS, 218 (24.1%) patients had advanced MDS or AML evolving from MDS in complete remission, and 227 (25.1%) patients were not in remission after treatment (in 12.3% information on stage of the disease was not available). Median time between diagnosis and transplant was 6.6 months (range 0.2-359.3). Matched related donor HSCT was performed in 574 patients (63.6%), and matched unrelated donor HSCT in 329 patients (36.4%). Bone marrow (35.4%) or peripheral blood (64.6%) served as stem cell graft. Myeloablative preparative regimens were used in 582 patients (64.5%), and a non-myeloablative regimen was given to 320 patients (35.4%). Impact of cytogenetic classification was analyzed in uni- and multivariate models regarding overall survival (OS) and relapse free survival (RFS) after HSCT. Predictive performance of the 2 classifications was compared by means of the cross-validated log partial likelihood. Results Estimated 5-year RFS and OS were 32% and 36% respectively. According to the 5-group cytogenetic classification 19 (2.1%) patients had very good risk cytogenetics, 204 (22.6%) normal risk cytogenetics, 438 (48.5%) intermediate risk cytogenetics, 178 (19.7%) poor risk cytogenetics, and 64 (7.1%) very poor risk cytogenetics. Good, intermediate, and poor risk cytogenetics according to IPSS were found in 192 (38.0%), 500 (40.2%), and 211 (23.7%) patients, respectively. In univariate analysis 5-group cytogenetic information was found to be strongly associated with OS and RFS (OS: log-rank test P

Hematological malignancies are successfully treated with chimeric antigen receptor (CAR) armed T cells. Despite the clinical success, CAR T cell therapy struggles still with some problems including the selection of tumor escape variants and on-target, off-tumor side reactions as well as massive cytokine release and uncontrollability of CAR T cell activity in the patients. In order to enable controllability of CAR T cells and to avoid unspecific side effects, we established a novel switchable, split and adaptable CAR platform technology, termed RevCAR system. The novel RevCARs lack the single chain variable fragment (scFv) commonly used as extracellular domain in conventional CARs. Instead of the scFv, RevCARs contain only a small peptide epitope as extracellular portion. This design reduces the CAR size, avoids unspecific antigen binding and prevents antigen independent tonic signaling caused by scFv dimerization. As RevCAR T cells do not recognize anything, they are per se inert. Only in the presence of a corresponding bispecific antibody based target module (RevTM) they can be specifically redirected to tumor cells. Therefor RevTMs consist of two scFvs. One recognizes the RevCAR peptide epitope and the other one simultaneously binds to a tumor associated antigen (TAA). By dosing of the RevTM, which has a very short half-life, the reactivity of RevCAR T cells can be switched on and off reversibly. Another advantage is that the RevCAR system can be flexibly adapted to any tumor antigen simply by exchanging the RevTM. Furthermore, the small RevCAR size is favorable for inserting more than one RevCAR in the same T cell thus facilitating the mode of gated targeting which is a highly attractive approach to minimize the risk for on-target, off-tumor toxicities against healthy tissues and to increase tumor specificity of conventional CAR T cells. For 'AND' gate targeting via the RevCAR system, two different RevCARs were constructed and expressed simultaneously in the same T cell. The two RevCARs differed with respect to the extracellular peptide epitope and the intracellular signaling domain. Moreover, the respective transmembrane domain was selected to isolate the respective RevCAR signal. The first RevCAR is designed to transmit the activation signal, the second RevCAR to deliver a costimulatory signal. For efficient RevCAR T cell activation, both RevCARs must be engaged via their respective RevTM which on the one hand binds to one of the two RevCAR epitopes and on the other hand to one of two TAAs expressed on the same target cell. Here, we present two RevCAR/RevTM systems for retargeting of AML cells as well as solid tumor cells including via gated targeting. In summary, we show proof of concept for a novel switchable RevCAR system that can be used for retargeting of AML cells as well as solid tumors. The novel modular RevCAR platform is characterized by small size, lacks unwanted tonic signaling effects, allows the control of RevCAR T cell activity, enables gated targeting strategies, and can be adapted to any tumor antigen and tumor type. Disclosures Koristka: Intellia Therapeutics: Employment. Bachmann:GEMoaB Monoclonals: Equity Ownership, Patents & Royalties.

To assess the impact of spleen status on engraftment and early morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT), we analyzed 9,683 myeloablative allograft recipients from 1990 to 2006; 472 had prior splenectomy (SP), 300 splenic irradiation (SI), 1,471 with splenomegaly (SM), and 7,440 with normal spleen (NS). Median times to neutrophil and platelet engraftment were 15 vs. 18 days and 22 vs. 24 days for the SP and NS groups, respectively (p5.7x106/kg improved platelet engraftment at day+28. After adjusting variables by Cox regression, the incidence of graft-versus-host disease (GVHD) and overall survival were not different among groups. Splenomegaly is associated with delayed engraftment while splenectomy prior to HCT facilitates early engraftment without impact on survival.

Glucocorticoids (GCs) regulate various physiological processes, including bone remodeling. Whereas physiological amounts of GCs are required for proper human osteoblast differentiation, prolonged exposure to GCs leads to substantial bone loss in vivo predominantly by inhibiting osteoblast functions. Compound A (CpdA) is a novel GC receptor modulator with the potential of an improved benefit/risk profile. Here we tested the osteoimmunological effects of CpdA on primary human osteoblasts and their paracrine interactions with osteoclasts. To assess the antiinflammatory potential of CpdA in human bone marrow stromal cell (BMSC)-derived osteoblasts, cells were stimulated with lipopolysaccharide and cytokine expression was determined. Similar to dexamethasone (DEX), CpdA profoundly suppressed lipopolysaccharide-induced TNF-α (−63%), IL-1β (−38%), and IL-6 (−36%) (P < 0.05) mRNA levels. Of note, CpdA failed to induce osteogenic differentiation of BMSCs, whereas DEX and budesonide enhanced matrix mineralization an d increased runt-related transcription factor 2 and alkaline phosphatase mRNA levels up to 5-fold in a dose-dependent manner. Interestingly, each substance promoted cell proliferation by 7–10% and suppressed apoptosis by 25–30% at low concentrations and early differentiation stages, whereas high concentrations (1 μm) suppressed proliferation and stimulated apoptosis in mature osteoblasts. Finally, CpdA did not increase the receptor activator of nuclear factor-κB ligand to osteoprotegerin mRNA ratio as compared with DEX and did not stimulate the formation of osteoclasts in coculture with BMSCs. In summary, CpdA displays dissociated osteogenic and immunological effects in human BMSCs that are distinct from those of conventional GCs. Whether the specific osteoimmunological profile of CpdA translates into a relevant in vivo effect needs to be further explored.

Allogeneic stem cell transplantation (SCT) is the most effective treatment option for many hematologic malignancies, but graft-versus-host disease (GVHD) remains a major cause of treatment failure. Along with well-established risk factors for transplantation outcomes, recent single-center studies have identified a birth order effect in HLA-identical sibling SCT, with lower rates of acute and chronic GVHD and improved overall survival when the donor is younger than the recipient. One hypothesized mechanism for this effect is microchimerism due to fetomaternal and transmaternal sibling cell trafficking during pregnancy as the donor is exposed to recipient antigens in utero. The aim of the present study was to validate previously reported single-center data in a large, multicenter cohort provided by the Center for International Blood and Marrow Transplantation. All adult and pediatric patients (n = 11,365) with a hematologic malignancy who underwent allogeneic SCT with a graft from an HLA-identical sibling donor between 1990 and 2007 were included. When donors were younger than recipients, there was a significantly lower rate of acute GVHD grade II-IV and chronic GVHD in children, as well as a lower rate of chronic GVHD in adolescents. However, the hypothesized overall positive effect of lower relapse and better survival when donors are younger than recipients was not observed. Our data suggest that if otherwise equally matched, a graft from a younger sibling may be superior to a graft from an older sibling for children and adolescents undergoing SCT.

Purpose The role of unrelated allogeneic stem-cell transplantation in acute lymphoblastic leukemia (ALL) patients is still not clear, and only limited data are available from the literature. We analyzed factors affecting clinical outcome of ALL patients receiving a related or unrelated stem-cell graft from matched donors. Patients and Methods The total study population was 264 adult patients receiving a myeloablative allogeneic stem-cell transplant for ALL at nine bone marrow transplantation centers between 1990 and 2002. Of these, 221 patients receiving a matched related or unrelated graft were analyzed. One hundred forty-eight patients received transplantation in complete remission; 62 patients were in relapse; and 11 patients were refractory to chemotherapy before transplant. Fifty percent of patients received bone marrow, and 50% received peripheral blood stem cell from a human leukocyte antigen–identical related (n = 103), or matched unrelated (n = 118) donor. Results Disease-free survival (DFS) at 5 years was 28%, with 76 patients (34%) still alive (2.2 to 103 months post-transplantation), and 145 deceased (65 relapses, transplant-related mortality, 45%). We observed an advantage regarding DFS in favor of patients receiving transplantation during their first complete remission (CR) in comparison with patients receiving transplantation in or after second CR (P = .014) or who relapsed (P < .001). We observed a clear trend toward improved survival in favor of B-lineage ALL patients compared with T-lineage ALL patients (P = .052), and Philadelphia chromosome–positive patients had no poorer outcome than Philadelphia chromosome–negative patients. Total-body irradiation–based conditioning improved DFS in comparison with busulfan (P = .041). Conclusion Myeloablative matched related or matched unrelated allogeneic hematopoietic stem-cell transplantation in ALL patients should be performed in first CR.

Objective. To assess the outcome of intensive care unit (ICU) treatment in patients with hematological malignancies. Design and setting. Retrospective cohort study in the medical ICU of a university hospital. Patients. 104 critically ill patients after receiving conventional chemotherapy or autologous hematopoietic stem cell transplantation. Interventions. We analyzed demographic data, underlying disease, intensity of antineoplastic regimen, cause of admission, need for mechanical ventilation, and hemofiltration, ICU survival, and survival after discharge, furthermore neutrophil count, C-reactive protein (≤150 vs. >150 mg/l), antithrombin III, prothrombin time, and SAPS II (≤50 vs. >50) at ICU admission. All recorded variables were evaluated for prognostic relevance by univariate and multivariate analyses. Measurements and results. Overall ICU mortality was 44%, with significantly higher mortality in ventilated patients (74% vs. 12% in nonventilated patients, p

Systemic mastocytosis (SM) is a rare hematologic neoplasm characterized by abnormal growth and accumulation of tissue mast cells (MC) in various organ systems, including bone marrow (BM). Indolent and advanced forms of SM have been described. Whereas patients with ISM have a normal or near normal life-expectancy, patients with advanced SM, including those suffering from mast cell leukemia (MCL) have a poor prognosis. In these patients, neoplastic MC are usually resistant against conventional drugs and various targeted drugs. In rapidly progressive aggressive SM (ASM) and MCL, polychemotherapy followed by allogeneic hematopoietic stem cell transplantation (alloHCT) has been proposed. However, outcome of alloHCT in advanced SM is unknown, and it also remains uncertain whether clinically relevant graft-versus-SM (GVSM) effects may occur in these patients, as only sporadic case reports have been published. We performed a retrospective multi-center analysis to evaluate the outcome of alloHCT in patients with advanced SM. Fifty-four advanced SM patients receiving SCT in 32 transplantation centers in Europe and America were identified between 1990 and 2013. The median patient age was 45 years. Donors were: HLA identical siblings (31), unrelated donors (URD) (15), umbilical cord blood donors (UCB) (2), and haploidentical donors (1). In 5 patients, stem cell source was not defined (5). Thirty-four patients received myeloablative conditioning (MAC) and 18 received reduced intensity conditioning regimens (RIC). In 2 patients, conditioning regimen was not specified. Indications for alloHCT were SM with an associated clonal hematologic non-mast cell lineage disease (SM-AHNMD) (n=32), MCL (n=13, including one with MCL-AHNMD), 8 with ASM and 1 with myelomastocytic leukemia (MML). The most prevalent AHNMD was acute myeloid leukemia (AML, n=16). With follow-up of 35-6180 (median 365) days, SM responses (defined as ≥50% decrease in BM mast cells ± decrease in serum tryptase ± regression of other organ manifestations) were observed in 39 patients (72%), including complete responses (CR) documented in 12 patients (22%). Eleven patients had stable disease, whereas 4 patients (7%) progressed immediately after alloHCT (primary resistance). In addition, 10 patients progressed (5 of them within 100 days) after an initial response. Progression was most frequently seen in MCL patients (n=6, 50%). In the AHNMD group, only 8 patients relapsed/progressed (25%). The overall survival (OS) and SM progression-free survival (PFS) at 1 year were 63% and 50% for all patients, 77% and 68% for SM-AHNMD, 63% and 50% for ASM, and 25% and 17% for MCL, respectively. The strongest predictive variable associated with inferior survival was a diagnosis of MCL. Other factors associated with poor outcome were: Karnofsky performance status ≤70%, ≥2 SM regimens given before alloHCT (e.g., steroids, cladribine, chemotherapy, tyrosine kinase inhibitor), donor source (alternative donors-UCB and haploidentical compared to sibling or URD), SM progression within the first 100 days, normal cytogenetics (compared to t(8;21) (q22;q22), and RIC (compared to MAC). The following variables were not associated with poor outcome: patient and donor age, recipient-donor sex match status, graft source (BM vs. peripheral stem cells), BM mast cell percentage at time of alloHCT, and CR status of AML or SM response at time of alloHCT. This largest multi-center analysis of results in advanced SM provides evidence for clinical efficacy of alloHCT, presumably because of a GVSM effect of alloHCT (achieving CR, and response to donor-lymphocyte infusions and RIC alloHCT). However, responses varied among different SM categories: while patients with SM-AHNMD enjoyed excellent outcomes, the OS for MCL patients in general, was poor. Nevertheless it is remarkable that 3 of 13 patients with MCL – an otherwise fatal disease with a median survival of Disclosures: Vercellotti: Sangart Inc.: Research Funding; Seattle Genetics: Research Funding. Akin:Novartis: Consultancy. Valent:Novartis: Consultancy, Honoraria, Research Funding.

Abstract 3060 Background: Chromosome 5 abnormalities in patients (pts) with acute myeloid leukemia (AML) or advanced myelodysplastic syndrome (MDS) are mostly associated with a poor outcome. Although allogeneic hematopoietic stem cell transplantation (HCT) is the treatment approach with the highest curative potential, early relapse rates following HCT are still considerably high. Since therapeutic options in these pts are often limited, the prevention of relapse represents a major challenge. Lenalidomide (LEN) has been successfully used in MDS pts with del(5q) cytogenetic abnormalities. Besides a direct anti-proliferative effect on del(5q) progenitors, its immunmodulatory function might also enhance T-or NK cell mediated GVL effects. Therefore it could be an effective maintenance drug to prevent relapse after allogeneic HCT in pts with MDS or AML and del(5q) abnormalities. Methods: We report results of a prospective multicenter phase II clinical trial evaluating the efficacy to prevent relapse as well as safety of LEN maintenance following HCT in pts with MDS or AML and cytogenetic abnormalities including del(5q). Only pts achieving a complete hematological remission (CR) after HCT were eligible. In the absence of toxicity or relapse pts could receive up to 12 cycles of LEN maintenance at a dose of 10 mg/day orally, for 21 days, with 7 days rest (28 day cycle). Results: Ten pts with either MDS (n=1, RAEB-1) or AML (n=9) with a median age of 65 years (range 40–72) were included. The disease status prior to allogeneic HCT were CR in 4 pts, relapse/refractory disease in 3 pts, unknown in 2 pts while the MDS pt had not received any prior therapy. A complex aberrant karyotype including del(5q) was documented in 5 pts. Two pts had an additional cytogenetic abnormality besides del(5q) and 3 pts displayed single del(5q). While three pts underwent allogeneic HCT from a matched sibling donor, four pts were transplanted from a matched unrelated and three from a mismatched unrelated donor (single allele or antigen mismatch). Conditioning was of reduced intensity and was followed by the infusion of peripheral blood stem cells in all pts. LEN maintenance therapy was started after complete hematopoietic recovery with documented complete remission at a median of 2.5 months (range 2–4 months) following HCT. After a median of 4 cycles 8 of 10 pts (80%) had to discontinue LEN treatment due to relapse (n=4), development of severe GvHD grade 2–4 (n=2) or other adverse events (n=2). Altogether, 6 of 10 pts (60%) developed severe acute GvHD grade 3–4 within the first 2 cycles of LEN maintenance. All pts were still under systemic immunosuppression at the time of GvHD appearance. Other common adverse events were gastrointestinal side effects (nausea) and myelotoxicity. Reversible neutropenia grade 3/4 was documented in 3 of 10 (30%) of the pts while thrombocytopenia grade 3/4 occurred in 4 of 10 (40%) of them. During the treatment course specific T cell responses against different tumor/leukemia-associated antigens (TAAs/LAAs) using ELISpot analysis for Interferon alpha and granzyme B were measured. Sufficient T cells before and during LEN treatment were only available in one pt, who showed an increase of specific T cell responses against the TAAs/LAAs. With a median follow-up of 254 days (range 32–677) from the start of LEN maintenance, 5 of 10 pts (50%) are currently alive with four pts in continuous CR since the time of HCT. The study was stopped prematurely because of suspected induction of GVHD by LEN. Conclusions: Early LEN maintenance to prevent relapse following HCT in pts with MDS or AML and del(5q) may be associated with the induction of severe acute GVHD. Disclosures: Kobbe: Celgene: Consultancy, Research Funding; Ortho Biotec: Consultancy. Germing:Celgene: Consultancy, Research Funding. Bornhaeuser:Celgene: Honoraria. Platzbecker:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract 3486 Delayed myeloid engraftment after HCT is a risk for increased morbidity and mortality, especially in patients with splenomegaly (SM) at time of transplant. Time to engraftment and overall survival after HCT have not been well analyzed in patients with prior splenectomy (SP) or splenic irradiation (SI), when compared to patients with normal spleens (NS) or with SM. A total of 9,683 recipients with myeloproliferative diseases and/or myelodysplasia who were reported to CIBMTR after receiving a myeloablative allogeneic HCT between 1990 to 2006 were compared according to the spleen status at transplant: 472 SP; 300 SI; 1,471 SM and 7,440 NS. Recipients of cord blood grafts were excluded. The median age was 39 years for all groups, the SP group had a higher proportion of patients with Karnofsky performance score OR d21 WBC1 (95% CI2) p-value OR d28 Platelet3 (95% CI) p-value RR4 Overall Mortality (95% CI) p-value Normal spleen (NS) 1 - 1 - 1 - Splenectomy (SP) 2.25 (1.76–2.89) Disclosures: Maziarz: Millenium: Speakers Bureau; Genzyme: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Abstract 2154 Background: Myeloid Sarcoma (MS) is defined as an extramedullary mass composed of myeloid blasts occurring at an anatomical site other than the bone marrow. Furthermore, the term extramedullary manifestation (EM) is applied if it accompanies overt acute myeloid leukemia (AML) and represents non-effacing tissue infiltration. EM is reported to correspond often to the skin but can affect almost every site of the body. The prognosis of MS or EM has been discussed controversially in the past. EM at diagnosis of AML is generally thought to be a rare event. However, data defining the prevalence of EM at diagnosis of AML and its prognostic value are missing. The aim of this analysis was to provide data for estimating the prevalence of EM at diagnosis of AML and to determine its relevance by including clinical and laboratory data from patients being treated in the prospective AML96 trial of the Study Alliance Leukemia (SAL) study group. Patients and Methods: A total of 326 patients with AML (age 17 – 83 years) and EM were treated within the AML96 trial with a median follow up of 8.8 years (95% CI, 8.4 to 9.3 years). All patients received double induction chemotherapy. Consolidation therapy contained high-dose cytosine arabinoside and for patients ≤ 60 years of age the option of autologous or allogeneic hematopoietic stem cell transplantation (HSCT). Logistic regression analyses were used to identify prognostic variables for CR rates. The method of Kaplan-Meier was used to estimate OS and EFS. Confidence interval (CI) estimation for the survival curves was based on the cumulative hazard function using the Greenwood's formula for the SE estimation. Survival distributions were compared using the log rank test. Results: 17% of the AML patients entered into the AML96 trial were diagnosed with EM. In 313 of the 326 patients (96%) EM was evident at diagnosis. The majority of patients with EM were diagnosed with de novo AML (84%, n=273), whereas gingival infiltration (51%, n=166) displayed the main EM of AML with CNS involvement being less common (4%, n=14). The majority of patients had a cytogenetic intermediate risk profile (71%, n=221) with a total of 172 patients (56%) harboring a normal karyotype. Patients with EM had a statistically significant lower median CD34-positivity of bone marrow blasts, higher percentage of FAB subtypes M4 and M5, higher WBC counts and LDH at diagnosis and higher percentage of NPM1 mutations compared to those patients without EM (all p Conclusions: This analysis represents the largest study so far investigating the impact of EM AML. Patients with EM AML have distinct differences from AML patients without EM regarding their clinical and molecular characteristics at diagnosis. However these differences do not translate into differences in response to induction chemotherapy. Compared to patients without EM, survival analysis revealed differences according to the NPM1/FLT3-ITD mutation status which is also described for patients without EM AML. However, the prognosis for patients with EM who harbor a mutated NPM1 the prognosis at relapse seems to be dismal. Disclosures: No relevant conflicts of interest to declare.

Abstract 3328 Poster Board III-216 Introduction Relapse is a major cause of treatment failure after alloSCT against acute leukaemia, and no standard treatment has been established in this challenging situation. The introduction of reduced conditioning regimens, and the broader availability of alternative donors have increased the possibilities to perform a second alloSCT as salvage treatment, using different preparative regimen and/or different stem cell donors. Methods To evaluate the role of a second alloSCT (tx2) for the treatment of relapse after first alloSCT (tx1), we performed a nationwide retrospective analysis based on the German registry for stem cell transplantation (DRST). Datasets were completed by the reporting centres on request, following a specifically designed questionnaire. Results 212 patients (69% AML, 31% ALL), from 23 centres were included. Median age at tx1 was 37y. Donor at tx1 were HLA identical siblings (41%), matched unrelated (39%), mismatched family or unrelated (17%) or syngeneic donors (3%). Conditioning intensity at tx1 was standard (SIC, 62%), intermediate (intC, 25%) or reduced (RIC, 13%). Median remission after tx1 was 7 months, median time from relapse to tx2 was 74d. At tx2, patients were aplastic (4%), in CR (20%) or showed active disease (76%). In 59%, the same donor was used for tx1 and tx2, whereas a different donor was chosen in 41%. Conditioning at tx1/tx2 were SIC/SIC (14%), intC/intC (10%), (RIC/RIC (10%), less intensive at tx2 (mostly intC or RIC after SIC, 58%), or more intensive at tx2 (SIC after RIC or intC, 8%). Following tx2, CR was achieved in 56% of patients, out of which 81% relapsed again. Hence, leukemia was the most frequent cause of death. With a median FU of 23 months after tx2, median OS after tx2 is 117d. In a univariate analysis (log rank), OS after tx2 depended on stage at tx1 (CR vs. active disease, p12m (31%), p Conclusion Survival of acute leukemia after second allogeneic SCT is determined by the duration of remission after tx1. Using an alternative donor for tx2 did not improve the results in our series. Further analysis is required to evaluate the role of RIC regimen for tx2. Disclosures No relevant conflicts of interest to declare.

Abstract 2307 Poster Board II-284 Objectives: T-cell depletion (TCD) is controversial in patients with chronic lymphocytic leukemia (CLL). While TCD is a powerful tool to prevent graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT) concern is raised about an increased incidence of relapse. The aim of this retrospective analysis was to study the impact of in vivo TCD in patients with CLL registered with the EBMT database. Patients and Methods: Patients with CLL who received allogeneic HCT from a matched sibling (SIB) or unrelated donor (UD) and cyclosporine-based GVHD prophylaxis between 2001 and 2008 were eligible. Patients who received ex vivo T-cell depleted grafts were excluded. The outcome of three major groups of patients was compared: Patients who were transplanted without TCD, those who received anti-thymocyte globulin (ATG) and patients who received alemtuzumab (CAM). Baseline and follow-up data were downloaded from the EBMT database. Results: 413 patients were eligible. 73% of patients had SIB donors and 27% matched UD. Reduced intensity conditioning regimens were applied for the majority of patients (82%). No TCD was used in 234 patients, while 100 patients received CAM and 79 patients ATG. The median follow-up after HCT was 22 months (1 to 92 months). Univariate comparisons of GVHD and the rates of DLI are reported for patients with matched sibling donors only. After SIB-HCT the cumulative incidence of acute GVHD II-IV was 7% with CAM, 22% with ATG and 34% without TCD (gray test, p=0.0001). Subsequently, 28% of patients with CAM received prophylactic donor lymphocyte infusions (DLI) compared to 5% of patients with ATG and 9% without TCD (p=0.03). DLI for any reason was given to 45% of patients with CAM, 24% with ATG and 15% without TCD (p=0.009). As a result of late-onset GVHD the incidence of chronic GVHD after TCD with CAM increased from 20% at 1 year to 60% at 4 years after SIB-HCT. At 4 years after HCT comparable cumulative incidences of chronic GVHD were observed (70% without TCD, 59% after ATG and 60% after CAM; p=0.1). For the whole cohort of patients 4-year overall survival (OS) and progression-free survival (PFS) were 57% (95% CI, 50% to 64%) and 44% (95% CI, 37% to 51%). At 4 years the cumulative incidence of relapse was 28% (95% CI, 17% to 39%) and the incidence of non-relapse mortality was 28% (95% CI 18% to 38%). In multivariate Cox regression analysis of PFS when GVHD prophylaxis without TCD was used as reference category the adjusted hazard ratio for TCD with CAM was 1.4 (95% CI, 0.9 to 2.2, p=0.2) and for TCD with ATG 1.4 (95% CI, 0.9 to 2.3, p=0.1). For relapse incidence the adjusted hazard ratio for TCD with CAM was 1.4 (95% CI, 0.7 to 2.6, p=0.3) and for TCD with ATG 1.0 (95% CI, 0.5 to 2.1, p=0.9) and for non-relapse mortality the adjusted hazard ratio for TCD with CAM was 1.4 (95% CI, 0.8 to 2.8, p=0.3) and for TCD with ATG 1.9 (95% CI, 1.0 to 3.5, p=0.06). Conclusion: In patients with CLL the combination of in vivo TCD and DLI appears to result in comparable rates of chronic GVHD and PFS compared to T-cell replete HCT. Disclosures: Schetelig: Bayer Schering: Research Funding.

Introduction: 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 upfront allogeneic HSCT for remission induction as part of induction therapy has the potential to circumvent these problems and might furthermore reduce cumulative toxicity in high-risk patients. Therefore, in 2003 we started a prospective multicenter randomized trial that investigates both the feasibility and efficacy of upfront allogeneic stem cell transplantation for remission induction in high-risk AML patients. Methods: The AML2003 study compares in a randomized fashion an intensified treatment approach using upfront allogeneic transplantation in high risk patients as part of the induction therapy (IT) during marrow aplasia achieved by DA (daunorubicin 60 mg/ m2 – day 3–5; cytarabine 100 mg/m2 – day1–7) to a “conventional” treatment strategy, which allows for allogeneic transplantation only in patients achieving remission after two induction courses (DA). To do this, rapid analysis of cytogenetics, FLT3 status and HLA-DNA-typing of the patient and possible family donors is of utmost importance. This “fast search diagnostics” together with routine analyses of morphology and immunophenotyping is accomplished centrally in all enclosed patients. The dose-reduced preparative regimen for upfront allogeneic stem cell transplantation within induction therapy consisted of melphalan 150mg/m2 and fludarabine 150mg/m2. Results: Until the last update we recruited 679 patients Conclusions: These preliminary results show that rapid risk profiling and fast donor-search is feasible in a large multi-center study. This leads to a significant proportion of upfront allogeneic stem cell transplants as part of the induction therapy within the group of high-risk AML patients, which may improve the disastrous prognosis of this group of patients in the future.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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.

The administration of myelosuppressive chemotherapy followed by daily injections of granulocyte-colony stimulating factor (G-CSF) is the common procedure to mobilize autologous CD34+ peripheral blood stem cells (PBPC). Pegfilgrastim (NeulastaTM, Amgen Inc., Thousand Oaks, USA) is a covalent conjugate of filgrastim and polyethylene glycol with an increased elimination half-life due to decreased serum clearance. Whereas a single injection of pegfilgrastim (PEGFIL) has been shown to be equivalent to daily filgrastim in enhancing neutrophil recovery after chemotherapy, the experiences with PEGFIL in mobilization of PBPC are limited. We report 40 pts (22 male, 18 female, median age 53 years) who had a PBPC mobilization treatment for Hodgkin′s lymphoma (n=3), non-Hodgkin′s lymphoma (n=13), multiple myeloma (n=16), acute lymphoblastic leukaemia (n=3) or solid tumors (n=5). The mobilization regimen consisted of disease specific chemotherapy and a single subcutaneous injection of 6 mg PEGFIL administered 48 hours after the end of cytotoxic treatment (day 0). CD34+ cells in the peripheral blood (PB-CD34) were measured if white blood cells (WBC) exceeded 1.0 Gpt/L after nadir. PBPC collections started at a PB-CD34 cell count >10/μl and were performed as large-volume apheresis (4x blood volume) using a Cobe Spectra (Gambro BCT Inc.). Additional conventional filgrastim (FIL) was given at a dose of 2x5μg/kg if PB-CD34 count was found to be The median start of aphereses was on day +9 after the administration of PEGFIL and on day +15 after start of chemotherapy regimen, respectively. Median PB-CD34 peak was 74/μl (range 9–565/μl). The median PBPC yield was 7.6 x 10^6 CD34+ cells/kg (range 1.5–88.1). The target cell dose to be collected (≥ 2.5 x 10^6 CD34+ cells/kg) was achieved in 36 (90 %) pts, in 29 pts (72.5 %) ≥ 4.0 x 10^6 CD34+ cells/kg could be obtained with a single collection. Additional FIL administrations were necessary in 7 patients (17.5 %) for 2 to 6 days. All of them were heavily pretreated including a previous autologous transplant in two of these patients. PEGFIL was well tolerated except for moderate bone pain which occurred in all patients. The mean values (± SD) for peak plasma concentration of PEGFIL (cmax), time to reach the maximum plasma level (tmax) and elimination half-life (t1/2) were 154 (± 83) ng/ml, 56 (±21) hours and 23 (± 9) hours, respectively. We conclude that a single dose of 6 mg PEGFIL after chemotherapy is safe and highly effective in enhancing the mobilization of CD34+ PBPC for stem cell collection. Further investigations are warranted, including comparison with non-pegylated G-CSFs and in combination with antiadhesive agents.

Gemtuzumab ozogamicin (GO) has been successfully used in older patients with relapsed CD33+ acute myeloid leukemia. Retrospective analyses suggest that the use of GO within a few months before or after hematopoietic stem cell transplantation (HCT) is associated with a increased risk for sinusoidal obstruction syndrome (SOS) or veno-occlusive liver disease (VOD). Ongoing studies investigate the use of GO in the induction and post-remission therapy of AML patients. We hypothesized that GO might be safe and effective as part of a reduced intensity conditioning regimen containing fludarabine and total-body irradiation (TBI) Fifteen patients relapsing after conventional induction chemotherapy (n=9) or after previous transplantation (autologous n=3; allogeneic n=3) were included so far. The last twelve patients were treated within an ongoing phase II trial. The preparative regimen contained 6 mg/m2 and 3 mg/m2 GO on day −21 and day −14 before allogeneic transplantation. Patients who responded to GO treatment and were below the age of 60 (n=8) received fludarabine 120 mg/m2 and 800 cGy TBI (n=6) during GO-induced aplasia followed by allogeneic HCT. Patients older than 60 years or those with relapsing AML These data suggest, that GO can be combined with a TBI-containing, fludarabine-based conditioning regimen before allogeneic HCT in patients with relapsed AML. No increased incidence of VOD has to be expected even in recipients who had undergone previous conditioning therapy and transplantation. Nevertheless, additional chemotherapy during aplasia before allogeneic transplantation is required in 60–70% of patients who do not respond to GO. For responding patients the combined regimen is associated with minimal toxicity and a considerable chance for long-term cure.