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Background Human bone marrow contains hematopoietic stem cells and stroma cells known as mesenchymal stem cells (MSC). MSC are cells with the morphological features of fibroblasts, which, in addition to their nursing function for hematopoietic stem cells, retain the ability to differentiate into cartilage, bone, fat, muscle, and tendon and have an important immunmodulatory function. To understand in more detail hematopoietic engraftment and immune modulation after hematopoietic cell transplantation, we investigated the ability of donor MSC to engraft after hematopoietic cell transplantation in dependency to the conditioning regimen (myeloablative vs. reduced intensity) and source of the graft (bone marrow vs. peripheral blood). Methods Bone marrow MSC of 12 patients were analyzed, a median of 23.4 (range 0.9-137.8) months after human leukocyte antigen matched but gender mismatched bone marrow transplantation after myeloablative conditioning (n=4) or peripheral blood cell transplantation after myeloablative (n=4) or reduced intensity conditioning (n=4). MSC were characterized by morphology, positivity for CD 105+, CD73+, CD 44+, and CD 90+, and by their capacity to differentiate into adipocytic and osteogenic cells. Recipient and donor origins were determined by fluorescent in situ hybridization for sex chromosomes. Results While overall blood and bone marrow chimerism was 100% donor type, MSC remained in all patients of recipient origin (>96%). There was no difference between patients receiving bone marrow and peripheral blood grafts, nor was any difference observed between patients receiving full intensity in comparison with reduced intensity conditioning. Conclusions We conclude that MSC remain of host type irrespective of the conditioning regimen and graft source.

A 35-year-old female patient was diagnosed with acute myeloid leukemia with multiple genetic aberrations [48 XX, del(3)(q21), +6, t(11;15)(q23;q15), +21] including an 11q23/MLL abnormality. The patient achieved a complete remission after one induction chemotherapy cycle. After three courses of consolidation, a matched unrelated hematopoietic cell transplantation (HCT) was performed. Following an upper respiratory tract infection 7 years after transplant, her blood counts declined to leukocytes of 1 × 109/l, platelets of 51 × 109/l and hemoglobin of 7.5 g/dl. A bone marrow aspirate revealed 55% leukemic blasts carrying the unfavorable genetic aberrations seen at initial diagnosis (11q23/MLL). In the absence of any disease-specific treatment, the leukemic blasts cleared from the bone marrow within 6 days after diagnosis of relapse and peripheral blood counts returned to normal. Molecular analysis of the 11q23/MLL rearrangement was used to evaluate minimal residual disease, which became undetectable in repetitive FISH analyses. This is the first report of spontaneous remission in a patient with initially a multiaberrant leukemic cell clone and a proven 11q23/MLL abnormality at relapse after HCT.

The optimal consolidation chemotherapy in AML patients >60 years has yet to be defined in detail. Although age-adjusted induction chemotherapy results in CR rates comparable to those in younger patients, relapse remains the major hurdle to successful treatment. While the role of stem cell transplantation (HSCT) in elderly patients is currently being evaluated in randomized studies, we focus here on the intensity of consolidation chemotherapy. Patients data from the elderly AML trials OSHO 1997 (n=410) and OSHO 2004 (n=733) were pooled and analyzed. These protocols have identical inclusion/exclusion criteria and induction chemotherapy, but differ in the intensity of consolidation therapy. In the OSHO 1997 trial, Ara-C 120 mg/m2 bid was given from day 1-5 and mitoxantrone 10 mg/m2 from day 1-2 as consolidation. In the OSHO 2004 an intensified consolidation using Ara-C 500 mg/m2 bid on day 1/3/5 was applied together with mitoxantrone as used in the OSHO 1997 study. Of the 1143 patients, 689 entered CR (60% in the OSHO 1997 and 61% in the OSHO 2004) and 536 (OSHO 1997, n=242, OSHO 2004, n=294) did not receive HSCT as consolidation. The analysis concentrated on the dose of AraC used in the consolidation for this elderly population and on the cycles of consolidation applied. Patient characteristics were compared using chi-square test for categorical data and Wilcoxon rank sum test for continuous data. OS was analyzed using the Kaplan-Meier method, and univariate comparisons were made by means of the log-rank test. Cox regression was used to find any association between consolidation chemotherapy considered as a time-dependent covariate on Overall Survival (OS) or Relapse Incidence (RI). RI and Non Relapse Mortality (NRM) were calculated using the competing risk method, and the Gray test was applied to compare differences. Multivariate modeling was performed by Cox regression analyses with a forward selection method. Median ages in the AML studies were 66 (60-81) years and 69 (60-85) years for the OSHO 1997 and OSHO 2004, respectively. Patients characteristics were balanced except for age and Karnofsky score (p< .0005) and a trend towards more intermediate and high risk karyotypes, more female and less WBC in the OSHO 2004 compared to the OSHO 1997 study (p=0.06). OS at 15 years was 14±2% in all patients with no difference between the two consolidations, but strong dependence on cytogenetic risk factors. In multivariate analyses risk factors for survival were high/intermediate risk karyotypes, male gender, non de-novo AML and less than two consolidations. Patients with two consolidations had better OS than patients with one or no consolidations in the pooled group and in each of the two protocols with no difference between OSHO 1997 and OSHO 2004. Relapse incidence amounted to 79±2% and NRM 10±04% at 15 years with no difference between the two protocols. Relapse incidence was dependent upon cytogenetic risk and the number of consolidations applied in a multivariate model. There were no risk factors predicting TRM in multivariate analysis. Our analysis of patient characteristics according to the number of consolidations showed the distribution of consolidation therapies to be 15.2%, 28.0%, 56.6% and 14.2%, 32.3% and 53.4% for 0, 1 and 2 consolidations in the OSHO 1997 and OSHO 2004 respectively (n.s.). Higher age, higher risk cytogenetics, non-de novo AML type, less CR after one induction cycle and lower WBC count at diagnosis were characteristic of patients receiving none or one as compared to two consolidation therapies. The multivariate analysis revealed cytogenetics and gender as independent risk factors, but not the application of one as opposed to two consolidation treatments. The increase of AraC dose in the OSHO 2004 was unable to either increase survival or improve relapse incidence in the cohort of elderly patients. TRM was not different between the OSHO 1997 and 2004 studies. However, the application of one or two consolidation cycles had a significant impact on survival that was not due to decreased relapse incidence after normalization for risk factors. Interestingly, just above 50% of patients received 2 consolidations as proposed in the protocol with no statistically significant difference between OSHO 1997 and OSHO 2004. Patients receiving fewer consolidation therapy cycles are older, have more non-de novo AML and lower WBC count. Disclosures: Hochhaus: Novartis: Consultancy, Honoraria, Research Funding, Travel Other; BMS: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Ariad: Consultancy, Honoraria.

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=

Objective Bone marrow contains pluripotent mesenchymal stem cells (MSC) that form bone, cartilage, adipose tissue and muscle. These cells are not immunogenic and escape recognition by alloreactive T cells and natural killer cells. MSC can be readily isolated from bone marrow and expanded ex vivo without modification in phenotype or loss of function. They are capable of differentiating along multiple mesenchymal lineages, play a crucial role in the bone marrow microenvironment and have profound immunosuppressive properties. In the present investigation we analysed the antiproliferative capacity of MSC using primary and secondary immune responses in vitro. Methods MSC were isolated from heparinized bone marrow of healthy donors (n=5). Bone marrow aspirates were layered onto a Percoll cushion (density 1,073g/ml) and the MSC-enriched mononuclear fraction was collected, washed and resuspended in Dulbecco modified Eagle medium with low glucose and 10% fetal bovine serum. The cells were plated at 2x105/cm2 and maintained at 37°C in a humidified atmosphere and subcultured prior to confluency. MSC stained positive for specific markers as SH2, SH3 and SH4 but were negative for CD14, CD34 and CD45. Immunsuppressive effects were assessed by adding third party MSC to primary mixed lymphocyte reactions (MLR) in decreasing concentrations (10%, 1%, 0,1%) on days 0–5. Cell proliferation was measured on day 6 by means of an 18-hour pulse with 3H-thymidine (3H-TdR). Secondary MLRs were performed by restimulating MSC co-cultured MLRs with the primary PBMC on day 10. In addition secondary MLRs were also tested in presence of MSC and cultured for 1, 2, 3 and 6 days in 96-well plates and proliferation was detected by 3H-TdR uptake. Results Primary MLRs show significant inhibition of proliferation with 10% MSC added on day 0 (85% inhibition), 1 (69% inhibition) or 2 (70% inhibition) (p Conclusions Our findings emphasize the immunosuppressive effects of MSC. We observed that MSC-induced inhibition of primary MLRs was reversible in secondary MLRs. This clearly demonstrates that MSC do not induce tolerance and could be used in a clinical setting due to their immunomodulatory properties.

In the last years, focus of regenerational studies has pointed on mesenchymal stem cells (MSC) and their ability to differentiate into several mesenchymal tissues. MSC have been shown to play a pivotal role in the microenvironment of bone marrow cells and in the modulation of immune response as they can suppress lymphocytic proliferation in vitro. Moreover, some animal studies have suggested they could favor the proliferation of malignant cell clones in solid tumor models. Their role in hematological malignancies, however, remains to be further elucidated and little is known about the influence of MSC in the development and maintenance of the malignant clone in chronic myeloid leukemia (CML). This disease is characterized by the presence of the Philadelphia (Ph) chromosome, a fusion product generated by the reciprocal translocation between chromosomes 9 and 22. Previous reports showed that hepatocytes precursors, found in the liver of CML patients carry the Ph translocation. Our intent was to elucidate whether MSC isolated from patients with CML in different stages of the disease originate from the malignant clone. To this purpose bone marrow aspirates of 11 patients with CML were obtained after informed consent. Five patients were analyzed at diagnosis, two after allogenic stem cell transplantation, three on treatment with the tyrosine kinase inhibitor imatinib and one on treatment with interferon alpha in combination with hydroxyurea. MSC were then generated as previously described. Briefly, cells were isolated by density gradient methods, resuspended in RPMI1640 medium containing 10% fetal bovine serum and plated in culture flasks to adhere. After 4–5 weeks of culture cells were collected and characterized by the expression of several surface markers in a fluorescence activated cell sorter (FACS). The presence of the Ph chromosome was assessed by both fluorescence in situ hybridization (FISH) and polymerase chain reaction (nested PCR). Moreover whole bone marrow was analyzed and results compared with those obtained in the MSC population. MSC showed a typical morphological pattern, growing to confluence after a few weeks of culture and appearing as an adherent, spindle shaped cell layer. In FACS they stained positive for SH2 and SH3 and did not express CD34, CD45 and CD14. MSC were then analyzed by FISH using probes for BCR-ABL. We could not detect the Ph translocation in any of the analyzed patients, though it was present at variuos levels in the remnant bone marrow cells. Results did not change, if expression of BCR-ABL was measured by high sensitivity RT-PCR. Our results showh that MSC of patients with CML are Philadelphia negative irrespective of the stage of disease and the treatment given, suggesting that these cells are not involved in the development of the malignancy. However, their interactions with leukemic cells as well as their role in the immune response against the tumor remains to be further characterized.