We retrospectively analyzed outcomes of a CD34(+)-selected stem cell boost (SCB) without prior conditioning in 32 patients (male/22; median age of 54 years; range, 20 to 69) with poor graft function, defined as neutrophils ≤1.5 x 10(9)/L, and/or platelets ≤30 x 10(9)/L, and/or hemoglobin ≤8.5 g/dL). The median interval between stem cell transplantation and SCB was 5 months (range, 2 to 228). The median number of CD34(+) and CD3(+) cells were 3.4 x 10(6)/kg (.96 to 8.30) and 9 x 10(3)/kg body weight (range, 2 to 70), respectively. Hematological improvement was observed in 81% of patients and noted after a median of 30 days (range, 14 to 120) after SCB. The recipients of related grafts responded faster than recipients of unrelated grafts (20 versus 30 days, P = .04). The cumulative incidence of acute (grade II to IV) and chronic graft-versus-host disease (GVHD) after SCB was 17% and 26%, respectively. Patients with acute GVHD received a higher median CD3(+) cell dose. The 2-year probability of overall survival was 45%. We suggest that SCB represents an effective approach to improve poor graft function post transplantation, but optimal timing of SCB administration, anti-infective, and GVHD prophylaxis needs further evaluation.

NOX-A12 is a PEGylated mirror-image oligonucleotide (a so-called Spiegelmer) that binds to CXCL12 (stromal cell-derived factor-1, SDF-1) with high affinity thereby inhibiting CXCL12 signaling on both its receptors, CXCR4 and CXCR7. In animals, NOX-A12 mobilized white blood cells (WBCs) and hematopoietic stem and progenitor cells (HSCs) into peripheral blood (PB). In healthy volunteers, single doses of NOX-A12 had a benign safety profile and also dose-dependently mobilized WBCs and HSCs into PB. HSC peak mobilization reached a plateau at five times the baseline level at an i.v. dose of 5.4 mg/kg. In accordance with the plasma half-life of 38 h, the duration of the WBC and HSC mobilization was long lasting and increased dose-dependently to more than 4 days at the highest dose (10.8 mg/kg). In conclusion, NOX-A12 may be appropriate for therapeutic use in and beyond mobilization of HSCs, e.g., in long-lasting mobilization and chemosensitization of hematological cancer cells.

Abstract 4689 Introduction: Allogenic stem cell transplantation (allo SCT) offers a potential curative approach for many malignant and non malignant haematological diseases. Despite its therapeutic benefit, long term immunodeficiency, poor immune reconstitution and Graft vs. Host Disease (GvHD) can often be limiting drawbacks. Since the nineties, regulatory T cell subsets (Treg) have been described and several lines of evidence indicated their implication on GvHD occurrence and progression. We analysed the immune reconstitution of 184 patients who underwent allo SCT at our Transplant Center from 2007 till 2009. Patients, Materials and Methods: Differential lymphocyte subsets were analysed by flow cytometry. Antigens were stained by usage of the following mAb: CD3, CD4, CD8, CD19, HLA-DR, CD56/CD16, CD45RA, CD45RO, CD45, γδ TCR, CD25, and CD127. Tregs were evaluated on simultaneous expression of CD4/CD25hi/CD127low. Data were obtained in monthly intervals for the first six months and thereafter every six months for the next 3 years. Data were analysed for three different subgroups: Multiple Myeloma (MM: n=83), Myelofibrosis (PMF: n=22) and AML/MDS (n=51). Smaller number subgroups of patients with CML (n=11), NHL (n=10) and ALL (n=7) were included into the overall analysis but not evaluated separately. Results: The mean value of Treg cell number before allo SCT was 2,5% of the total leukocyte number in all patients. There was no significant difference in the Treg level in any of the three major groups (MM: 2,2%; PMF: 2,1% and AML/MDS: 2,03%). All patients exhibited a significant reduced number of Treg cells during the first 30 days after allo SCT (MM: 0,79%; p= 0,009; PMF: 0,41%; p= 0,01; MDS/AML: 0,6%; p=0,01). Between day 30 and 60 after allo SCT patients with MM had a transient Treg recovery to baseline level (2,4%) while Tregs of patients with PMF or MDS/AML remained significantly lower in comparison to baseline value (PMF: 0,72%, p=0,002 and MDS/AML 0,81%, p=0,01 respectively). One year after allo SCT a faster Treg recovery (1,3% and 1,8% respectively) was observed in MM and MDS/AML patients while patients with PMF still maintained a significant reduction (0,65%; p=0,01). Interestingly, in the second year after allo SCT, Treg cell levels were decreased in all investigated subgroups (MM: 1,1%, p=0,008; PMF: 0,7%, p=0,02 and MDS/AML: 0,7%, p Conclusion: A highly dynamic Treg cell recovery after allo SCT was observed in our group of patients. Even one year after allo SCT Treg reconstitution is still ongoing. Our data highlight that there is a distinctive difference in Treg recovery among the various fore mentioned diseases. Treg reconstitution appeared to be prolonged in patients with PMF in comparison to the other subgroups. Our data provide a basis for further analysis towards differential Treg reconstitution and its potential impact on allo SCT complications. Disclosures: No relevant conflicts of interest to declare.

We evaluated immune recovery in 67 patients with acute myeloid leukemia (AML) with a median age of 40 years (4-69) following allo-SCT after reduced (n = 35) or myeloablative (n = 32) conditioning. The following lymphocyte populations were determined on days +30, +90, +180, +270, and +365 by flow associated cell sorting: CD3+, CD3+CD4+, CD3+CD8+, CD3+CD4+/CD3+CD8+ ratio, CD3-CD56+, and CD19+ cells. Peripheral blast count >5% was related to lower number of CD3+CD4+ (day +30) and NK cells (day +180; p = 0.02). Intensity of conditioning did not have any significant impact on the kinetics of immune recovery. Patients with normal CD3+CD4+/CD3+CD8+ ratio (day +30) and NK cell count (day +90; p <0.05) experienced better survival than those with decreased parameters. Post-transplant sepsis/severe infections impaired CD3+CD8+ (day +90; p = 0.015) and CD19+ (day +90; p = 0.02) recovery. Relapse in patients following allo-SCT showed an association with decreased numbers of CD19+ (day +270) and NK cells (day +365). Acute GvHD (II-IV) was accompanied by reduced CD19+ and CD3+CD4+ cells. Thus, the evaluation of post-transplant immune reconstitution in patients with AML might improve risk stratification concerning either relapse or TRM and remains to be further explored.

Donor lymphocyte infusions (DLIs) are used for adoptive immunotherapy to prevent or treat relapse and infectious complications after allogeneic hematopoietic stem cell transplantation (HSCT). Unmanipulated DLIs are associated with a risk of graft-versus-host disease (GVHD), probably related to CD8(+) T cell activity. We investigated an automated clinical-scale human-CD4(+)-cell purification method to deplete CD8(+) cells. Twenty-four stem cell recipients received a total of 24 leukapheresis products being enriched for CD4(+) cells using magnetic associated cell sorting (MACS) with an automated device (CliniMACS(®)) before DLIs. MACS resulted in a mean CD4(+) cell count of 16 × 10(6)/kg bw corresponding to 3.4-fold CD4(+) cell enrichment. Mean yield and purity of CD45(+)CD3(+)CD4(+)CD14(-)7AAD(-) were 74% ± 23% and 82% ± 11%, respectively. Median initial dose of DLIs was 1.1 × 10(6) CD4(+)/kg. During a median follow-up of 25 months, 7 (30%) patients experienced GVHD (acute II-IV: n = 4, 17%; acute III-IV: n = 2, 8%; chronic limited: n = 2, 8%; chronic extensive: n = 1, 4%). Thirteen of 21 further evaluable patients (62%) showed measurable clinical response, 2 patients with therapy refractory infectious complications (HSV) showed remarkable immunologic improvement. Automated enrichment of CD4(+) by magnetic cell sorting provides an efficient and rapid method for processing donor lymphocytes. Additional studies should further investigate this approach in terms of efficacy and the risk of GVHD.

Abstract Abstract 2376 Allogeneic stem cell transplantation (HSCT) for multiple myeloma is a potential curative treatment approach. A high number of relapses after allogeneic stem cell transplantation after reduced intensity conditionings underlying the need of post transplant strategies to improve remission rates and disease free survival. Lenalidomide is an effective drug in treatment of multiple myeloma patients. The efficacy and the immunmodulatory properties on T- and NK- cells may augment the Graft versus Myeloma effect after HSCT, but myelosuppression as well as induction of GvHD is a concern of using the drug early after allogeneic stem cell transplantation. Study objective was to determine the maximal tolerable dose (evaluating three dose levels) of lenalidomid after HSCT in patients with multiple myeloma. A total of 18 patients with multiple myeloma were enrolled so far. Lenalidomide as single agent maintenance treatment was started between 100 and 180 days after HSCT. In the first subgroup three patients started with dose of 5 mg daily from day 1 till 21 for duration of four cycles. In this group dose limited toxicities not appear. The next higher dose level was 10 mg/d lenalidomide for a total of 6 patients. In this cohort of patients, 3 patients showed dose limiting toxicities, which were caused by an acute pancreatitis in one case, elevated liver enzymes (CTC grade III), attribute to liver GvHD in one case and renal insufficiency in one patient. Because 3 patients in 10 mg cohort developed DLT, the maximum tolerate dose has been declared as 5 mg. So far 6 additional patients were treated with this dose level (5 mg/d, day 1–21). In these cohort four patients experienced CTC grad III toxicity was observed: two cases with acute GvHD, one case with elevated liver enzymes and one case with intolerance and dizziness. Beside lenalidomide’s effect on myeloma cells, the drug is a known immune modulator. We therefore, analysed T- and NK cell subsets of patient peripheral blood by flow cytometry after lenalidomide treatment. An increase of 6% in activated CD3 cells was observed, as indicated by expression of HLA-DR molecules. Furthermore, except for the increase of whole CD8 cell number, we also observed increased proinflammatory CD8/INFg+ T cell numbers (1,5% to 4,5% p=ns). Along with the T cell date, NK cells expressed more activating receptors, like NKp44 and less inhibitory receptors, like NKG2A, support the immunmodulatory efficacy of lenalidomide. We concluded 5 mg lenalidomide as a single agent is the maximum tolerate dose if used early after allogeneic stem cell transplantation (day 100 – 180). Lenalidomide has high immune modulatory properties that might increase the risk of GvHD by activating CD8 cell. Combining lenalidomide with immunosuppressive drugs such as dexamethasone or bortezomib may reduce the risk of GvHD. Disclosures: Schonland: Celgene: Research Funding. Kröger: celgene: Research Funding.

Background: Demethylating agents like 5-Azacytidine (5-Aza) have entered treatment for myeloid dysplastic syndrome (MDS) and acute myeloid leukaemia (AML). Beyond the effect of differentiation, one suggested mechanism is that 5-Aza demethylates antigens such as Cancer-Testis Antigens (CTA) which will then be presented in order to induce immune response by immunocompetent cells. While a lot of attention was paid to the effects of 5-Aza in the myeloid cells, less is known about the impact of 5-Aza on immune cells. We therefore investigated the effects of 5-Aza on T cells. Materials and methods: T-cells were isolated from buffy coats using magnetic beads. Cells were stimulated and cultured for 1 week in the presence of IL-2. Thereafter cells were treated with or without 5 μM 5-Aza or 20 μM for 48h. mRNA was isolated and used for cDNA synthesis. qPCR was done under standard conditionings for expression of IL-10, FoxP3, and TGF-beta normalized for GAPDH. T cells were analyzed by fl ow cytometry using the following antibodies: CD3, CD4, CD8, HLA-DR, FoxP3, CD 127, and CD25. Functional killing of an AML cell line (HL60) was measured using a LDH release assay. Results: We observed a signifi cant (P = 0.03) growth delay of the 5-Aza treated T cells compared to untreated control cells. Only the highest dosage (20 μM) of 5-Aza increased the fraction of apoptotic cells. 5-Aza did not infl uence the expression of CD3, but caused a change in the distribution of T cell subpopulations: percentages of CD8 + T cells decreased from 46% to 35% whereas CD4 + T cells increased from 57% to 68%. When stained for activation marker HLA-DR, a reduced expression was observed after 5-Aza treatment (57%, 48%, and 44% respectively). By screening different mRNA levels we found that the mRNA expression of IL10, FoxP3, and TGF-beta was upregulated (P = 0.02, P = 0.003 and P = 0.02 respectively). Strikingly, we also found an upregulation of CD4 + FoxP3 + Treg cells from 4.8% to 9.6% to 10.9% respectively. In a cytotoxicity assay we observed a signifi cantly reduced capability of Aza-treated T cells to lyse target cells (P = 0.02). Conclusions: Our data indicate that 5-Aza inhibits the activity of T cells by: 1. reducing the number of CD8 + T killer cells, 2. upregulating the subpopulation of Tregs as well as increasing the expression of inhibitory cytokines like IL-10. We therefore conclude that 5-Aza treatment may constrain the distribution and function of immune cells. R1243 Dendritic-cell based tumour vaccination in prostate and renal cell cancer: systematic review and meta-analysis A. Draube (1), N. Klein-González (1), S. Rademacher (1), C. Brillant (2), M. Hellmich (3), M. von Bergwelt-Baildon (1) (1)Stem Cell Transplantation Program Cologne (Cologne, DE); (2)University Hospital of Cologne (Cologne, DE); (3)University of Cologne (Cologne, DE)

Objective. To investigate post-transplant immunotherapy with escalating donor-lymphocyte infusions (DLI) and novel agents (thalidomide, bortezomib, and lenalidomide) to target complete remission (CR).Materials and Methods. Thirty-two patients with multiple myeloma who achieved only partial remission after allogeneic stem cell transplantation were treated with DLI. If no CR was achieved, one of the novel agents was added to target CR.Results. CR defined either by European Group for Blood and Marrow Transplantation criteria, flow cytometry, or molecular methods as assessed by patient-specific immunoglobulin H-polymerase chain reaction or plasma cell chimerism polymerase chain reaction was accomplished in 59%, 63%, and 50% of patients, respectively. Achievement of CR resulted in improved 5-year progressive-free and overall survival, according to European Group for Blood and Marrow Transplantation criteria (53% vs 35%; p = 0.03 and 90% vs 62%; p = 0.06), flow cytometry (74% vs 15%; p = 0.001 and 100% vs 52%; p = 0.1), or molecular methods (84% vs 38%; p = 0.001 and 100% vs 71 %; p = 0.03).Conclusions. Our finding demonstrates the clinical relevance of posttransplantation therapies to upgrade remission, and of remission's depth for long-term survival in myeloma patients. (C) 2009 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.

We investigated efficacy and toxicity of lenalidomide in 24 heavily pretreated myeloma patients with a median age of 59 years (range: 37–70) and relapse after allo-SCT. Lenalidomide was given at a dose of 15 mg (n=4), or 25 mg (n=20), orally once daily on day 1 to day 1 every 28 days, with (n=20) or without (n=4) DHAP. The median number of lenalidomide cycles was five (range: 2–17). Major side effects were leukopenia (grade 4: 4%, grade 3: 21% and grade 2: 17%) and thrombocytopenia (grade 3: 17% and grade 2: 29%); infectious complications were observed in 50%. Non-hematological toxicity consisted of muscle cramps (n=9), fatigue (n=5) and constipation (n=2). Mild grade I–II GVHD was seen in three patients. Response was achieved in 66%: CR in 8%, VGPR in 8%, PR in 50% and SD in 13%. The median time to progression was 9.7 months (95% confidence interval (CI): 7.5–11.9), and median OS was 19.9 months (95% CI: 17.3–22.5). Immunomonitoring after lenalidomide showed significant increase of activated NK (NKp44+) and T (HLA-DR+) cells, as well as regulatory T cells (CD4+, CD25+, CD127lo), supporting an immunomodulating anti-myeloma effect of lenalidomide.

Mannan-binding lectin (MBL) deficiency is determined by MBL gene polymorphisms and is associated with an increased infection risk. To clarify the role of MBL in Allo-SCT, 131 recipients–donors were analysed. MBL genotypes were determined by PCR and heteroduplex analyses, MBL serum levels by ELISA, and MBL oligomers by western blotting. MBL levels <400 ng/ml were associated with increased susceptibility to fungal pneumonia (7/12 vs 35/111; P=0.04, adjusted P=0.002), HSV/VZV (7/12 vs 26/111; P=0.03), CMV reactivation and acute GVHD. Donor genotypes had no influence. Pre-SCT MBL levels corresponded to recipients’ genotypes (P<0.001), changed significantly post-SCT, but were not influenced by donors’ genotypes. MBL oligomer profiles were similar pre-/post-SCT. Cultured CD34+ cells were found not to synthesise MBL. In conclusion, low MBL levels pre-transplant predisposed patients to sepsis, fungal and viral infection. Donors’ MBL genotypes did not influence infection rates. Prospective studies should clarify the importance of MBL as a prelude for MBL replacement after SCT.