Combination Of G-CSF Research Articles

A human promyelocytic-like population is responsible for the immune suppression mediated by myeloid-derived suppressor cells

AbstractWe recently demonstrated that human BM cells can be treated in vitro with defined growth factors to induce the rapid generation of myeloid-derived suppressor cells (MDSCs), hereafter defined as BM-MDSCs. Indeed, combination of G-CSF + GM-CSF led to the development of a heterogeneous mixture of immature myeloid cells ranging from myeloblasts to band cells that were able to suppress alloantigen- and mitogen-stimulated T lymphocytes. Here, we further investigate the mechanism of suppression and define the cell subset that is fully responsible for BM-MDSC–mediated immune suppression. This population, which displays the structure and markers of promyelocytes, is however distinct from physiologic promyelocytes that, instead, are devoid of immuosuppressive function. In addition, we demonstrate that promyelocyte-like cells proliferate in the presence of activated lymphocytes and that, when these cells exert suppressive activity, they do not differentiate but rather maintain their immature phenotype. Finally, we show that promyelocyte-like BM-MDSCs are equivalent to MDSCs present in the blood of patients with breast cancer and patients with colorectal cancer and that increased circulating levels of these immunosuppressive myeloid cells correlate with worse prognosis and radiographic progression.

Novel strategies for blood stem cell mobilization: special focus on plerixafor

Introduction:More than 98% of autologous stem cell transplants are now performed with the support of mobilized blood stem cells, and the proportion of allogeneic blood stem cell transplants has risen to more than 70%. Blood stem cell mobilization strategies are therefore important components of all transplant programs.Areas covered:Stem cell mobilization strategies are evaluated based on current literature, with special focus on the use of plerixafor, a CXCR4 chemokine receptor antagonist. Mobilization methods in autologous settings include the use of G-CSF alone or following chemotherapy (chemomobilization), and the use of G-CSF alone in allogeneic transplants. A combination of G-CSF + plerixafor has been shown to be effective in patients who have failed a previous mobilization. This combination has also been found to be superior to G-CSF alone in Phase III studies in myeloma and non-Hodgkin lymphoma patients as the first-line mobilization.Expert opinion:Addition of plerixafor to chemomobilization or G-CSF mobilization may be more cost-effective than its routine use, and it is worth considering in predicted or proven poor mobilizers. Novel mobilization strategies have allowed more successful stem cell collection in autologous setting, although the effect of plerixafor on graft content and long-term patient outcomes needs further investigation.

PLERIXAFOR IN POOR STEM CELL MOBILIZERS: THE BELGIAN COMPASSIONATE USE PROGRAM

Introduction: Currently available stem cell mobilizing regimens (G-CSF +/- chemotherapy) show high failure rates, especially in heavily pretreated patients. Plerixafor, a new stem cell mobilizing agent blocking the CXCR4-SDF-1 interaction, offers a new strategy for stem cell mobilization, especially in poor mobilizers. This study reports on the outcome of the Belgian compassionate use program (CUP).Materials and methods: Between July 2008 and July 2009, 14 Belgian transplant centres participated in plerixafor CUP. In total, 22 poor stem cell mobilizers were included. Patients who previously failed stem cell mobilization received a combination of G-CSF (morning of Day 1-5) and plerixafor (evening of Day 4). Apheresis was performed on Day 5. G-CSF, plerixafor and apheresis were continued until at least 2 × 106/kg CD34+ cells were obtained in a maximum of 3 collections.Results: A mean of 2 plerixafor administrations was needed to reach ≥ 2 × 106/kg CD34+ cells. The overall cumulative success rate (defined as the proportion of patients achieving a successful collection after a maximum of 3 apheresis days) was 64%. Half of the heavily pretreated patients (≥ 3 prior chemotherapy regimens) could be mobilized successfully. Patients who received ≤ 2 prior chemotherapy regimens mobilized successfully in 75% of the cases. Thirteen patients (59.1%) underwent autologous stem cell transplantation with normal neutrophil and platelet recovery times.Conclusion: For patients failing previous mobilization attempts, the combination of plerixafor and G-CSF is a successful mobilizing strategy, even in poor mobilizers who received ≥ 3 prior chemotherapy regimens.

Pretreatment of IL-2 influences the proliferation and polarization of mice splenic lymphocyte following mobilization with G-CSF

Objective To explore the influence of IL-2 pretreatment on splenic lymphocyte following mobilization with G-CSF, which may provide a new approach to attenuate acute graft-versus-host disease (GVHD). Methods Splenic cells and na(i)ve CD4+T cells from C57BL/6N mice receiving G-CSF-mobilized were pretreated with IL-2(50 U/ml) , and cocultured with the allogeneic antigens from BALB/c mice. The proliferation responses and the polarization of T cells were determined. C57BL/6N mice were randomly divided into 4 groups: G-CSF + IL-2 group, G-CSF group, IL-2 group , control group. Results Compared with the control group, IL-4 increased obviously while IFN-γ decreased significantly in the group of G-CSF + IL-2. The proliferation responses were also suppressed in vitro. Conclusion The proliferation responses of splenic cells and naive CD4 + T cells from C57BI/6N mice receiving G-CSF-mobilized to the allogeneic antigens were significantly abrogated by the pretreatment with IL-2, T cells were polarized toward the production of type-2 cytokines. The combination of G-CSF and IL-2 is potentially synergetic in the induction of T lymphocyte immune tolerance. Key words: Interleukin-2; Granulocyte colony-stimulating factor; Cell proliferation; Cell polarization; Mixed lymphocyte reaction

G-CSF, erythropoietin, oxymetholoneの併用により造血3系統の回復を認めた重症再生不良性貧血の1小児例

G-CSF, erythropoietin, oxymetholoneの併用により造血3系統の回復を認めた重症再生不良性貧血の1小児例

Formation of an adherent hematopoietic expansion culture using fucoidan

Expansion of transplantable cord blood (CB) progenitors using a stroma requires provision of an exogenous cell source because of the low frequency of stromal precursor cells in CB. A simpler approach from a clinical regulatory perspective would be to provide synthetic extracellular matrix. The aim of this study was to characterize the effect on hematopoietic cell culture of fucoidan. The modulation of cytokine-driven hematopoietic cell expansion by fucoidan was investigated using two-level fractional and full factorial experimental designs. Mobilized peripheral blood (PB) CD34(+) cells were grown over 10days in various combinations of FL, SCF, TPO, G-CSF, and SDF-1. Cultures were analyzed by immunophenotype. The effect of fucoidan on the divisional recruitment of CD34(+) cells was studied by CFDA-SE division tracking. Fucoidan was adsorbed by polystyrene to tissue culture plates and promoted formation of an adherent hematopoietic culture. Factorial design experiments with mobilized PB-CD34(+) cells showed that fucoidan reduced the production of CD34(+) cells and CD34(+)CXCR4(+) ratio but did not affect the production of monocytic, granulocytic, or megakaryocytic cells. The inhibitory effect of fucoidan on expansion of CB-CD34(+) cells was greater than mobilized PB. Division tracking analysis showed that CD34(+) cell generation times were lengthened by fucoidan. Fucoidan binds growth factors via their heparin-binding domain. The formation of an adherent hematopoietic culture system by fucoidan is most likely mediated by the binding of L-selectin and integrin-αMβ2 on myeloids. Fucoidan deserves further investigation as glycan scaffold that is suitable for immobilization of other matrix molecules thought to comprise blood stem cell niche.

Preparation of granulocyte concentrates by apheresis: collection modalities in the USA

Preparation of granulocyte concentrates by apheresis: collection modalities in the USA

Optimizing the Efficacy of Plerixafor as a Salvage Option In Poor Mobilizers Following Chemotherapy Plus G-CSF Mobilization

Recent data of the combination of G-CSF (G) with Plerixafor (P) has established its efficacy in stem cell mobilization for autologous transplants. However, there is limited data about the utility of plerixafor in patients who are being mobilized with chemotherapy and G.

Importance of blood graft characteristics in auto-SCT: implications for optimizing mobilization regimens

Although blood stem cells have been widely used to support high-dose therapy in the autologous setting, limited data are available on the effects of graft characteristics in patient outcomes other than haematopoietic engraftment. Retrospective studies suggest that patients who mobilize more CD34(+) cells have better outcomes than do patients who mobilize less well. Furthermore, immunological reconstitution may be important in terms of post-transplant outcome and is apparently affected by graft composition. There is accumulating evidence that the mobilization regimen used may be an important determinant of graft content. Plerixafor has been recently introduced combined with G-CSF in patients who mobilize poorly. In addition to enhancing mobilization of CD34(+) cells, there are indications that plerixafor may also affect other graft components. A combination of chemotherapy plus G-CSF with plerixafor has been shown to be very effective in stem-cell mobilization, but more data are needed in regard to other graft characteristics in this setting. Prospective studies are needed to evaluate whether higher CD34(+) doses or other modifications to graft composition translate into better long-term outcomes in the autologous setting. These studies are not only important in regard to defining the optimal stem-cell graft in the autologous setting, but also in identifying the optimal mobilization regimen.

Plerixafor is effective and safe for stem cell mobilization in heavily pretreated germ cell tumor patients

Up to 10% of germ cell tumor patients require salvage high-dose chemotherapy with stem cell support, achieving cure rates in the range of 10-60%. Stem cell mobilization may be difficult in these patients because of multiple lines of treatment known to seriously hamper stem cell recovery. Plerixafor significantly enhances the success of the CD34+ cell harvest, even in cases where prior mobilization attempts have failed. Six germ cell tumor patients provided informed consent and were included in the compassionate use program. All patients were heavily pretreated, with a median of 3.5 prior lines of therapy. All failed prior mobilization with G-CSF in combination with chemotherapy. Five patients yielded a median of 2.6 × 10(6) CD34+ cells per kg body weight in a median of 4 apheresis days when plerixafor was used. Three patients underwent subsequent high-dose chemotherapy with autologous stem cell support. Median time to leukocyte engraftment was 11 days. Median time to platelet engraftment was 12.5 days, both of which are comparable to previous historical data. Accordingly, plerixafor seems to be safe and effective in germ cell tumor patients who have failed prior mobilization therapy. Larger prospective studies are warranted to further assess its use in germ cell cancer.

CD26 Inhibition Preferentially Enhances In Vitro Migration of G-CSF + Plerixafor (AMD3100) Mobilized PB as Compared to G-CSF Mobilized PB In Multiple Myeloma Autografts.

AbstractAbstract 3714Hematopoietic stem and progenitor cell (HSC/HPC) trafficking into the bone marrow during hematopoietic stem cell transplantation (HSCT) is thought to be an important biological step that can be targeted to improve transplant efficiency and subsequent immune reconstitution. It has been reported that loss/inhibition of the peptidase CD26, also known as DPPIV (dipeptidylpeptidase IV), increases the transplant efficiency of mouse HSC/HPC (Christopherson, KW 2nd, et al. Science 2004). In a similar manner, inhibition of CD26 on CD34+ or lin− cord blood (CB) cells improves in vitro cell migration and in vivo engraftment into immunodeficient mice (Christopherson, KW 2nd, et al. Stem Cells and Dev 2007). There is currently controversy as to whether inhibition of CD26 on mobilized peripheral blood (mobPB) has benefit since it has been reported that inhibition of CD26 does not improve migration of G-CSF mobPB CD34+ cells in response to SDF-1 (Kawai K, et al. Stem Cells and Dev 2007. 16:361–70) but does improve the migration of G-CSF mobPB mononuclear cells (MNC) (Frank R, et al. ASH Abstract #372, 2009). Additionally, it has been reported that AMD3100 mobPB c-kit+ mouse cells have high CD26 expression similar to BM in contrast to G-CSF mobPB c-kit+ mouse cells which have very little CD26 expression (Bonig H, Exp Hem, 2009). The combination G-CSF + AMD3100 (a CXCR4 antagonist also known as plerixafor or Mozobil ®) has been recently introduced into the clinic for use in obtaining mobPB from Multiple Myeloma patients. We therefore investigated whether CD26 on human mobPB MNC obtained from Multiple Myeloma autografts mobilized with either G-CSF or G-CSF + plerixafor would have an effect on SDF-1 induced cell migration. To do this, non-adherent MNC from mobPB were obtained from discarded bags following the completion of stem cell infusion of previously frozen autografts. CD26 expression was detected by flow cytometery. CD26 activity was determined using the substrate Gly-Pro P-nitroanilide (Gly-Pro-pNA). To assess migration, chemotaxis assays were performed by loading 5×104 cells into the upper chamber of a 96 well transwell plate, adding human SDF-1α (0–400 ng/mL) in the lower chamber, and incubating for 2 hours at 37°C, 5% CO2. By flow cytometry, CD26 expression was 21.61±2.19% and 21.65±3.68% of the CD45+ cell population for G-CSF and G-CSF + plerixafor mobPB MNCs respectively. CD26 activity was 15.4±1.2 pmol pNA/minute and 16.3±0.4 pmol pNA/minute for G-CSF and G-CSF + AMD3100 MNCs. During functional in vitro assays, cells were cultured with or without a 5mM concentration of the CD26 inhibitor Diprotin A (DpA). DpA treatment of G-CSF + plerixafor mobPB resulted in a significant increase in cell migration as compared to untreated cells. Specifically, the percent migration at 400ng/mL SDF-1α was 21.3±2.4% and 31.6±3.0% for untreated and DpA treated G-CSF + plerixafor mobPB cells respectively (p=<0.01, n=10). The percent migration in response to 400ng/mL SDF-1α was 19.8±2.0% and 25.3±2.6% for untreated and DpA treated G-CSF mobPB cells respectively (p=0.21, n=7). These data indicate that CD26 inhibition, by treatment with DpA, results in a preferential increase in SDF-1 induced migration of G-CSF + plerixafor mobPB cells as compared to G-CSF mobPB cells. The preferential response does not appear to correlate to any change in CD26 expression or activity since both types of mobPB have equivalent expression and activity prior to the migration assay. Our data suggest that un-fractionated mobPB collected following mobilization using the combination of G-CSF + plerixafor (AMD3100) may have inherently different cell trafficking regulatory properties when compared to mobPB collected following G-CSF alone and that additional regulatory mechanisms allow the G-CSF + plerixafor mobPB to have an enhanced response to SDF-1 in the absence of CD26 suppression. Additionally, our data suggest that inhibition of CD26 peptidase activity may be more clinically relevant in the G-CSF + plerixafor mobilized patient population with regard to achieving improved stem cell trafficking. Disclosures:Fung:Genzyme: Honoraria, Speakers Bureau.

First Results of a Phase-II Study with the New CXCR4 Antagonist POL6326 to Mobilize Hematopoietic Stem Cells (HSC) In Multiple Myeloma (MM)

AbstractAbstract 824In multiple myeloma (MM), the second most common hematological malignancy, high-dose therapy followed by autologous CD34+ stem cell transplantation (ABSCT) is therapy of choice for younger patients. Standard treatment to mobilize hematopoietic stem cells (HSC) is either G-CSF alone or combined with chemotherapy. In the last years the antagonism of the CXCR4 receptor has been identified as a potent mechanism of HSC release from the bone marrow compartment. This mobilization by CXCR4 antagonists is more direct and more rapid than by G-CSF given over 4 to 6 days, and the combination of G-CSF plus a CXCR4 antagonist is superior to G-CSF alone. Furthermore, HSC mobilized using G-CSF and a CXCR4 antagonist have been shown to result in a rapid and sustained engraftment post-transplantation.POL6326 is a novel, potent and selective CXCR4 antagonist based on the PEM (Protein Epitope Mimetics) technology for intravenous application. In a previous Phase I study in 52 healthy volunteers POL6326 has been demonstrated to effectively mobilize CD34+ stem cells and was very well tolerated.The goal of the Phase II study in newly diagnosed myeloma patients reported here was to test to what extent POL6326 can be used as monotherapy, infused over 1 or 2h, to mobilize sufficient HSC for subsequent autologous transplantation. In our study the minimum number of HSC required for ABSCT was 2 × 106 CD34+ cells/kg body weight (BW). All patients also received CAD/G-CSF (cyclophosphamide, doxorubicin and dexamethasone) about 10 days after POL6326 and leukapheresis.The second goal was to determine tumour cell mobilization by polymerase chain-reaction after POL6326 or CAD/G-CSF respectively.Here we report the first data of this ongoing study. In all doses tested, up to 1200 μg/kg BW over 2h, POL6326 was safe and very well tolerated. During a total number of 38 infusions the only minor adverse event possibly related to study drug was a discrete pruritus of CTC grade 1 at the infusion site during POL6326 administration on two consecutive days. It resolved spontaneously and did not require any medication. This excellent safety and tolerability profile warrants further dose escalation in the ongoing trial.After first diagnosis, patients received 3 cycles of induction chemotherapy with bortezomib (or thalidomide respectively in one single subject) plus doxorubicin and dexamethasone. 3 weeks after the last injection of induction treatment HSC were mobilized with POL6326 given as a 1 or 2h infusion on up to 4 consecutive days according to an intra-individual dose escalation scheme (from 600 to 1200 μg/kg) with consecutive leukapheresis 30 min after end of infusion.In all patients (n=16) we observed an elevation of leukocytes and HSC compared to baseline. In 66% of patients sufficient stem cells were mobilized with POL6326 for ABSCT (with a mean of 2.5 × 106 CD34+ cells/kg BW). In 75 % of these subjects 2 leukapheresis cycles were sufficient to reach the minimum number of HSC.In all patients undergoing ABSCT with HSC mobilized by POL6326, the engraftment was successful. The time to reconstitution ranged from 10 to 19 days (mean 14 days) and was comparable to ABSCT with CAD/G-CSF-mobilized HSC (historical mean of 14 days and a range from 6 to 31 days).Five patients were evaluated for the presence of tumour cells in peripheral blood before mobilization and in leukapheresis products after treatment with POL6326 and CAD/G-CSF. In 2 of these patients we could not detect any tumour cells, neither in all leukapheresis products nor in all peripheral blood samples. In 2 patients a minor contamination with less than 0.0001% of tumour cells was detected both in the peripheral blood before POL6326 and in the leukapheresis products. Finally, 1 patient showed a similarly minor (<0.0001%) percentage of tumour cells only in the apheresis product after POL6326 treatment. However, a significant mobilization of tumour cells (0.001%) was detected after CAD plus G-CSF in this patient.Due to the excellent safety and tolerability profile observed for POL6326 in this study the next steps include further dose escalation. These initial data with low doses of POL6326 indicate that this novel CXCR4 antagonist holds the promise to be eventually used as a stand alone therapy not requiring the application of G-CSF. The preliminary findings of the absence or very low counts of tumour cells in the leukapheresis product generated with POL6326 warrant further investigation. Disclosures:Off Label Use: POL6326 as new CXCR4 antagonist for experimental CD34+ hematopoietic stem cell mobilization. Dembowsky:Polyphor Ltd: Employment. Braun:Cytonet Heidelberg GmbH: Employment. Ludin:Polyphor Ltd: Employment.

Updated Results of Combination Cytokine Immunotherapy In the Treatment of Aplastic Anemia and Myelodysplastic Syndrome (MDS)

AbstractAbstract 2920Immunosuppressive therapy using antithymocyte globulin (ATG) and cyclosporine has been successfully used in the treatment of cytopenias in patients (pts) with severe aplastic anemia (AA). Laboratory and clinical data suggest that dysregulated immune mechanisms may also contribute to the pathogenesis of ineffective hematopoiesis in some subsets of early MDS and result in cytopenias. Rabbit ATG (rATG) is active and well tolerated in pts have recurrent/persistent disease after treatment with equine ATG. Here we present our updated results with a combination of rATG, cyclosporine, prednisone, and G-CSF in pts with aplastic anemia or myelodysplastic syndrome.This is a single-arm phase II study evaluating the efficacy and safety of the following combination in pts with aplastic anemia and low or intermediate-1 risk MDS (by the international prognostic scoring system) : rATG 3.5 mg/kg/day (or 2.5 mg/kg/day for age ≥ 55 yrs) intravenously (IV) for 5 days, methylprednisone 1mg/kg/day IV daily for 5 days (given prior to each dose of ATG), followed by oral prednisone tapered off over 1 month, G-CSF (filgrastim daily or pegfilgrastim every 2 weeks) subcutaneously for up to 3 months, and cyclosporine (5 mg/kg) daily for up to 6 months. Dose reductions or interruptions for toxicities were allowed at the discretion of the treating physician. Responses were assessed at 3 months after initiating therapy.Of the 51 pts enrolled, 46 have received treatment including 23 (50%) pts with AA and 23 (50%) pts with MDS. The median age overall was 60 yrs (range, 19–82), 54 (19-82) in the AA and 63 (48-79) in the MDS cohorts respectively. Of the 43 pts evaluable for response (21 AA, 22 MDS), there were 13 (62%) responders (4 CR, 8 PR, 1 HI) in AA cohort and 6 (27%) responders (2 CR, 4 HI) in the MDS cohort. Median age of responders was 59 (19-79). The median time to response (TTR) was 3.2 months. Of the 46 pts who received treatment, 20 pts (43%) were HLA-DR15 positive, 22 (48%) were negative, and 4 (9%) were not checked. In the MDS cohort, 9 (39%) pts were HLA-DR15 positive and none responded. In the AA cohort, 11 (48%) were HLA-DR15 positive, out of which 6 (55%) responded. Among the 19 responding pts described above, 6 (32%) were HLA-DR15 positive and all had AA. The median disease free survival for responders (CR & PR) is 26 months. The median overall survival of responders has not been reached, compared to 20 months for nonresponders (p<0.001). Overall the treatment was tolerable. The most common grade 3/4 toxicities were thrombocytopenia (43%), neutropenia (41%), transaminase elevation (13%), hyperbilirubinemia (7%), infection/fever (7%), and hyperglycemia (4%). Grade 3/4 dyspnea, abdominal pain, and hypertension occurred in 1 patient each. Twenty pts (43%) had infusion related reactions including fever, chills, and dyspnea that was manageable with supportive care.The combination of rATG, cyclosporine, steroids, and G-CSF is a well tolerated, effective regimen in the treatment of aplastic anemia and also has activity in a subset of pts with MDS. The rate of serious infections was relatively low, but infusion reactions were common and should be managed with premedication and supportive care. HLA-DR15 positivity correlated with response in patients with AA, but not in MDS. Responses were slow to occur, but durable, and associated with improved survival. Disclosures:Kadia:Genzyme: Research Funding. Ravandi:Genzyme: Honoraria.

Peripheral Blood Stem-Cell Mobilisation and Autologous Stem Cell Transplantation In HIV-Related Malignancies.

AbstractAbstract 4592 Introduction:Patients (pts) with HIV-infection are generally excluded from clinical trials that evaluate the role of high dose chemotherapy (HDCT) in malignant lymphoma or relapsed germ cell tumor (GCT). However, recent data indicate that HDCT followed by autologous peripheral blood stem cell transplantation (ASCT) may be effective in relapsed HIV-related lymphoma. Methods:This is an observational cohort study including patients with HIV-related lymphoma or HIV-related GCT who have peripheral blood stem cells mobilized by a combination of chemotherapy (CT) and G-CSF. Pts did or did not undergo consecutive ASCT. The primary outcome measure is feasibility. High-dose BEAM was used as a conditioning regimen in pts with HIV-related lymphoma while high-dose carboplatin/etoposide (CE) was chosen for pts with GCT. Results:From 07/05 to 03/10 peripheral blood stem cells (PBSC) were successfully harvested in 10 of 11 HIV-infected pts with diffuse large B-cell lymphoma (DLBCL) [n=4], Burkitt's lymphoma (BL) [n=3], plasmablastic lymphoma (PL) [n=2], Hodgkin lymphoma (HL) [n=1] and testicular GCT [n=1]. The mean number of collected stem cells was 15.7×106/kg CD34+ cells (range, 6.3 – 33). PBSC-mobilisation failed in one pt with relapsed BL. 7 of 11 pts were mobilized following salvage CT for DLBCL [n=4], BL [n=1], HL [n=1] or GCT [n=1] while 4 pts were under primary CT for BL or PL. So far, 5 of 10 pts received HDCT + ASCT. Pt 1 (44 yrs, CDC C3; HIV-RNA< 50 cop/ml at time of SCT) received HDCT as 3rd salvage therapy for DLBCL. A total of 9.2 × 106/kg CD34+ cells were transplanted. Neutrophil engraftment occurred on day +14. The pt achieved a partial remission but died of progressive lymphoma 6 months after ASCT. Pt 2 (60 yrs, CDC B3; HIV-RNA< 50/ml) underwent HDCT + ASCT (13.8 × 106/kg CD34+ cells) for a 1st relapse of HL. Neutrophil engraftment was observed on day +10. The pt is well and disease free 25 months after ASCT. Pt 3 (26 yrs, CDC C3, HIV-RNA< 50/ml), a hepatitis C co-infected haemophiliac, received HDCT + ASCT for refractory DLBCL but died of liver cirrhosis and neutropenic sepsis with multi-organ failure on day +16. Pt 4 (25 yrs, CDC A3, HIV-RNA< 50/ml) received 3 sequential courses of HD-CE followed by ASCT in 3-week intervals for a 3rd relapse of a nonseminomatous GCT. Neutrophil engraftment occurred on day +10, + 12 and +14, respectively. A complete remission (CR) was achieved. However, the pt suffered another relapse involving the central nervous system and died of progressive GCT 15 month after the 3rd transplant. Pt 5 (41 yrs, CDC C3, HIV-RNA 220/ml) underwent HDCT in 2nd complete remission after successful salvage-CT for a first sensitive relapse of DLBCL. A total of 12.9 × 106/kg CD34+ cells were transplanted. The pt is currently alive and neutropenic (day +3). ASCT was not performed in the other 6 pts because of refractory BL [n=1], ongoing first remission following induction CT for BL [n=2] and PL [n=2] and concomitant histoplasmosis necessitating antifungal therapy [n=1]. Conclusions:Successful mobilisation of PBSC is feasible in the majority of pts with HIV-related malignancies. ASCT seems effective in selected pts with chemo-sensitive relapse of malignant lymphoma or GCT. HIV-infected pts should no longer be excluded from HDCT-programs. Disclosures:No relevant conflicts of interest to declare.

Nonanthracycline Induction Chemotherapy (T-FLAG) for Elderly Patients with Newly Diagnosed Acute Myeloid Leukemia

AbstractAbstract 4356 Introduction:Prognosis of elderly patients (age>60 years) with acute myeloid leukemia (AML) is poor as compared to younger patients with a higher incidence of cardiac co-morbidities. With standard induction therapy, complete remission (CR) is achieved in 35% to 50% of elderly patients. However, the induction anthracycline containing chemotherapy related mortality is in this population has been reported to be approximately 30%. We attempted to decrease this by replacing the anthracycline with topotecan. Alternative therapies are needed that are less toxic but equally efficacious. In a phase I/II trial of the combination of topotecan, fludarabine, cytarabine and G-CSF (T-FLAG) in elderly AML and MDS, there was 50% CR and 15% treatment related deaths. In this study, we report patients >60 years of age treated with T-FLAG as an initial therapy for AML at our institute. Methods:We retrospectively reviewed the charts of elderly patients with AML treated with T-FLAG at our institution. Elderly patients were defined as more than 60 years of age. IRB approval was obtained for the study. All patients received topotecan 2mg/m2, fludarabine 30 mg/m2, and cytarabine 2000 mg/m2 all given over 30 min from day 1 to day 4 and G-CSF 400 μ g/m2/day from the day 5 to neutrophil counts recovery. International working group criteria was used to assess responses. All statistical analysis was done using SAS statistical software. Descriptive statistics were used. Progression free survival was defined from the time of diagnosis to progression, death or last follow up, whichever comes first. Overall survival and progression free survival was calculated using Kaplan and Meier method. Results:Thirteen elderly patients were treated with T-FLAG. The median age of diagnosis was 73 years (Range: 60–86 years). There were 10 male and 3 female patients. Ten (77%) patients had complex cytogenetics. Cardiomyopathy (EF <50%) or elevated liver function tests precluding anthracycline use was present in 54% of the patients. Response rate was 69% with 7 (54%) patients achieving CR and 2 (15%) patients achieving partial remission. Out of 10 patients with abnormal cytogenetics at baseline, 7 (70%) patients had cytogenetic CR. Eleven (85%) patients were alive at day 30. Median progression free survival and overall survival was 36 weeks and 54 weeks respectively. The median time for the recovery of neutrophil counts (>500/μ L) was 22 days (Range: 10–47 days). Conclusions:In this study of unfavorable risk group elderly patients, T-FLAG induction chemotherapy results in comparable remission rates with low induction chemotherapy related mortality. It may be an alternative regimen for elderly patients with complex cytogenetics or patients who cannot tolerate anthracyclines. More studies are needed to confirm these results. This however, may be useful to bridge patients to transplant or maintenance demethylating agents to improve survival. Disclosures:No relevant conflicts of interest to declare.

Efficacy of the coadministration of granulocyte colony-stimulating factor and stem cell factor in the activation of intrinsic cells after spinal cord injury in mice

Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic cytokine that induces undifferentiated stem cells from the bone marrow (BM) into the peripheral blood. Stem cell factor (SCF) is also a hematopoietic cytokine that stimulates the differentiation and proliferation of neural stem cells and has neuroprotective effects. In cerebrally infarcted mice, the combination of G-CSF and SCF promotes the differentiation of BM-derived cells into neural cells, stimulates the proliferation of intrinsic neural stem cells, and improves motor function. The object of this study was to investigate the effects of these cytokines on BM stem cells, intrinsic cells, and motor function recovery in spinal cord-injured mice. For marking BM-derived cells, the authors induced contusive spinal cord injury in mice transplanted with BM cells from green fluorescent protein (GFP)-transgenic mice after whole-body irradiation. These mice were treated with G-CSF and SCF in the subacute injury phase. Bromodeoxyuridine (BrdU) was injected into these mice to label proliferating cells. The cell numbers and phenotype of the BM-derived cells were evaluated, and the change in intrinsic cells (proliferation, accumulation, and differentiation) was noted using immunohistological analysis at 4 weeks postinjury (wpi). A behavior analysis was conducted until 12 wpi using the Basso, Beattie, Bresnahan locomotor rating scale. In the SCF + G-CSF group, improvement in hindlimb motor function was significantly greater than in the SCF group, G-CSF group, and sham-treatment (vehicle) group after 8 wpi. At 4 wpi, the number of GFP+ BM-derived cells induced in the lesion did not significantly differ between groups. At 4 wpi, the authors evaluated perilesional GFP− intrinsic spinal cord cells. The number of GFP− and F4/80+ cells was significantly greater in the SCF + G-CSF group than in the other 3 groups. As compared with the sham group, the number of NG2+/BrdU+ cells was significantly increased in the SCF + G-CSF group. In this study, the combined administration of SCF and G-CSF in traumatic spinal cord injury not only improved motor function, but also induced the accumulation of intrinsic microglia and the active proliferation of intrinsic oligodendrocyte precursor cells.

Plerixafor (Mozobil) for stem cell mobilization in patients with multiple myeloma previously treated with lenalidomide

Lenalidomide and other new agents have considerable activity in multiple myeloma (MM) and have changed the landscape of treatment. Data suggest that lenalidomide therapy before autologous hematopoietic stem cell transplantation has a detrimental effect on stem cell mobilization. This retrospective study examined the efficacy of plerixafor in combination with G-CSF among patients with MM previously treated with lenalidomide (median, 4 cycles; range, 1-20 cycles). Data were analyzed for 60 patients who received plerixafor plus G-CSF for frontline mobilization in a phase 3 clinical trial or an expanded access program (n=20) or for remobilization in a compassionate use program (n=40). The overall median number of CD34+ cells collected was 5.6 × 10(6) per kg (range, 0.45 × 10(6)-37.2 × 10(6)). The minimum number of CD34+ cells (2 × 10(6) per kg) was collected from 86.7% of patients in a median of 1 day. This minimum was collected from 100% of patients who underwent frontline mobilization and 80% of patients who underwent remobilization. These data suggest that CD34+ hematopoietic stem cells can be successfully and predictably collected with combination plerixafor plus G-CSF for primary or secondary mobilization in the majority of patients with MM who have been previously treated with lenalidomide.

Levels of follicular G-CSF and interleukin-15 appear as noninvasive biomarkers of subsequent successful birth in modified natural in vitro fertilization/intracytoplasmic sperm injection cycles

To explore oocyte competence for subsequent birth. The modified natural IVF/intracytoplasmic sperm injection (ICSI) cycle was used as an experimental model by measuring levels of cytokines, chemokines, and growth factors in individual follicular fluids (FF). A retrospective blinded study. European network of research, Embryo Implantation Control (EMBIC). Single FF from 83 women were analyzed during a modified natural IVF/ICSI cycle, and reproducibility of follicular composition was evaluated over two cycles for 15 patients. Each FF sample was blindly tested to assess levels of 26 factors by bead-based immunoassays. Each mediator was evaluated as a potential biomarker of subsequent birth by multivariate regression analysis. A combination of both FF G-CSF and IL-15 was the optimal model to predict birth (AUC(ROC), 0.85). Birth rates per cycle were 48.9% (16/33) if two good-prognosis criteria were present (FF G-CSF>12 pg/mL and IL-15<7 pg/mL) and 8% (3/36) and 0% (0/14) if, respectively, one or none were present. FF G-CSF was significantly correlated over two cycles (r=.71), suggesting a possible prognostic value of its documentation. Combined follicular G-CSF and IL-15 quantification appears as an efficient and noninvasive method to define oocyte competence for subsequent successful conception in modified natural IVF/ICSI cycles.

Phase III Randomized Stem Cell Mobilization Trial Comparing Standard Vs Double-Dose G-CSF Vs Standard Doses of G-CSF Plus GM-CSF in Hard to Mobilize Patients Undergoing An Autologous Hematopoietic Stem Cell Transplant (HSCT).

Abstract 3228Poster Board III-165 IntroductionUsing standard dose G-CSF (10 μg/kg) for stem cell mobilization, 25-40% of patients, deemed to be hard to mobilize based on prior therapy, will not collect sufficient HSC (> 2-2.5 × 106 CD34/kg) to proceed to a prompt autotransplant. Strategies to improve CD34/kg yields have included dose escalating G-CSF up to 30 μg/kg or combining G-CSF and GM-CSF. While dose escalated G-CSF is effective in increasing CD34 yields in normal donors as is the combination of G-CSF and GM-CSF, their comparative value in pre-treated patients has not been tested. To determine the value of these strategies, we performed a randomized comparison of high dose G-CSF (30 μg/kg as 2 doses 12 hours apart), to the combination of simultaneous single daily doses of G-CSF (10 μg/kg) plus GM-CSF (5 μg/kg), to a control group receiving G-CSF at an equivalent total dose of cytokine to the combination arm (15μg/kg) as a single dose. Patients and MethodsPatients were eligible if heavily pre-treated, defined as: a minimum of 10 total cycles of combination chemotherapy and two prior regimens, or a total of 6 chemotherapy cycles if the patient also received RT to marrow bearing sites, platinum-based chemotherapy or 2 or more cycles of any BCNU or fludarabine containing regimen. Baseline WBC had to be > 3000/μl, ANC > 1500/μl and a platelets > 100,000/μl. Twelve liter aphereses began on day 5 of mobilization, and continued until ≥ 4 × 106 CD34/kg were collected or a maximum of 5 aphereses. Patients typically proceeded to transplant if they had ≥ 2.5 × 106 CD34/kg collected and were always re-mobilized if they collected < 2.0 × 106 CD34/kg. CD34 subsets (CD34+/CD33- and CD34+/CD38-) were also assessed for the 3 groups to determine if more primitive HSC were mobilized by the 2 novel strategies. The sample size was calculated based as follows: 60% of the control group would collect 2.5 × 106 CD34/kg and this would rise to 90% in one or both study arms. The detection of such differences with a power of 80% and a 2-sided alpha level of 0.025 required a total sample of 120 patients. ResultsA total of 120 patients were randomized; 119 were eligible. Patient demographics, shown in the Table, were matched among the three groups:Standard Dose G N = 40High Dose G N = 40G + GM N = 39Age50 (20-71)50 (21-66)49 (22-69)Sex M:F26:1421:1916:23Disease-NHL19 (47.5%)20 (50.0%)17 (43.6%)-HD9 (22.5%)8 (20.0%)7 (17.9%)-Multiple Myeloma4 (10.0%)4 (10.0%)5 (12.8%)-Other8 (20.0%)9 (22.6%)9 (23.0%)Remission Status-CR1/PR15 (13.9%)5 (13.5%)8 (27.6%)-CR2/PR27 (19.4%)10 (27.0%)6 (20.7%)-IF/Ref24 (66.7%)22 (59.6%)15 (51.7%)# Prior Radiotherapy1087Median # of Prior Chemo Regimens2 (1-6)2 (1-6)2 (1-6)Median # of Total Cycles9 (4-22)10 (1-18)10 (4-34)Time from DX to Entry (months)21 (7-207)25 (9-295)23 (10-129)Baseline ANC K/μl3.0 (1.7-6.8)4.0 (1.6-10.7)3.7 (1.5-9.7)Baseline platelets K/μl213 (131-413)213 (84-358)206 (37-432)The % of patients collecting ≥2.5 × 106 CD34/kg was: standard G: 60%, high dose G: 57% (p = 1.0), G + GM: 41% (p = 0.1). Median CD34 collected in first mobilization were, 3.6 × 106/kg, 3.0 × 106/kg (p = 0.22) and 2.0 × 106/kg (p = 0.05) respectively in a median of 4, 4, and 5 aphereses (p = NS). Re-mobilization rates: standard G; 37.5%, high dose G: 35%; G + GM: 50% (p = NS). Total median CD34 collected from first and any second mobilizations were: standard G: 4.8 × 106/kg, high dose G: 3.9 × 106/kg, and G + GM: 3.5 × 106/kg. One patient in the standard G arm and 3 in high dose G did not proceed to transplant due to poor initial mobilization and progression in 2, and one each for progression or poor mobilization alone. There were no significant differences in median engraftment times: for ANC, 10, 11 and 15 days respectively for the standard G-, high dose G- and G + GM arms and for platelets, 11, 13 and 14 days respectively. The overall survivals @ the median f/u time of 37 months were 59.8%, 61.8% and 48.1% respectively (p = 0.272) for the three groups. The % primitive HSC (CD34+/CD33- and CD34+/CD38-) from the first mobilization were identical in the 3 patient groups. ConclusionsWe found no advantage to dose escalated G-CSF nor to the combination of G-CSF and GM-CSF to mobilize HSC for autotransplantation in heavily pre-treated patients. We also did not find higher numbers of more primitive CD34 subsets mobilized by these newer strategies. Alternative approaches, e.g. the combination of plerixifor + standard dose G-CSF (Stiff et al: BBMT; 15:249-56, 2009) would appear to be the preferred method of initial HSC mobilization for heavily pre-treated patients. DisclosuresStiff:Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

An In Vivo Mouse Model for Testing the Effect On Hematopoietic Stem Cell Mobilization of Bortezomib Alone and in Combination with G-CSF.

Abstract 2156Poster Board II-133 Introduction:Hematopoietic stem cell (HSC) collection for autologous transplantation is a standard of care for lymphoma and myeloma patients due to old age and extensive prior chemotherapy. However, about 25% of patients fail to collect adequate amount of HSC sufficient for a transplant. Hence, new strategies are being sought to increase HSC collection in these patients. Cell adhesion and chemotaxis are involved in retaining HSC in the marrow. The CXCR4 receptor and its specific ligand, SDF-1 are involved in retaining HSC in the marrow, hence, blocking of CXCR4 results in egress of HSC from the marrow to the peripheral blood. We and others have previously shown that the small bicyclam molecule AMD-3100, by itself can induces >a 5-fold increase in HSC mobilization to the peripheral blood and a 2-3 fold increase in blood HSC over G-CSF alone in myeloma and lymphoma patients in phase 2 and 3 studies. Recently, this drug was approved by the FDA as a mobilizing agent in combination with G-CSF under the name Mozobil. Bortezomib, a proteasome inhibitor is widely used for the treatment of MM and mantle cell lymphoma. However, in addition to blocking of the proteasome, bortezomib has also many other ensuing activities such as immune modulation, blocking of the CXCR4/SDF-1 axis and cell adhesion. Therefore, we hypothesized that Bortezomib will have similar effect as AMD3100 as a mobilizer of HSC. Mice and methods:Phase 1 was designed for optimization of HSC mobilization by Bortezomib. Mice (C57/Bl; x10/group) were injected with saline, or Bortezomib (IV,0.1 to 0.8 mg/kg). Mice were sacrificed after 1 hour,2,4,8, 16 and 24 hours and blood was collected by cardiac puncture. Red cell were lysed and white blood cells (WBS) were counted and cells used for CFU-G, CFU-M and CFU-GM colonies in a standard 14-day, methyl cellulose colony assay (Stem Cell Technologies). Cells were seeded at 200,000/plate in triplicate assay. In phase2 –Bortezomib was used in combination with G-CSF for HSC mobilization. Mice (C57/Bl; x10/group) were injected with saline, or Bortezomib (IV,0.8 mg/kg). Mice were sacrificed after 16 hours and blood was collected and analyzed as above. Two other groups of mice were injected SC with r-hG-CSF (2ug/mouse/dayx4days), or with G-CSF (x4days) followed by 16 hours of Bortezomib (IV,0.8 mg/kg). Mice from these 2 groups were sacrificed and blood was collected by cardiac puncture and processed for WBC and colony assays as above. Cells from each mouse were seeded at 100,000 cells/plate (x5 mice) and 40,000 cells/plate in triplicate assay. Results of phase1:A peak increase in total WBC (p<0.0001, vs. saline) was observed at 8 and 24 hours after Bortezomib. Similarly, significant increase of ∼3 fold and ∼4 fold was observed in the number of blood CFU-GM and BFU-E colonies/plate, respectively after 8 and 24 hours of mobilization with Bortezomib. In a similar study with fixed amount of Bortezomib (0.8mg/kg) administered for 8, 16 and 24 hours, a peak increase in peripheral blood WBC, CFU-GM and BFU-E of 3.5 fold, 4 fold and 8 fold, respectively was observed at 16 hour of Bortezomib (saline vs. Bortezomib, p<0.0001). Results of phase2:An increase of 2 fold, 4 fold and 5 fold in the mean WBC/μl, over saline was observed in the peripheral blood of mice treated with 0.8mg/kg of Bortezomib, G-CSF, or Bortezomib+ G-CSF, respectively. Similarly, an increase of 3 fold, 4.8 fold and 12.9 fold was observed in the number of blood CFU-GM colonies/plate in mice treated with Bortezomib, G-CSF, or Bortezomib+ G-CSF, respectively. Similar to CFU-GM colonies, we observed a 5.7 fold, 7.8 fold and 15 fold increase in the number of BFU-E progenitors in mice treated with Bortezomib, G-CSF, or G-CSF+ Bortezomib, respectively. One way ANOVA revealed a p value <0.0001 between the 4 groups for all comparisons. Conclusions:Bortezomib is a potent mobilizer of HSC and augment G-CSF mobilization of HSC in an additive fashion. Based on these results a pilot study has been initiated to study HSC mobilization in lymphoma and myeloma patients with the combination of bortezomib and G-CSF. Disclosures:Gazitt:millennium: Research Funding.