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Introduction: For allogeneic stem cell donation it is current practice to mobilize with G-CSF bid for 5 days. Here, the feasibility of mobilizing with a single injection of pegfiligrastim was examined in a phase II study with an emphasis on donor safety and product composition. Results: 28 sibling donors (14 female, 14 male) of median age 50 (range 22 - 69) years were included in the study. The median donor weight was 80 (range 45 - 119) kg and that of recipients was 75 (range 47 - 127) kg. Large volume apheresis (4 x total blood volume) was possible in 26 donors 4 days after the administration of pegfilgrastim 12 mg s.c. The aphereses were performed when CD34+cells exceeded 10/µl peripheral blood, median CD34+concentration prior to apheresis being 66.25 (range 22.8 - 136.6) cells/µl. White blood cell (WBC) concentration peaked on day 4 of stimulation with a median 52.55 (range 22.8 - 85) GPT/l. The lactate dehydrogenase (LDH) and C-reactive protein (CRP) levels increased to a median 2.55 (range 1.36 - 6.02) and 3.23 (range 0.47 - 15.13) x upper limit of normal (ULN) respectively. There were no changes in creatinine, electrolytes or transaminases. Two donors required filgrastim in addition to the pegylated G-CSF, with the aphereses being performed on day 5 after pegfilgrastim. Six donors required more than one apheresis. In 25/28 donors, a median of 6.5 (range 4.6 - 14.5) x106CD34+/kg recipient body weight were collected. In three donors the yield was below 4x106CD34+/kg recipient body weight, i.e. 3, 3.4 and 3.6 respectively. The yield of CD34+/CD133+cells was median 4.45 (range 2.74 - 12.5) x106/kg recipient body weight. In 27/28 cell concentrates, the number of CFU-GM amounted to median 126 (range 65 - 323) /105mononuclear cells. The yield of CD3+cells was median 3.25 (range 1.4 - 6.2) x108/kg recipient body weight. The CD3+/CD8+and CD3+/CD4+ratios were 0.92 (range 0.33 - 2.58) and 2.14 (range 0.85 - 4.16) x108/kg, respectively. The yield of natural killer (NK) cells was median 0.4 (range 0.2 - 1) x108/kg recipient body weight. On the first collection day, 27/28 donors reported through a standardized questionnaire that the stimulation was well tolerated, although 26 experienced bone pain, which was strong in 11 cases. Other side effects such as cephalea, tiredness or inability to work were reported only in single cases. Hematopoietic Stem Cell Transplants were performed without complications. WBC recovery occurred on median day 14 (range 8 - 34) after HSCT. Acute GvHD > Gr. 3 was noted in one patient only. No rejection was observed. Conclusion: Pegfilgrastim is a feasible alternative to conventional bid filgrastim for stem cell mobilization. A single application was able to mobilize sufficient CD34+/CD133+cells into the peripheral blood for successful collections. The most frequent adverse effects were elevation of CRP and/or LDH and bone pain. Disclosures Niederwieser: Miltenyi: Speakers Bureau; Novartis: Research Funding.

The antitumor activity of natural killer (NK) cells has recently been shown to be assessable at a single-cell level via flow cytometric detection of CD107a. We used this novel method to prospectively quantify and characterize tumor-reactive NK cells in patients undergoing myeloablative or nonmyeloablative conditioning and allogeneic hematopoietic stem cell transplantation (HSCT). Degranulation of NK cells in the peripheral blood of 34 patients after HSCT (day +30, day +90) was determined by evaluating CD107a expression after coincubation of the cells with tumor targets. The percentage of degranulating NK cells was higher after nonmyeloablative conditioning than after myeloablative conditioning (P < .001), indicating a higher activation state and increased antitumor activity of NK cells after nonmyeloablative conditioning. We were able to analyze NK cell subsets separately and found that CD56bright NK cells following HSCT are functionally different from CD56bright NK cells in healthy donors, as indicated by a high percentage of degranulating NK cells in response to tumor targets in patients after HSCT. The CD107a assay is a new and feasible method to quantify and characterize tumor-reactive NK cells after HSCT. Using this method, we found that NK cells had high antitumor cytotoxic activity after nonmyeloablative conditioning, which may contribute to the effectiveness of nonmyeloablative conditioning.

We previously reported results in 71 patients with advanced hematologic malignancies given HLA-matched unrelated granulocyte colony-stimulating factor–mobilized peripheral blood mononuclear cell (G-PBMC) grafts after fludarabine 90 mg/m2, 2 Gy of total body irradiation, and postgrafting mycophenolate mofetil (MMF) 15 mg/kg twice daily and cyclosporine 6.25 mg/kg twice daily orally. Graft rejection was 15%; the cumulative probability of acute graft-versus-host disease (GVHD) was 52%. According to MMF pharmacokinetic studies, which showed a short half-life of its active metabolite, mycophenolic acid, we increased MMF dosing from 15 mg/kg twice daily to 15 mg/kg 3 times daily to increase immunosuppression and reduce the incidence of both graft rejection and acute GVHD. Among 103 patients so treated, graft rejection occurred in 5%, whereas acute GVHD remained at 53%. Outcomes were compared with results of previous G-PBMC recipients given MMF twice daily. Infection rates were slightly higher with MMF 3 times daily than with MMF twice daily. Nevertheless, 2-year nonrelapse mortality and overall and progression-free survivals were similar for MMF 3-times-daily and twice-daily patients (19%, 58%, and 49% versus 20%, 48%, and 37%, respectively). Nonmyeloablative conditioning with postgrafting cyclosporine and MMF given 3 times daily allowed 95% durable engraftment of unrelated donor G-PBMC grafts.

Between 1996 and 2004, a total of 708 patients were enrolled in the acute myeloid leukaemia (AML) '96 and '02 studies of the East German Study Group (OSHO). Of these, 138 patients (19.5%) had unfavourable cytogenetics defined as complex karyotype, del (5q)/-5, del (7q)/-7, abn (3q26) and abn (11q23). In all, 77 (56%) achieved complete remission 1 (CR1) after induction chemotherapy and were eligible for haematopoietic cell transplantation (HCT). HCT was performed after a median of two cycles of consolidation chemotherapy (CT) in the AML '96 and one cycle in the AML '02 study (P=0.03). After a median follow-up of 19 months, overall survival (OS) at two years was significantly better in the donor group (52+/-9%) versus the no-donor group (24+/-8%; P=0.005). Differences in outcomes were mainly because of a lower relapse incidence in patients after HCT (39+/-11%) compared with a higher relapse incidence in patients undergoing CT (77+/-10%; P=0.0005). Treatment-related mortality was low and not statistically significantly different between the two treatment groups (15+/-7 and 5+/-5% for HCT and chemotherapy, respectively; P=0.49).We conclude that early HCT from related or unrelated donors led to significantly better OS and leukaemia-free survival compared with chemotherapy in patients with unfavourable karyotype.

Treatment of steroid-resistant acute GVHD with OKT3 and high-dose steroids results in better disease control and lower incidence of infectious complications when compared to high-dose steroids alone: a randomized multicenter trial by the EBMT Chronic Leukemia Working Party

In a 64-year-old man suffering from hypoblastic myelodysplastic syndrome a secondary acute myeloid leukaemia developed. After induction chemotherapy with resulting partial remission he received an allogenic (related) peripheral blood stem cell transplantation conditioned with 2 Gy total body irradiation. After haematopoietic reconstitution chest pain and dyspnoea appeared. Computer tomography revealed diffuse bilateral infiltrates which were considered to be suspicious for an invasive pulmonary aspergillosis of the left upper lobe. Respiratory and circulatory insufficiency occurred. In bronchoalveolar lavage fluid Aspergillus antigen was detected. In addition, Aspergillus flavus was isolated on Sabouraud-dextrose agar. Ambisome (liposomal encapsulated amphotericin B) was applied in high dosages. On the skin of the sides and the back five livid red stained nodular lesions with haemorrhagic infarctions appeared. Pathohistologically, both in PAS (periodate acid Schiff) and Grocott-Gomori staining conglomerates of septated hyphae were detected in corium and subcutis. In addition, Aspergillus flavus grew from skin tissue. Despite antifungal treatment the patient died from Aspergillus pneumonia and generalized aspergillosis with dissemination to heart, brain, and skin.

Abstract 2148 Poster Board II-125 Introduction: The standard procedure for obtaining peripheral blood stem cells (PBSC) is donor mobilization with G-CSF. Pegfilgrastim is a covalently bound conjugate of filgrastim and monomethoxypolyethylene glycol with longer half-life elimination due to decreased plasma clearance and could represent an alternative approach for PBSC mobilization in healthy donors. Design and Methods: From July 2006 till August 2009 28 related healthy donors (50% male, 50% female) were treated with single dose of 12 mg pegfilgrastim for mobilization of allogeneic PBSC. The harvests were performed as large-volume, continuous-flow collections using a Cobe Spectra blood cell separator on day 4 and if necessary on day 5 of the mobilization regimen. In case of inadequate CD34+ counts (less than 4×106/kg body weight of recipient on day 5), stimulation was continued with filgrastim. In addition, the serum level of filgrastim was determined twice daily. Results: We present the results of 27 donors (the results of the 28th donor are still pending). In all 27 cases the harvests were successful. In 22 out of 27 donors (82%) only a single apheresis was needed to reach the target. Two of the donors required additional treatment with non-pegylated filgrastim. The maximal concentration of circulating CD34+ cells was achieved on day 4 (median 74.3/μl; range 24.6-136.6). The median yield of CD34+ cells was 5.9×106/kg of the recipients body weight (range 3-14.5), and the median CD3+ count was 9.1×108/kg of the recipient body weight (range 1.4-6.2). Serum filgrastim level peak was on day 2 of the mobilization regimen with a median level of 226 ng/ml (range 35 to 1123 ng/ml), thus preceding the increase of CD34+ cells in blood. The main adverse events were WHO grade 1 and included headaches, bone pain and transient elevations of alkaline phosphatase and lactate dehydrogenase. Conclusion: PBSC mobilization with a single dose of pegfilgrastim is feasible for healthy donors. The graft composition was comparable to that obtained with the conventional regimen of short-term G-CSF. Long-term follow-up of healthy donors treated with pegfilgrastim should be further investigated. Disclosures: No relevant conflicts of interest to declare.

NK-cells have been shown to play a pivotal role in haploidentical hematopoietic cell transplantation (HHCT) for engraftment, GvL effects and to combat infectious complications. Different strategies have been employed to hasten NK-cell recovery after HHCT. Here we compare the immune recovery of 17 patients after CD34 selected HHCT receiving additional adoptive CD3-depleted CD56-enriched NK cells 2 days after HHCT (adoptive NK-cells), with 18 patients receiving CD3/CD19 depleted grafts (CD3/CD19) for HHCT. Transplantations were performed at two different institutions with a median follow-up of >1 year. Conditioning consisted of 12 Gy TBI, thiotepa (10mg/kg), fludarabine (150 mg/m2) and OKT3 (day −4 to +2) in the group receiving CD34 selected grafts and adoptive NK-cell transfusions. All patients in the CD3/CD19 group received conditioning with fludarabine (150–200 mg/m2), thiotepa (10 mg/kg), melphalan (120 mg/m2) and OKT-3 (day −5 to +14). No postgrafting immunosuppression was used in both groups. Seven out of the 17 patients in the adoptive NK-cell group received IL-2 activated NK cells. Median age was 37 years in the adoptive NK-cell group compared to 40 years in the CD3/CD19 group. Diagnoses in the adoptive NK-cell group included AML (n=10), ALL (n=3), CML (n=2), and Hodgkin’s disease (n=1) and MDS (n=1). Diagnoses in the CD3/CD19 group were AML (n=10), ALL (n=5), NHL (n=1), CML (n=1) and multiple myeloma (n=1). The grafts contained a median of 12.5x10E6 CD34+ cells/kg and 1.1×10E4 CD3+ cells/kg in the CD34 selected group versus 9.2×10E6 CD34+cells/kg and 2.3×10E4 CD3+cells/kg in the CD3/CD19 group. The number of transferred CD56+ cells was 8.3×10E6/kg in the adoptive NK-cell group and 7.2×10E7/kg cells in the CD3/CD19 group. Hematopoietic recovery was similar in both groups. Among the patients receiving adoptive NK-cells we observed a striking difference in immune recovery between the patients receiving IL2-activated and those treated with non-activated NK cells: patients receiving activated NK cells showed significantly lower numbers of NK- and T cells during the first months post transplant (p=

To improve the antileukemic effectiveness of haploidentical stem cell transplantation (HSCT) and strengthen the cell mediated immune response, we have adoptively transferred high numbers of alloreactive donor NK cells during the early phase after transplantation. In addition, we activated the transferred NK-cell population ex vivo in short term cultures with high doses of IL-2 in order to enhance the antileukemic activity and inhibit the occurrence of severe GvHD. Method: In a phase-II study, 17 patients (10 AML, 1 MDS, 1 HD, 2 CML, 3 ALL, median age 37 yrs, range 17–48 yrs) were transplanted in late phases of their disease (6 pts. as 2nd transplantation) and received purified NK cells from their haploidentical donors at day +2 after HSCT. Conditioning consisted of 12 Gy fTBI, Thiotepa (10mg/kg), Fludarabine (5 x 30 mg/qm) and OKT3 (day −4 to +2). NK cells were isolated from the CD34- fraction using an automated two-step procedure of CD3+ depletion and subsequent CD56+ selection. Seven patients received activated NK cells and 10 patients received unstimulated NK cells. Cells were activated by 16h incubation with IL-2 (500U/1x107 cells). Results: After selection and subsequent overnight activation of the NK cells with IL-2 (7 out of 17 patients), a mean number of 8.3 x 106/kg CD56+CD3− NK cells was transferred at day 2 after transplantation. The purity was 76%, due to contaminating CD56+ monocytes. The mean number of contaminating CD3+ cells in the transfused NK product was 2.1 x 104/kg. Activation of donor NK cells with IL-2 resulted in an increase of cytotoxic activity, when cells were tested against target cell lines. No differences in yield or number of contaminating T cells were observed between IL-2 activated and not activated NK cells. No severe acute toxicity attributable to NK cell infusion was observed in both groups of patients. Comparing the rate of high-grade GvHD revealed an interesting result. Whereas only one patient developed GvHD ≥ grade II after treatment with IL-2 activated NK cells, seven out of ten patients showed GvHD ≥ grade II after transfer of non-activated NK cells (p

NK cell alloreactivity can mediate strong graft versus leukemia (GvL) effects following haploidentical hematopoietic stem cell transplantation (HSCT). In an attempt to further improve the antileukemic effectiveness of this approach, we have adoptively transferred high numbers of alloreactive donor NK cells during the early phase after transplantation. Method: In a phase-II study, 10 patients (6 AML, 1 MDS, 1 HD, 1 CML, 1 ALL, median age 38 yrs, range 17–48 yrs) were transplanted in late phases of their disease (5 pts. as 2nd transplantation) and received purified NK cells from their haploidentical donors at day +2 after HSCT. Conditioning consisted of 12 Gy fTBI, Thiotepa (10mg/kg), Fludarabine (5 x 30 mg/qm) and OKT3 (day −4 to +2). Two patients received a reduced conditioning with Fludarabine and OKT3 alone. NK cells were isolated from the CD34- fraction using an automated two-step procedure of CD3+ depletion and subsequent CD56+ selection. Results: No severe technical problems occurred and a mean of 12,1 x 10E8 (16,74 x10E6/kg, range 6,12 to 27,2 x10E6/kg) CD34+ cells was selected in high purity (95,9 %) with a very low T-cell content (mean 1,83 x10E4/kg CD3+ cells, 4,5 Log depletion). A mean of 5,7 x10E8 (6,7 x 10E6/kg) CD56+CD3− NK cells was transferred (yield 70,36%, purity 70%). The mean number of contaminating CD3+ cells was 3,2 x10E4/kg (3,6 Log depletion).No severe acute toxicity attributable to NK cell infusion was observed. Hematopoietic recovery was fast with leukocytes > 1/nl between day 5 and 11. Seven patients developed early grade II GvHD of the skin which promptly resolved after CSA and steroids. One patient developed late graft rejection five months after reduced conditioning, received a 2nd graft from a different donor, engrafted but unfortunately died due to pneumonia one month after the second transplantation. One pt with CML died due to adenovirus infection at day 140. One pt. with HD in 5th CR died due to pneumonia at day 85. One patient showed a relapse of the AML three months after transplant. She received a DLI with NK cells form the donor but died due to disease progression one month after. Interestingly, leukemic cells from this patient proved to be resistant to donor NK cell mediated lysis. Five of 9 patients are alive and in CCR with a median follow up of 192 days, the two patients with the longest follow up are in very good condition and free of GvHD at day +1271 and +1019. Our data show for the first time that the early adoptive transfer of high numbers of HLA-mismatched NK cells is safe and feasible.