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Natural killer (NK) cells are part of a phylogenetically old defense system, which is characterized by its strong cytolytic function against physiologically stressed cells such as tumor cells and virus-infected cells. Their use in the treatment of hematological malignancies may be more advantageous in several ways when compared with the already established T lymphocyte-based immunotherapy. Given the different mechanisms of action, allogeneic NK cell products can be produced in a non-personal based manner without the risk of the formidable graft-versus-host disease. Advanced manufacturing processes are capable of producing NK cells relatively easily in large and clinically sufficient numbers, useable without subsequent manipulations or after genetic modifications, which can solve the lack of specificity and improve clinical efficacy of NK cell products. This review summarizes the basic characteristics of NK cells and provides a quick overview of their sources. Results of clinical trials in hematological malignancies are presented, and strategies on how to improve the clinical outcome of NK cell therapy are discussed.

Neurodegenerative disorders present a broad group of neurological diseases and remain one of the greatest challenges and burdens to mankind. Maladies like amyotrophic lateral sclerosis, Alzheimer's disease, stroke or spinal cord injury commonly features astroglia involvement (astrogliosis) with signs of inflammation. Regenerative, paracrine and immunomodulatory properties of human mesenchymal stromal cells (hMSCs) could target the above components, thus opening new therapeutic possibilities for regenerative medicine. A special interest should be given to hMSCs derived from the umbilical cord (UC) tissue, due to their origin, properties and lack of ethical paradigms. The aim of this study was to establish standard operating and scale-up good manufacturing practice (GMP) protocols of UC-hMSCs isolation, characterization, expansion and comparison of cells' properties when harvested on T-flasks versus using a large-scale bioreactor system. Human UC-hMSCs, isolated by tissue explant culture technique from Wharton's jelly, were harvested after reaching 75% confluence and cultured using tissue culture flasks. Obtained UC-hMSCs prior/after the cryopreservation and after harvesting in a bioreactor, were fully characterized for "mesenchymness" immunomodulatory, tumorigenicity and genetic stability, senescence and cell-doubling properties, as well as gene expression features. Our study demonstrates an efficient and simple technique for large scale UC-hMSCs expansion. Harvesting of UC-hMSCs' using classic and large scale methods did not alter UC-hMSCs' senescence, genetic stability or in vitro tumorigenicity features. We observed comparable growth and immunomodulatory capacities of fresh, frozen and expanded UC-hMSCs. We found no difference in the ability to differentiate toward adipogenic, osteogenic and chondrogenic lineages between classic and large scale UC-hMSCs expansion methods. Both, methods enabled derivation of genetically stabile cells with typical mesenchymal features. Interestingly, we found significantly increased mRNA expression levels of neural growth factor (NGF) and downregulated insulin growth factor (IGF) in UC-hMSCs cultured in bioreactor, while IL4, IL6, IL8, TGFb and VEGF expression levels remained at the similar levels. A culturing of UC-hMSCs using a large-scale automated closed bioreactor expansion system under the GMP conditions does not alter basic "mesenchymal" features and quality of the cells. Our study has been designed to pave a road toward translation of basic research data known about human UC-MSCs for the future clinical testing in patients with neurological and immunocompromised disorders. An industrial manufacturing of UC-hMSCs next will undergo regulatory approval following advanced therapy medicinal products (ATMP) criteria prior to clinical application and approval to be used in patients.

The care for younger patients with acute myeloid leukaemia (AML) requires fully equipped medical facilities that have routine experience with the management of this aggressive disorder. Two indepen...

Introduction: Daratumumab (Dara) is an anti-CD38 monoclonal antibody representing a novel treatment agent for multiple myeloma (MM). Nonetheless, several studies have reported a Dara-related impairment of CD34+ hematopoietic stem cell (HSC) mobilization and post-autologous stem cell transplantation (ASCT) complications, including low yields of mobilized HSCs and delayed neutrophil engraftment. Impact of Dara on the mobilization process and HSCs remains poorly understood even though sufficient yields of CD34+ cells are necessary for a successful ASCT and subsequent patient recovery. Aims: To compare the effect of the Dara-containing (Dara-Bortezomib-Dexamethasone [D-VCd]) and conventional (Bortezomib-Thalidomide-Dexamethasone [VTd]) therapy on CD34+ HSCs. Methods: Transplant eligible MM patients were treated with D-VCd or VTd induction regimen followed by a cyclophosphamide + G-CSF mobilization and a high-dose melphalan D -1 before ASCT. Flow cytometry (FCM) screening of CD34+ subsets was performed in the bone marrow (BM) or apheresis product (AP) at three consecutive time points: 1) diagnostic BM (DG), 2) mobilization AP (MOB), 3) a day prior ASCT BM (D-1). Furthermore, RNA sequencing (RNAseq) of sorted CD34+ cells was performed on total RNA with ribo-depletion protocol in AP after the induction. D-VCd samples had lower RNA yields thus the D-VCd or VTd groups were processed as independent batches. Results: Clinical data revealed no significant differences in mobilization (p >0.050) likely due to a small cohort sizes (D-VCd n=5 vs VTd n=9), though a trend towards worse performance in D-VCd was observed. Median CD34+ cell yield was 3.08 vs 10.56 x 10 6/kg. Platelet recovery of >20x10 9/L was D+14 vs D+12 (range: 11-18 vs 10-16). Neutrophil recovery of >0.5x10 9/L was D+12 in both groups (range: 11-17 vs 11-12). In FCM analysis, DG (n=14), MOB D-VCd (n=5) vs VTd (n=9), D-1 D-VCd (n=7) vs VTd (n=15) were compared. CD34+ frequency (Fig. 1A) difference in MOB D-VCd vs VTd was insignificant (median: 1.15% vs 1.89%), whereas CD34+ fraction dropped in D-1 D-VCd (median: 0.52% vs 0.72%, p=0.027), albeit there was no significant reduction in D-1 D-VCd vs initial DG (median: 0.52% vs 0.45%). Differences in the distribution of certain HSC subsets were detected in the CD34+ pool (Fig. 1B-E). Frequency of multipotent progenitors (MPPs) (Fig. 1B) was increased in MOB D-VCd (median: 82.1% vs 66.2%, p=0.004). Frequency of lympho-myeloid-primed progenitor + granulocyte-monocyte progenitor (LMPP+GMP) (Fig. 1C) subset was reduced in D-VCd in both MOB (median: 1.7% vs 16.9%, p=0.042) and D-1 (median: 5.3% vs 14.0%; p=0.026). Erythro-myeloid progenitors (EMPs) (Fig. 1D) were reduced in MOB D-VCd (median: 10.7% vs 19.5%, p=0.042), while the frequency of EMPs increased in D-1 D-VCd (median: 20.8% vs 12.4%, p=0.045). No considerable differences were found in the expression of adhesion molecules CD44/HCAM or CD184/CXCR4. CD38 was strongly diminished in the whole D-VCd CD34+ fraction of MOB and D-1. To understand whether the differences in the mobilization efficacy after D-VCd induction were reflected in the expression profile of mobilized CD34+ cells, differential expression analysis was performed. Overall 133 significantly deregulated genes (p(-)1) between cohorts (D-VCd n=5 vs VTd n=5) were revealed (Fig. 2). Pathway analysis showed cellular response and localization as the most deregulated categories. The list of deregulated genes contained 25% of non-coding RNAs, some of which were linked to a protein localization in the cell (RN7SL1/2). The expression of adhesion molecules was inspected independently. Out of 59 HSC hallmark genes, only 8 were significantly altered in D-VCd. Interestingly, the main homing molecule CXCR4 seemed to be downregulated in D-VCd, while integrins A3 and B4 were upregulated. Conclusions: Despite the limited cohort sizes, a prospective trend of delayed neutrophil and platelet recovery was observed after D-VCd therapy. FCM analysis revealed a significant reduction of CD34+ subsets responsible, among others, for a reconstitution of neutrophils and megakaryocytes. A strong signal in transcriptome data which would potentially explain differential mobilization in D-VCd cohort was not detected, nevertheless, several genes with adhesive/homing and stem cell differentiation function were indeed altered. The results warrant further investigation. Figure 1 Figure 1. Disclosures Hajek: BMS: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Novartis: Consultancy, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharma MAR: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

I ntroduction Extramedullary disease (EMD) is a less frequent manifestation of multiple myeloma (MM), where MM plasma cells become independent of the bone marrow (BM) microenvironment and infiltrate other tissues and organs. The incidence of EMD is increasing and is associated with worse prognosis and drug resistance. The specific and efficient treatment is lacking. Therefore, a better understanding of EMD pathogenesis is desperately needed. Aims To identify biological pathways leading to EMD development and to evaluate therapeutic targets in EMD plasma cells with further focus on EMD tumor microenvironment to reveal presence of effector immune cells that are crucial for immunotherapy. Methods To identify EMD specific genes, FACS/MACS sorted aberrant plasma cells were collected from: i) fresh 11 EMD relapse tumors for which we had ii) 7 corresponding cryopreserved paired BM samples from the time of MM diagnosis (NDMM), iii) 9 unpaired fresh NDMM without EMD confirmed by PET-CT and iv) 6 unpaired fresh relapsed MM (RRMM). For library preparation, we used total RNA with rRNA depletion protocol and Illumina sequencing. Residual rRNA was filtered out by SortMeRNA. Differential expression analysis was performed using Salmon for read mapping and quantification and Deseq2 package. For single-cell RNAseq we used 10x Genomics technology for sequencing and CellRanger and Seurat for data processing and analysis. Results To better understand the aggressive nature of EMD, we have analyzed bulk RNA samples (7 EMD samples plus 7 corresponding cryopreserved paired BM samples from the time of MM diagnosis). Our preliminary analysis revealed a unique EMD profile (Fig 1A) with 423 up-regulated and 421 down-regulated genes in EMD samples (adjusted p-value < 0.1; absolute fold change > 1.5), with G2M checkpoint proteins being the most enriched hallmark pathways pointing to higher proliferation of EMD cells. EMD down-regulated genes mainly belong to genes of the adaptive immune response which together with lower immunoglobulin production suggest loss of mature plasma cell function. Among the top genes uniquely overexpressed in EMD (versus RRMM or NDMM) were SCD and ELOVL6 that regulate crucial steps in unsaturated fatty acids synthesis. Also their transcription factor SREBF1 was significantly up-regulated. The importance of these genes in EMD pathogenesis can be supported by the involvement of SREBP1 in stem cell differentiation and mediation of bortezomib resistance by ELOVL6 (Yi et al. 2018, Lipchick et al. 2021). Our dataset also revealed several deregulated lncRNA in EMD compared to NDMM. MALAT1 was highly expressed, however, we did not confirm results by Handa et al. 2017 showing lncRNA MALAT1 as upregulated in EMD. Furthermore, we aimed to evaluate expression of known immunotherapy MM targets being currently in use or under investigation. We compared the information about expression level in EMD vs paired NDMM, with unpaired NDMM without EMD lesion confirmed by PET/CT, and with RRMM. The analysis revealed a decrease in the expression of several antigens commonly used in anti-MM immunotherapy (e.g. CD38, SLAMF7, BCMA or PDL1) on EMD PCs (Fig 1B). Intriguingly, our data show EMD specific elevated expression of EZH2 gene being promising target in preclinical MM investigation which can prove efficient especially for the aggressive MM stage - EMD. Effective immunotherapy depends on the presence of effector immune cells. Therefore, we have evaluated immune cell types and their proportion in EMD tumors. Using flow cytometry we identified T and NK cells as the only immune cell subsets present in EMD tumors (median 0.9% and 0.5%, respectively). Single-cell RNAseq analysis of two EMD samples supported these findings. Conclusions Here, we present up to our knowledge the worldwide largest cohort of 11 EMD samples (including 7 longitudinal pre-EMD/EMD samples) analysed using RNAseq with focus on biological pathways and dysregulation of particular genes leading to EMD development. Drop of expression of several known drug targets may suggest limited efficacy of the modern treatment in EMD as already presented by Jelinek et al., 2021. Importantly, we are also providing the initial insight into the microenvironment (including single-cell RNA analysis) of EMD tumors, where we detected presence of T cell and NK cells in very limited numbers. Figure 1 Figure 1. Disclosures Hajek: Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Novartis: Consultancy, Research Funding; Pharma MAR: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Cyclophosphamide (Cy) plus granulocyte-colony stimulating factor (G-CSF) is currently a standard regimen for hematopoietic stem cell (HSC) mobilization in patients with multiple myeloma (MM). However, cytarabine (AraC) in intermediate doses plus G-CSF seems to have a higher mobilization efficacy. The aim of this study was to retrospectively compare mobilization using AraC and Cy. Thirty consecutive MM patients were mobilized by Cy + G-CSF, and the subsequent 40 patients by AraC + G-CSF. Both groups were comparable. The target yield of 10 × 106 CD34+ cells/kg (for tandem and 2 additional transplantations) was achieved in 98% (AraC) and 57% (Cy) of patients (p

Loss of heterozygosity is considered to be the most common type of tumour-specific somatic mutation of the human leucocyte antigens (HLA) genes in patients with haematological malignancies. Nevertheless, subtle DNA sequence changes, namely short insertions/deletions, may also abolish the expression of HLA molecules and interfere with routine HLA typing. Two male patients with acute myelogenous leukaemia (AML) were indicated for the search of a suitable donor for allogeneic haematopoietic stem cell transplantation (aHSCT). The patients and their relatives were initially HLA typed by serological and DNA techniques at a low-resolution level. The HLA high-resolution (HR) type was obtained by means of sequencing-based typing (SBT). In both cases, anomalous frameshifts in the sequence were observed in the HLA-B gene, namely in exon 3 (Case 1, heterozygous deletion of two bases) and exon 4 (Case 2, heterozygous insertion of two bases). In the second case, the insertion variant was associated with a loss of HLA-B8 expression. To reveal whether these sequence patterns may be caused by somatic mutations in the malignant cells, blood sample in remission (Case 1) and buccal swab sample (Case 2) were collected from the patients. In an important manner, the SBT in these germline samples revealed common HLA-B*07:02,*15:01 (Case 1) and HLA-B*08:01,*35:02 (Case 2) types with no evidence for the sequence alteration observed in the initial samples. In conclusion, the insertion/deletion sequence variants of the HLA-B gene in two patients were limited to the initial blood samples with a substantial proportion of AML cells and thus may be attributed to the somatic mutation in the malignant cells. HLA somatic mutations should be taken into account in patients with haematological malignancies to prevent HLA mistyping and inappropriate selection of an aHSCT donor.

Autologous haematopoietic SCT with PBSCs is regularly used to restore BM function in patients with multiple myeloma or lymphoma after myeloablative chemotherapy. Twenty-eight experts from the European Group for Blood and Marrow Transplantation developed a position statement on the best approaches to mobilising PBSCs and on possibilities of optimising graft yields in patients who mobilise poorly. Choosing the appropriate mobilisation regimen, based on patients' disease stage and condition, and optimising the apheresis protocol can improve mobilisation outcomes. Several factors may influence mobilisation outcomes, including older age, a more advanced disease stage, the type of prior chemotherapy (e.g., fludarabine or melphalan), prior irradiation or a higher number of prior treatment lines. The most robust predictive factor for poor PBSC collection is the CD34(+) cell count in PB before apheresis. Determination of the CD34(+) cell count in PB before apheresis helps to identify patients at risk of poor PBSC collection and allows pre-emptive intervention to rescue mobilisation in these patients. Such a proactive approach might help to overcome deficiencies in stem cell mobilisation and offers a rationale for the use of novel mobilisation agents.

Introduction: Medical care has recently been moving from wards to outpatient clinics due to a growing number of patients, convenient therapeutic approaches and rising emphasis on quality of life. There is an increased need for intensive monitoring of haemato-oncological patients undergoing long-lasting treatment especially when serious infectious complications are expected. Telemedicine is a great tool to keep an eye on patients spending most of their treatment at home, where reaction time plays an important role in reducing the impact of severe adverse events. Methods: We plan on enrolling 100 haemato-oncological patients undergoing treatment who are at high risk of febrile neutropenia and/or sepsis. In the comfort of their homes, selected patients will measure their body temperature, blood pressure and pulse regularly every morning, plus whenever they feel unwell. Contactless measuring is provided by a digital blood pressure monitor and an infrared thermometer. Both devices communicate via bluetooth with a mobile HUB (cell phone), which then encrypts the information and forwards it to the National Monitoring Center. Physicians have real-time access to the measured values through a web portal using any device with an internet connection. Importantly, values registered outside the individually set-up range are immediately sent by SMS to a designated physician, who can then promptly react. Results: In our pilot project, we enrolled 21 patients (33-80 y; median 60 y) including 9 treated for acute leukemia, 5 for multiple myeloma, 4 for chronic lymphocytic leukemia, 1 for myelodysplastic syndrome, 1 for malignant lymphoma and 1 for aplastic anemia. The median duration of monitoring was 6 months (1-16). One thousand, six hundred and twenty-three critical values of systolic (91 mmHg) blood pressure in 19 patients were registered. High blood pressure in patients with inadequate antihypertensive therapy was the most frequent warning. Forty-four critical values of body temperature (>37.9°C) in 6 patients were registered, two cases with concomitant low blood pressure. After a telephone conversation with a physician, ambulatory care was recommended 12 times. Two cases of febrile neutropenia were detected, with the patients instantly hospitalized without progression into sepsis. Four cases of infectious complications (genital herpes, upper respiratory tract infection) with the need for anti-infective therapy and 6 cases of viral infections with only symptomatic treatment indicated were also addressed. Conclusion: Even in a small cohort of patients we demonstrated that the remote monitoring of body temperature, blood pressure and pulse at home enables the early detection of infections and prompt provision of adequate treatment. An evaluation of the financial savings on medical expenses might also be a useful aspect to revise. Disclosures Hajek: Celgene: Honoraria, Other: Consultant or advisory relationship, Research Funding; AbbVie: Other: Consultant or advisory relationship; Bristol-Myers Squibb: Honoraria, Other: Consultant or advisory relationship, Research Funding; Novartis: Other: Consultant or advisory relationship, Research Funding; PharmaMar: Honoraria, Other: Consultant or advisory relationship; Takeda: Honoraria, Other: Consultant or advisory relationship, Research Funding; Janssen: Honoraria, Other: Consultant or advisory relationship, Research Funding; Amgen: Honoraria, Other: Consultant or advisory relationship, Research Funding.

BACKGROUND: Plerixafor with granulocyte–colony-stimulating factor (G-CSF) has been shown to enhance stem cell mobilization in patients with multiple myeloma and lymphoma with previous mobilization failure. In this European named patient program we report the experience in insufficiently mobilizing patients diagnosed with nonhematologic diseases. STUDY DESIGN AND METHODS: Thirty-three patients with germ cell tumor (n = 11), Ewing sarcoma (n = 6), Wiscott-Aldrich disease (n = 5), neuroblastoma (n = 4), and other nonhematologic diseases (n = 7) were included in the study. Plerixafor was limited to patients with previous or current stem cell mobilization failure and given after 4 days of G-CSF (n = 21) or after chemotherapy and G-CSF (n = 12) in patients who mobilized poorly. RESULTS: Overall, 28 (85%) patients succeeded in collecting at least 2 × 106/kg body weight (b.w.) CD34+ cells (median, 5.0 × 106/kg b.w. CD34+ cells; range, 2.0 × 106-29.5 × 106/kg b.w. CD34+ cells), and five (15%) patients collected a median of 1.5 × 106/kg b.w. CD34+ cells (range, 0.9 × 106-1.8 × 106/kg b.w. CD34+ cells). Nineteen patients proceeded to transplantation. The median dose of CD34+ cells infused was 3.3 × 106/kg b.w. (range, 2.3 × 106-6.7 × 106/kg b.w. CD34+ cells). The median numbers of days to neutrophil and platelet engraftment were 11 (range, 9-12) and 15 (range, 10-25) days, respectively. CONCLUSION: These data emphasize the role of plerixafor in combination with G-CSF or chemotherapy and G-CSF as an effective mobilization regimen with the potential of successful stem cell collection. Accordingly, plerixafor seems to be safe and effective in patients with nonhematologic diseases. Larger prospective studies are warranted to further assess its use in these patients.

A proportion of patients with multiple myeloma (MM) who have already undergone autologous stem cell transplantation (autoSCT) might benefit from a further transplantation. For this, they might need to undergo another round of stem cell mobilization. We analyzed retrospectively the outcomes of stem cell mobilization with plerixafor and granulocyte colony-stimulating factor (G-CSF) in a group of 30 patients who had undergone autoSCT previously, and in 46 other patients. The previously transplanted patients were significantly different from the remaining patients with respect to the intensity and number of previous therapies. We observed that the median peripheral blood concentration of CD34+ cells after the first administration of plerixafor was lower in previously transplanted (19 cells/μL) than in other patients (30 cells/μL, P < 0.05). Despite a comparable number of apheresis sessions being performed, the median total yield of CD34+ cells was significantly lower in the previously transplanted than in the remaining patients (2.8 × 10(6) cells/kg vs. 4.2 × 10(6) cells/kg, P < 0.05). However, successful collection of at least 2.0 × 10(6) CD34+ cells/kg was achieved finally in a similar proportion of previously transplanted and other patients (70% vs. 82.6%). Our data suggest that stem cell mobilization with plerixafor and G-CSF might overcome the negative effect of prognostic factors for poor stem cell mobilization in patients with MM who have undergone autoSCT previously.

Objectives: The purpose of this study was to determine the impact of autologous transplantation of mononuclear bone marrow cells on myocardial function in patients with left ventricular (LV) dysfunction due to an acute myocardial infarction. Methods: The randomized study included 82 patients with a first acute myocardial infarction treated with a stent implantation. This presentation is a subanalysis of 47 patients with left ventricular dysfunction–EF (ejection fraction) ≤ 40%. Group H patients (n = 17) received higher number (100,000,000) of cells; Group L patients (n = 13) received lower number (10,000,000) of cells. The patients of control Group C (n = 17) were not treated with cells. The Doppler tissue imaging and single photon emission computed tomography were performed before cell transplantation and 3 months later. Results: At 3 months of follow-up, the baseline EF of 35%, 36%, 35% in Groups H, L, and C increased by 6% (P < 0.01 vs. baseline), 5% (P < 0.01 vs. baseline), and 4% (P = NS vs. baseline), respectively, as assessed by single photon emission computed tomography (P = NS between groups). The baseline number of akinetic segments of 6.9, 7.0, and 6.2 in H, L, and C groups decreased by 1.7 (P < 0.01 vs. baseline), 1.5 (P < 0.01 vs. baseline), and 0.7 (P = NS vs. baseline, P = NS between groups), respectively, as demonstrated by echocardiography. Conclusion: In our study, the statistically important effect of transplantation of mononuclear bone marrow cells on myocardial function was not found. Only an insignificant trend toward the improvement of global LV EF fraction was found at 3-month follow-up.

Aims: Limited regenerative potential of cardiomyocytes (CMs) causes irreversible changes in heart tissue during pathological processes. However bone marrow mononuclear cells (BM-MNCs) can migrate to this tissue, incorporate to the area of dead or missing myocytes, and improve the global heart function. The mechanism of BMMSCs’ incorporation and interaction with CMs is not clear. Our aim was to create an in vitro model which would enable to study the interaction of BM-MNCs with CMs and to make a microscopy description of these interactions. Methods and Results: CMs were isolated from adult and newborn rats. BM-MNCs were isolated from bone marrow. BM-MNCs were added to the myocyte culture. Cell-to-cell adherence and Cx43 expression were evaluated by fluorescence microscopy, Ca2+ transients were evaluated in cardiomyocyte-BMC communication under electrical stimulation by fluo-4 fluorescence measurement. Analysis of calcein transport from BM-MNCs to CMs was performed using fluorescence microscopy. Conclusions: The adherence of BM-MNCs to CMs occurred quickly and was stable. Cx43 was detected in contact zones between BM-MNCs and CMs; pairs which displayed Cx43 positivity represented less than 1% from all BM-MNC-cardiomyocyte pairs in the coculture. Conductive structures between CMs and BM-MNCs were formed and verified by imaging calcein transfer and synchronous Ca2+ transients.

We treated 40 patients with poor prognosis lymphomas. Patients with non-Hodgkin's lymphoma (NHL, n = 14) received MINE chemotherapy (mesna, ifosfamide 1330 mg/m2 and etoposide 65 mg/m2 by i.v. infusions on days 1-3, mitoxantrone 8 mg/m2 i.v. on day 1), and those with Hodgkin's disease (HD, n = 26) received VIM chemotherapy (mesna, ifosfamide 1200 mg/m2 by i.v. infusion on days 1-5, etoposide 90 mg/m2 by i.v. infusion on days 1, 3 and 5, and methotrexate 30 mg/m2 i.v. on days 1 and 5). Chemotherapy was followed by G-CSF (10 or 16 microg/kg in two divided doses daily) to mobilize PBSC. We performed 134 aphereses (median three leukaphereses per patient) starting on either day 13 (median; VIM) or day 12 (median; MINE). The median yield was 9.9x10(6) CD34+ cells/kg and 53.2x10(4) CFU-GM/kg for VIM, and 13.5x10(6) CD34+ cells/kg and 53.4x10(4) CFU-GM/kg for MINE. Except for predictable myelosuppression, no serious toxicity was seen. Response rate using MINE was 63% (18% CR, 45% PR) and using VIM 50% (17% CR, 33% PR). We conclude that VIM and MINE are effective and well-tolerated salvage regimens in patients with lymphomas and, followed by G-CSF, they also exhibit good capacity to mobilize stem cells in a predictable time interval.

The effectiveness of the novel hematopoietic stem cell mobilizing agent plerixafor was evaluated in nationwide compassionate use programs in 13 European countries. A total of 580 poor mobilizers with non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma (HL) and multiple myeloma (MM) were enrolled. All patients received plerixafor plus granulocyte CSF with or without chemotherapy. Overall, the collection yield was significantly higher in MM patients (>2.0 × 10(6) CD34+ cells/kg: 81.6%; >5.0 × 10(6) CD34+ cells/kg: 32.0%) than in NHL patients (>2.0 × 10(6) CD34+ cells/kg: 64.8%; >5.0 × 10(6) CD34+ cells/kg: 12.6%; P2.0 × 10(6) CD34+ cells/kg: 81.5%; >5.0 × 10(6) CD34+ cells/kg: 22.2%) than in NHL patients (P=0.013). In a subgroup analysis, there were no significant differences in mobilization success comparing patients with diffuse large B-cell lymphoma, follicular lymphoma and mantle cell lymphoma. Our data emphasize the role of plerixafor in poor mobilizers, but further strategies to improve the apheresis yield especially in patients with NHL are required.

Higher BMI is not a barrier to stem cell mobilization with standard doses of plerixafor and G-CSF.

The introduction of plerixafor has enabled successful collection of stem cells in the majority of patients with lymphoma or myeloma in whom previous attempts at mobilization have failed. However, a proportion of patients have been shown to be resistant to this mobilization regimen. To identify the factors that impair stem cell mobilization and collection with plerixafor, we reviewed the data for 197 patients who had undergone mobilization with plerixafor and granulocyte-colony stimulating factor in Central Europe. Predictors of mobilization failure were evaluated using logistic regression analysis. Among the 197 patients mobilized, the target of >2.0 x 10(6) CD34+ cells/kg was collected from 133 (67.5%). Our analysis revealed that previous treatment with lenalidomide, bortezomib, melphalan, radiotherapy, or autologous stem cell transplantation and regimen of plerixafor use in combination with chemotherapy had no significant effect on the efficiency of collection. In contrast, an age >= 65 years (odds ratio 0.331, 95% CI: 0.112-0.977, P < 0.05), a diagnosis of non-Hodgkin's lymphoma (odds ratio 0.277, 95% CI: 0.124-0.622, P < 0.01), and treatment with >= four chemotherapy regimens (odds ratio 0.366, 95% CI: 0.167-0.799, P < 0.05) were associated significantly with failed mobilization. The rate of successful mobilizations was decreased in patients treated with purine analogues (odds ratio 0.323, 95% CI: 0.096-1.094, P = 0.07) but increased in female patients (odds ratio 1.961, CI: 0.943-4.080, P = 0.07). Patients who are characterized by the above negative features could benefit potentially from further improvement in the mobilization strategy. Am. J. Hematol. 86:550- 553, 2011. (C) 2011 Wiley-Liss, Inc.

Plerixafor can rescue the outcome of failing chemotherapy-based stem cell mobilization. However, the optimal time for plerixafor injection in this setting has not been determined. This was investigated by retrospective analysis of data from 48 mobilizations with plerixafor, chemotherapy, and granulocyte-colony stimulating factor (G-CSF). The required yield of 2.0 x 10(6) CD34+ cells/kg was collected from 71% of patients ; the median total yield was 4.1 x 10(6) CD34+ cells/kg. Patients to whom plerixafor was administered late (>= 15 days) after chemotherapy, after a long duration (>= 13 days) of treatment with G-CSF, or when the white blood cell count was high (>= 20 x 10(9)/L) were mobilized as efficiently as other patients. Plerixafor was shown to rescue mobilizations at a comparable rate in patients with critically low levels of peripheral blood CD34+ cells (53/mL) and those with higher concentrations. These data suggest that late administration of plerixafor in the course of chemotherapy-based mobilization does not contribute to the failure of this strategy.

The immune systems of multiple myeloma patients are suppressed by the disease itself, and this immunosuppression is further enhanced by standard therapies. The aim of our study was to evaluate the effects of initial chemotherapy and a peripheral blood mobilisation regimen on T-cell population diversity. Reverse transcription–polymerase chain reaction (RT–PCR) with a new set of primers, in combination with capillary electrophoresis, was established. The methodology was used to analyse the relative expression of 27 T-cell receptor beta variable gene families (BV families) in multiple myeloma patients undergoing peripheral blood stem cell harvest. We found that the overall BV family usage in these patients was restricted; the relative expression of 10 BV families was significantly depressed in patients compared to healthy donors. These findings demonstrate that the preparative regimen for autologous stem cell transplantation affects the T-cell population in terms of the restriction of its T-cell receptor diversity.

Fludarabine has been recognized as effective treatment in patients with follicular lymphoma (FL), but can induce myelotoxicity of unknown mechanism.Myelotoxicity was assessed by cultivation of two types of hematopoietic progenitor cells: colony-forming units granulocyte-macrophage (CFU-GM) and long-term culture-initiating cells (LTC-IC). Pretreatment amounts of CFU-GM and LTC-IC were correlated to age, gender, stage of disease, bone marrow involvement, and previous therapy. Posttreatment comparison of CFU-GM and LTC-IC was performed after different regimens of chemotherapy: fludarabine-based (FND +/- R), procarbazine-based (COPP +/- R), and CHOP(cyclophosphamide, doxorubicin, vincristine, prednisone) +/- R(Rituximab).One-hundred patients (median age 55 years; 21 patients relapsed) treated for FL were analyzed. The total number of progenitor hematopoietic cells in both types of cultures varied in wide ranges; for LTC-IC between 0 and 874 cells/mL with a median of 77.71 cells/mL and for CFU-GM between 0 and 531 x 10(2) cells/mL with a median of 30.58 x 10(2) cells/mL. Bone marrow involvement, gender, stage of disease, or previous therapy had no influence on LTC-IC and CFU-GM counts. We identified an increase in LTC-IC, but not CFU-GM, associated with age (p = 0.01). Median figures for CFU-GM and LTC-IC were found to be significantly lower after FND +/- R and COPP +/- R than after CHOP +/- R therapy, compared to baseline values (p0.01).Fludarabine and procarbazine have a dramatic influence, especially on the most immature hematopoietic cells, mirrored in reduced numbers of LTC-IC. This finding is consistent with clinical observations (poor mobilization after fludarabine) and offers an insight into the mechanism of fludarabine-induced myelotoxicity.

International audience; The role of autologous hematopoietic stem cell transplantation (autoHSCT) in adult acute lymphoblastic leukemia (ALL) is still unclear. We retrospectively analyzed the results of the autoHSCT and maintenance therapy, with oral 6-mercaptopurine and methotrexate, in comparison to conventional-dose chemotherapy in the consolidation treatment of adult ALL and lymphoblastic lymphoma (LBL). The patients, with HLA identical sibling donor, underwent allogeneic transplantation, while the others were treated with autoHSCT and maintenance therapy with oral 6-mercaptopurine and methotrexate, or by conventional-dose chemotherapy (patient's decision, no autologous hematopoietic stem cells harvest). Sixty consecutive adult patients (median age 35.2 years; range 17.3 to 70.7) with ALL ( = 52), LBL ( = 7), and acute biphenotypic leukemia ( = 1) were treated in our center from 1997 to 2007. Patients treated with chemotherapy alone ( = 35) had a shorter median progression-free survival (PFS) compared to patients who underwent autoHSCT plus maintenance therapy ( = 18), 8.4 and 46.8 months, respectively ( = 0.017). Patients treated with chemotherapy alone had also a shorter median overall survival (OS) compared to patients treated with autoHSCT: 13.0 vs. 46.8 months ( = 0.046). The differences remained statistically significant even after excluding patients with Ph positivity. We can conclude that, in our case, autoHSCT followed by maintenance chemotherapy is a good option for adult patients with ALL and, in standard-risk and high-risk patients, provides more favorable OS and PFS rates compared to patients treated by chemotherapy alone. However, we are aware of the fact that our analysis may have been distorted by the fact that the analysis is retrospective, that treatment with autoHSCT was based on patient's decision, and that chemotherapy may have been administered to negatively selected patients.

Peripheral blood stem cell (PBSC) harvesting in the smallest children (weight

Megakaryocyte colony-forming unit growth is enhanced by alemtuzumab: in vitro experiments and a case report of acquired amegakaryocytic thrombocytopenic purpura

Abstract 590 Background: Myeloblative conditioning (MAC) or reduced intensity conditioning (RIC) followed by autologous or allogeneic stem cell transplantation (ASCT or AlloSCT) is established and lifesaving treatment in selected indications. The quality of life (QoL) is then very important issue for long term surviving patients. The majority of data is often based on single center evaluation with limited number of patients. Therefore we have started the cross-sectional QoL project and this analysis is based on data collected from eight transplant centers. Methods: Altogether data from 1399 patients are included in the study. The FACT-G questionnaire (Q) was used for this analysis. The questionnaire consists of four parts - physical well-being (PWB), social/family well-being (SWB), emotional well-being (EWB), functional well-being (FWB). The patients completed the Q before the transplantation (at the time of indication or at the time of admission to SCT) n=304, after ASCT n=662 and after AlloSCT n=433. Patients were divided into 7 groups – before SCT, day +100, up to 1y, 1–2y, 2–3y, 3–5y and more than 5y. The clinical characteristics were obtained from national transplant registry; the data was cleaned and updated. Wilcoxon and Kruskall-Wallis tests were used for statistical analysis. Patient′s characteristic: The ASCT and AlloSCT groups (grp.) consist of 869 and 530 pts resp. including 207 pts before ASCT and 97 before AlloSCT. There were 52.8% and 55.7% men in ASCT and AlloSCT grp. resp. The median age in ASCT and AlloSCT grp. resp. was: 55.2 and 43.2y resp., the median follow-up 4.4 and 4.5y resp. The most frequent diagnosis of ASCT group were: Non-Hodgkin′s lymphoma (NHL) 46.1%, multiple myeloma (MM) 36.6%, Hodgkin′s lymphoma (HL) 8.5%. In AlloSCT: acute myelogenous leukemia 29.4%, acute lymphoblastic leukemia 15.7%, chronic myeloid leukemia 11.5% and myelodysplastic syndrome 10.0%. Disease progression/relapse was observed in 148 ASCT (22.4%) and 61 AlloSCT (14.1%) pts. In AlloSCT group MAC was used in 33% pts and matched unrelated donor (MUD) in 59.8% pts., aGVHD gr I-II was observed in 40.3% and gr III-IV 4.2% pts, cGVHD in 37.9% pts. Results: Significant differences in overall QoL before, during and after the AlloSCT (p Conclusion: We herein demostrate on large cohorts of pts that long term survivors have significantly better QoL compared to QoL in the time of indication of the transplantation and the improvement starts from 1y after ASCT and from 2y after AlloSCT. AlloSCT survivors report better QoL compared to the ASCT survivors. The most important factors affected QoL are age, cGVHD (AlloSCT) and diagnosis (ASCT), the borderline factors are relapse after SCT and type of donor (AlloSCT). Disclosures: No relevant conflicts of interest to declare.

Complex care in the hemato-oncological ICU sometimes requires the use of CRRT. As the hemato-oncological patients have some specifications we retrospectively followed up the results of our effort in patients with acute renal failure due to veno-occlusive disease (VOD) after allogeneic hematopoietic stem cell transplantation. VOD is one of the most severe early complications in this type of patients and its mortality is high.

Background: Several populations of adult stem cells have been identified in bone marrow: hematopoietic stem cells, which are able to regenerate hematopoiesis in all of its lineages, mesenchymal stem cells, which can give rise to connective tissues (bone, cartilage and fat), and endothelial progenitor cells, which can initiate angiogenesis. Adult stem cells are found within the mononuclear cells compartment of bone marrow. Recent reports describe the effect of mononuclear bone marrow cells in reparation of ischemic tissue damage. Methods: The authors designed the experimental protocol of cellular therapy for patients after acute myocardial infarction. Inclusion criteria were: first myocardial infarction treated with primary angioplasty and stent implantation, confirmed non-viability of myocardium by USG, PET and SPECT, elevated CK-MB, and age below 70 years. Patients with intervention on other coronary artery, in unstable condition at day 4 through 6, and with serious non-cardiac disease were excluded. Patients undergoing coronary angioplasty, who signed informed consent, were randomized into three arms: A - high dose of 1•108 mononuclear cells, B - low dose of 1•107 cells, C - no cells. The autologous bone marrow was collected within day 7 after infarction. The mononuclear cells were separated, cultured for 24 hours in serum-free medium, and implanted through catheter via coronary artery into the damaged heart muscle in 7 subsequent injections. Mononuclear cells were analyzed with multicolor flow cytometry and culture assays of CFU-GM and CFU-Meg. The cardiac perfusion, metabolism and function were evaluated with SPECT, PET and echocardiography at 3 months after cell implantation. Results: 31 patients enrolled into the study underwent the protocol (9 in group A, 11 in groups B and C, respectively), and their cardiac functions were evaluated afterwards. There were no serious adverse effects of cell therapy procedure observed in each group. The analysis at 3 months interval showed an improvement in metabolism in the cellular therapy arms detected by PET. Left ventricle ejection fraction improved from 38 to 44%, although this improvement in global heart function was not statistically significant. However, regional heart function at the infarction site (peak systolic velocity of the infarcted wall) improved from 4.1 to 5.0 cm/s in the arm A (p Conclusion: Mononuclear bone marrow cells as a potential source of adult stem cells can be enriched, cultured ex vivo, and safely used in the cellular therapy protocols for ischemic heart disease. The functional benefit of dose of 1•108 mononuclear cells can be detected in a group of patients after acute myocardial infarction.

Background: Hematopoietic stem cells are able to regenerate hematopoiesis in all of its lineages. They are clinically used in transplantation of bone marrow or peripheral blood stem cells (PBSC) in patients with diagnosis of leukemia or lymphoma. While amount of hematopoietic stem cells is critical for the long-term engraftment, the amount of progenitor and precursor cells can influence time to engraftment, and it is critical for duration of neutropenia after transplantation. The methods of expansion of hematopoietic stem cells could reduce time to engraftment and decrease the risk of early post-transplant complications. Methods: Authors analyzed expansion of enriched hematopoietic stem cells (HSC), selected by immunomagnetic separation of Lin− cells from PBSC, in the culture of serum-free medium in vitro with all combinations of 5 cytokines (SCF, Flt-3-L, IL-3, IL-6, TPO) with and without G-CSF. Cell counts, morphology, immunophenotyping, and CFU-GM and CFU-Meg cultures were performed. Clinical transplantation protocol based on these results was developed. 10 patients with diagnosis of non-Hodgkin’s or Hodgkin’s lymphoma indicated for autologous transplantation, who signed the informed consent, were enrolled into the protocol. Except of standard PBSC graft, additional cells were collected, Lin− cells were selected by immunomagnetic separation, frozen and stored at Tissue bank. At day -14, Lin− cells were thawed and expanded in culture of serum-free medium with cytokines SCF, Flt-3-L, IL-3, IL-6 and G-CSF. Patients received high-dose chemotherapy regimen BEAM from day −7 to day −2. Progenitor cells expanded ex vivo in culture from Lin− cells were infused at day 0, following transplantation of PBSC. Patients were monitored, blood counts were performed, side effects were observed, and times to engraftment in granulocytes and platelets were calculated. Results: In experiments, the highest number of CFU-GM colonies was observed at day +14 with cytokine combination SCF+IL-3+Flt-3-L+ IL-6. The highest number of CFU-Meg colonies was observed in cytokine combination SCF+IL-3+TPO. G-CSF increased count and maturation of cells. Number of total cells grew 200 to 350 times at day +14. In the clinical protocol, 2 patients were excluded for technical reasons and 1 patient was excluded because of disease progression. 7 patients completed the protocol. The clinical procedure was free of serious adverse effects in all patients. Patients received doses from 5•107 to 3•109 cells. The group of patients was compared with historical controls - 142 patients treated at our department with autologous PBSC transplantation after BEAM regimen for diagnosis of lymphoma. Control group received higher average dose of CD34+ cells than experimental group: 7.7•106 / kg versus 6.6•106 / kg. Engraftment in granulocytes > 1000 / μl was shortened from 11 to 7.3 days in experimental group. Engraftment in platelets was not changed significantly (12 versus 11 days). Duration of neutropenia was shortened from 8 to 5.6 days. In patients, who received higher doses of infused cells > 2•109, average engraftment in granulocytes occurred at 6.3 days and duration of neutropenia was 5 days. Conclusions: HSC can be enriched from PBSC grafts, cultured and expanded ex vivo, and safely used in the cellular therapy protocols. The procedure resulted in significant shortening of critical period of neutropenia.

Abstract 2993 High-dose chemotherapy followed by autologous stem cell transplantation is an approved therapeutic intervention in relapsed Hodgkin-lymphoma (HL) and Non-Hodgkin lymphoma (NHL). In multiple myeloma (MM) it remains standard of care in first remission. Unfortunately, a significant portion of patients fail to mobilize and collect a sufficient amount of hematopoietic stem cells, being considered as “poor-mobilizers”. The effectiveness of the hematopoietic stem cell mobilizing agent plerixafor was evaluated in nationwide compassionate use programs in 13 European countries and reported to the European Consortium of Stem Cell Mobilization (ECOSM). Here we describe the mobilization success of 580 proven poor-mobilizers (304 male, 276 female) with NHL, HL and MM in Europe between May 2008 and August 2009. Furthermore, we analyzed the mobilization of stem cells in major NHL subgroups. All patients received plerixafor plus granulocyte colony-stimulating factor in standard doses with or without chemotherapy. Two-hundred seventy patients with NHL (138 male, 132 female) with a median age of 56 years (range 12 – 75 years) and a median of two prior chemotherapy regimens (range 0 – 10) were enrolled. Median cell yield was 2.56 × 10 ^6 CD34+ cells/kg BW (range 0 – 17.37). The general accepted minimum of 2.0 × 10 ^6 CD34+ cells / kg bodyweight (BW) for transplantation was reached by 175 patients (64.8%) in a median of two apheresis sessions (range 1 – 4). Thirty-four patients (12.6%) yielded more than 5.0 × 10 ^6 CD34+ cells/kg BW. There were no significant differences in in stem cell harvests regarding number of prior mobilization attempts or number of prior chemotherapeutic regimens, as well as in comparing patients with diffuse large B cell lymphoma (n=28), follicular lymphoma (n=15), and mantle cell lymphoma (n=24), respectively. Fifty-four HL patients (24 male, 30 female) with a median age of 36 years (range 19 – 76) and a median of three prior lines of therapy (range 1 – 5) were enrolled. Median cell yield was 3.14 × 10^6 CD34 cells/kg BW (range 0 – 32.6). Forty-four patients (81.5%) collected the minimum of 2.0 × 10^6 CD34+ cells/kg BW in a median of two apheresis sessions (range 1 – 4). Twelve patients (22.2%) collected more than 5.0 × 10 ^6 CD34+ cells/kg BW. A total of 256 patients (148 male, 108 female) with a median age of 60 years (range 28 – 76) diagnosed with MM were enrolled. Patients had received a median of two prior lines of treatment and collected a median of 3.60 × 10 ^6 CD34+ cells/kg BW (range 0 – 15.27) in a median of two apheresis sessions (range 1 – 5). The minimum of 2.0 × 10 ^6 CD34+ cells/kg BW was collected by 209 patients (81.6%). Eighty-two patients (32.0%) yielded more than 5.0 × 10 ^6 CD34+ cells/kg BW allowing tandem transplantation. Overall, the CD34+ cell yield was significantly higher in MM patients than in NHL patients (p Disclosures: Duarte: Genzyme: Membership on an entity's Board of Directors or advisory committees. Kröger:Genzyme: Membership on an entity's Board of Directors or advisory committees. Mohty:Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees. Hübel:Genzyme: Membership on an entity's Board of Directors or advisory committees.

Background. Alemtuzumab (Ale) induces destruction of CD52 antigen-bearing cells by antibody-dependent cell-mediated lysis, complement-mediated lysis, and induction of apoptosis. As a potent T cell depleting agent, Ale is used also for GvHD prophylaxis, given either i.v. around the time of transplantation, or directly "in the bag" to the cell suspension containing hematopoietic stem cells and lymphocytes. Contrary to bone marrow-derived grafts, the experience with addition of Ale to the peripheral blood progenitor cells (PBPC) suspension is limited. Therefore, we decided to study the effect of short-time incubation of PBSC samples with Ale using flow-cytometry and cultivation assays. Methods. After an informed consent, a sample of PBSC product (15 mL) was obtained from the collection bag immediately after a leukapheresis (COBE Spectra) in 8 healthy donors mobilized by G-CSF. Cells were washed using 4% human albumin and then incubated at 37°C with 1) Ale at the dose of 5 μg/106 cells, and 2) water for injection as a control. Autologous plasma as a source of complement was added after 15 min of incubation and cells suspensions were further incubated for 15 minutes. Both cell suspensions were then washed, resuspended in 4% human albumin, and samples were collected for blood count, flow-cytometric analysis, and cultivation assays (CFU-GM, BFU-E, CFU-Meg). Results. As expected, Ale caused profound T-cell (by a median of 99.3% vs. 12.3% in control) as well as marked B-cell (by a median of 90.6% vs. 8.3% in control) depletion. CD4+ and CD8+ lymphocytes were eliminated equally by a median of 99.5%, and 99.1%, respectively. Ale also produced a moderate reduction of CFU-GM and BFU-E (by a median of 37.7% vs. 3.3%, and 49.7% vs. 10.8%, respectively). NK-cells were not significantly affected as well as granulocytes and monocytes. Surprisingly, incubation with Ale clearly enhanced the growth of CFU-Meg; in median 8-times more CFU-Meg were observed in samples after incubation with Ale comparing to control samples (from 3 to 21-times, p Conclusion. Our results demonstrate that megakaryopoiesis is physiologically suppressed by lymphocytes. To the best of our knowledge, this is the first description that profound lymphocyte depletion by Ale enhances megakaryopoiesis in vitro and that Ale can also be used in vivo for the treatment of AATP.

Abstract 1931 Mobilization of peripheral blood stem cells (PBSC) for autologous transplant in children can be challenging especially in those with prior intensive chemotherapy and radiotherapy, and with increasing use of sequential high-dose therapy that requires high PBSC collection doses. Plerixafor is currently not licensed for use in children, but is approved for PBSC mobilization in adults. We present a series of 40 children from 19 centers worldwide who had PBSC mobilised with plerixafor off-licence, mostly on the North American or European Compassionate Use Programs. Thirteen of these cases were included in a series presented in abstract form at ASH 2010, and a further 6 were presented at EBMT 2011, and these cases are now presented with updated clinical data; the remaining 21 cases have not been presented elsewhere at the time of submission. Median age was 10 years (range 3 months–17 years); median weight was 31 kg (range 6–78 kg). Diagnoses were neuroblastoma (n=13), Ewing's sarcoma (n=10), medulloblastoma (n=5), lymphoma (n=5), PNET (n=4), multicentric Castleman's disease (n=1), Osteosarcoma (n=1) and Desmoblastic Small Round Cell Tumor (n=1). Most patients had received extensive multi-agent chemotherapy prior to plerixafor mobilization, and 13 had also received radiotherapy. For 7 patients, plerixafor had been introduced on a “rescue” basis in the course of the patient's first PBSC mobilization attempt, because of poor peripheral CD34+ counts predicting a high likelihood of mobilization failure without plerixafor use. The remaining 33 patients had undergone 45 prior failed conventional PBSC mobilization attempts: no apheresis in 31 cases; 8 were suboptimal (CD34+ dose < 2×10^6/kg); 5 procedures had yielded insufficient cells for planned sequential high-dose therapy. Plerixafor was used in 44 mobilization episodes (4 patients had undergone two mobilizations using plerixafor). In 29 cases, the drug was given after G-CSF 10 mcg/kg/day for 4 days, without prior mobilizing chemotherapy. In the remaining 15 cases, plerixafor was introduced to chemomobilization protocols at the time of initial neutrophil recovery, with exact protocols varying between centers. Three plerixafor mobilisation episodes were entirely unsuccessful (no apheresis); 10 episodes were suboptimal (CD34+ dose of 2×10^6/kg). For the 41 episodes where apheresis was undertaken, a median of 3.5×10^6/kg CD34+ cells (range 0.26–11.8) was collected in a median of 2 aphereses (range 1–5) after a median of 2 plerixafor doses (range 1–5). A total of 16 possible plerixafor toxicities were reported, all graded mild or moderate: these were injection site reactions (3), nausea (3), bone pain (3), vomiting (2), diarrhoea (2), insomnia (2) & headache(1). Seven patients were lost to follow-up after plerixafor due to transfer to other centers; of the remainder, 22 patients (67%) remain alive and 16 (48%) remain progression-free at a median of 9 months' follow-up post-plerixafor (range 3–63 months). Autologous PBSC transplant was performed in 27 patients & allograft in 1 patient. All transplanted patients achieved neutrophil engraftment (median of 11 days to neutrophils>1.0×10^9/litre; range 10–23 days). There was one case of secondary AML at >3 years post-autograft. Plerixafor is safe and highly effective in this difficult-to-mobilize patient group. Disclosures: Douglas: Genzyme Europe: Honoraria. Duarte:Genzyme Europe: Honoraria.