G-CSF Administration Research Articles

Size-controllable striatal lesion model for evaluation of neuroprotective agents in rats

ObjectivesVarious focal brain lesions are primarily caused by excessive glutamate release. An easy and convenient in vivo striatal lesion model was developed in which reliable and controllable lesion sizes were created by glutamate for evaluation of neuroprotective agents. Materials and MethodsDose-dependent striatal lesions were created by intrastriatal infusion of sodium glutamate in anesthetized male Sprague-Dawley rats (250–350 g). The lesion sizes were estimated with a 2% triphenyltetrazolium chloride stain. Motor disturbances were evaluated by the rotarod test and rotational behavior test. This experimental model was attested with neuroprotective effects of granulocyte colony-stimulating factor (G-CSF, 200 μg/kg), a cytokine growth factor, and estradiol (2 mg/kg), a female sex hormone. ResultsIntrastriatal infusion of 2–6 μmol of 1 M (i.e., 2 Osm) sodium glutamate produced dose-dependent (or controllable) increases in sizes and volumes of striatal lesion in which unpredictable data were also included in analyses. The infusion rate appeared not to affect the lesion size or volume. Although the 2 Osm (1 M) glutamate solution had a much higher osmolarity than brain extracellular fluid (0.3 Osm), control infusions with the 2 Osm of glucose or sodium chloride (NaCl) produced only negligible lesions. The striatal lesion or volume induced by glutamate (4 μmol, 1 M) was reduced by pretreatment with the neuroprotective agents G-CSF and estradiol. The motor disturbance caused by glutamate infusion was also improved by G-CSF administration. ConclusionLocal intrastriatal infusions with controlled doses of glutamate can create a reliable, controllable striatal lesion size. This model is easy and convenient for evaluation of neuroprotective agents.

Daily vs every other day administration of G-CSF following autologous peripheral stem cell transplantation: A prospective randomized study

The purpose of the study was to evaluate whether every other day administration of G-CSF was as safe and efficient as daily administration of G-CSF on neutrophil engraftment following autologous peripheral stem cell transplantation (APSCT). Duration of G-CSF administration, incidence of blood stream infections, duration of febrile neutropenia, duration of non-prophylactic antibiotic therapy, transfusion requirements, duration of hospitalization and G-CSF costs were also studied. Forty-seven patients with diagnosis of lymphoma and multiple myeloma undergoing APSCT were randomized to receive post-transplant daily or every other day G-CSF therapy both beginning on day +1. Both groups were comparable with regard to patient characteristics. There was no significant difference in time to neutrophil engraftment (p=0.31). The duration of G-CSF administration was significantly less in the every other day group (p<0.001). There were no detectable differences seen in the number of febrile days, duration of non-prophylactic antibiotics, the incidence of blood stream infections, transfusion requirements and the duration of hospitalization. There was a trend towards a faster platelet recovery in the every other day group, although the difference was not statistically significant (p=0.059). The number of doses of G-CSF used per transplant is significantly reduced, resulting in a significant reduction in drug costs.

Analysis of chemotherapy-induced neutropenia and optimal timing for prophylactic use of G-CSF in B-cell non-Hodgkin lymphoma patients treated with R-CHOP

Febrile neutropenia (FN) is one of the serious complications of chemotherapy. However, the hematological nadir after chemotherapy and the timing of prophylaxis for FN remain unclear, especially for outpatients. We prospectively analyzed laboratory data from outpatients treated with a single chemotherapy regimen, rituximab (R)-CHOP, on three consultation days (days 8, 10, and 15) after chemotherapy to identify any factors that might predict the onset of the hematological nadir and the optimal timing of G-CSF prophylaxis. A total of 100 courses of chemotherapy (total 33 patients) were analyzed. Onset of the hematological nadir was not predictable in any of the patients who had a white blood cell count (WBC) of >5,500 × 10(6)/L and/or monocyte count of >80 × 10(6)/L on day 8, and thus there was little opportunity for G-CSF prophylaxis in each treatment course. Among patients who had a WBC count of 1,500-5,500 × 10(6)/L on day 8, the monocyte count on day 8 was significantly associated with the hematological nadir. Patients who had a monocyte count of <5 × 10(6)/L on day 8, were identified as a high-risk group for neutropenia for whom G-CSF administration during the current treatment course should be considered. Our results indicate that, in outpatients receiving R-CHOP chemotherapy, the monocyte count on day 8 is a useful marker of the hematological nadir, allowing an opportunity for G-CSF prophylaxis.

Acute toxicities of unrelated bone marrow versus peripheral blood stem cell donation: results of a prospective trial from the National Marrow Donor Program

AbstractAlthough peripheral blood stem cells (PBSCs) have replaced bone marrow (BM) as the most common unrelated donor progenitor cell product collected, a direct comparison of concurrent PBSC versus BM donation experiences has not been performed. We report a prospective study of 2726 BM and 6768 PBSC donors who underwent collection from 2004 to 2009. Pain and toxicities were assessed at baseline, during G-CSF administration, on the day of collection, within 48 hours of donation, and weekly until full recovery. Peak levels of pain and toxicities did not differ between the 2 donation processes for most donors. Among obese donors, PBSC donors were at increased risk of grade 2 to 4 pain as well as grade 2 to 4 toxicities during the pericollection period. In contrast, BM donors were more likely to experience grade 2 to 4 toxicities at 1 week and pain at 1 week and 1 month after the procedure. BM donors experienced slower recovery, with 3% still not fully recovered at 24 weeks, whereas 100% of PBSC donors had recovered. Other factors associated with toxicity included obesity, increasing age, and female sex. In summary, this study provides extensive detail regarding individualized risk patterns of PBSC versus BM donation toxicity, suggesting donor profiles that can be targeted with interventions to minimize toxicity.

Comparision of benefits of early, delayed, and no administration of G-CSF after autologous peripheral blood stem cell transplantation in lymphoma patients

Autologous peripheral blood stem cell transplantation (PBSCT) is commonly used in the treatment of lymphoma patients. G-CSF is widely used to boost white blood cell recovery. However, there are no clear data indicating which strategy of using G-CSF provides the most benefit. The aim of our study was to compare 3 strategies of G-CSF administration: from day +1, from day +5, and no administration. Data from 211 patients treated at 3 centers were gathered retrospectively. The patients in the 3 analyzed groups were not different in regard to type of disease, age, sex, and number of CD34+ cells received. The 3 strategies of G-CSF dosage had very similar results. G-CSF boosted the recovery of white blood cells and shortened the time of neutropenia. However, there were no differences in confirmed infections and the duration of hospitalization after transplantation. Our results question the use of G-CSF in a post-PBSCT setting, as it does not provide significant benefits in reducing the number of infections or shortening the duration of hospitalization.

G-CSF Reduction: The Equilibrium Solution for the DDE and a Sufficient Condition for the Global Stability

paper, we introduce delay differential equation (DDE) models of the hematopoietic system designed for the study of the effects of Granulocyte-Colony Stimulating Factor (G- CSF) administration. G-CSF is used clinically for treating subjects presenting low numbers of white blood cells, a condition referred to as Neutropenia that can result from different causes. The aim of this paper is to study alternative treatment that would minimize the use of G-CSF drug using a mathematical modeling. We propose is a parameter estimation model that considers G-CSF administration for Cyclical Neutropenia (CN), a dynamical disorder characterized by oscillations in the circulating neutrophil count. The model develops the dynamics of circulating blood cells before and after the G-CSF treatment. The model develops the equilibrium solution for the DDE and a sufficient condition for the global stability. The model focuses on the effects of two compartments forms of G-CSF for the treatment of CN (Fast Fourier Transform simulations). For each model, we use a combination of mathematical analysis and numerical simulations (linear chain trick) to study alternative G-CSF treatment that would be efficient while reducing the amount of drug. This reduces the quantity of G- CSF required for potential maintenance. This model gives us good result in treatment. The changes would be analytical and reduce the risk side as well as the cost of treatment in G-CSF.

Stem cell compartmentalization in diabetes and high cardiovascular risk reveals the role of DPP-4 in diabetic stem cell mobilopathy

Bone marrow (BM) derived stem and progenitor cells contribute to cardiovascular homeostasis and are affected by cardiovascular risk factors. We devised a clinical data-driven approach to test candidate stem cell mobilizing mechanisms in pre-clinical models. We found that PB and BM CD34+ cell counts were directly correlated, and that most circulating CD34+ cells were viable, non-proliferating and derived from the BM. Thus, we analyzed PB and BM CD34+ cell levels as a two-compartment model in 72 patients with or without cardiovascular disease. Self-organizing maps showed that disturbed compartmentalization of CD34+ cells was associated with aging and cardiovascular risk factors especially diabetes. High activity of DPP-4, a regulator of the mobilizing chemokine SDF-1α, was associated with altered stem cell compartmentalization. For validation of these findings, we assessed the role of DPP-4 in the BM mobilization response of diabetic rats. Diabetes differentially affected DPP-4 activity in PB and BM and impaired stem/progenitor cell mobilization after ischemia or G-CSF administration. DPP-4 activity in the BM was required for the mobilizing effect of G-CSF, while in PB it blunted ischemia-induced mobilization. Indeed, DPP-4 deficiency restored ischemia (but not G-CSF)-induced stem cell mobilization and improved vascular recovery in diabetic animals. In conclusion, the analysis of stem cell compartmentalization in humans led us to discover mechanisms of BM unresponsiveness in diabetes determined by tissue-specific DPP-4 dysregulation.

Abstract 18814: Autologous CD133+ Immunoselected Cells for the Treatment of "no-option" Critical Limb Ischemia: A Pilot Monocentric Study

Background: critical limb ischemia (CLI) is characterized by chronic ischemic rest pain, ulcers or gangren. CLI results in major amputation in about 50% of patients. Recently, it has been shown that bone marrow-derived (BM) CD34+/CD133+ circulating cells promote new vessel growth in experimental models of CLI. Accordingly, we investigated the feasibility and safety of local intramuscular administration of autologous immunoselected CD133+ (AI-CD133+) cells in “no-option” patients. Methods: the protocol was approved by the ethical committee of our hospital and 10 “no-option” patients suffering CLI were enrolled. BM-derived mononuclear cells were mobilized by G-CSF administration and collected by leukapheresis. CD133+ cells were selected with Clini-MACS (Miltenyi Biotec). AI-CD133+ cells were administered intramuscularly by direct injection. FACS analysis and Hill assay (CFU-Hill colonies) were performed both before and after G-CSF administration. Clinical examination, pain assessment, Ankle Brachial Index (ABI) and transcutaneous oxygen tension (TcPO2) measurements were performed before the treatment and 3, 6 and 12 months thereafter. At the same time points, patients underwent Contrast Enhanced UltraSound (CEUS) to assess muscle perfusion. Results: mobilization of CD34+/CD133+ cells was successful in 8 out of 10 patients. Of relevance, 6 out of 8 treated patients showed relevant clinical improvements. In particular, the treatment drastically ameliorated local pain and favored the healing of the ulcers (figure). The 2 non-responders, both diabetics, underwent major amputation. An overall increase in ABI rate was documented by CEUS (ABI at 12 months increased from 91,45 to 136,82; p&lt;0.05). TcPO2 increased by 45.5±34.1% after 1 year (p&lt;0.05). The number of CFU-Hill colonies was significantly lower (p&lt;0.05) in CLI patients compared with healthy donors; after G-CSF stimulation, the number of CFU colonies significantly increased, except in those patients who did not respond to the treatment. Conclusions: our results demonstrate the feasibility and safety of G-CSF mobilized AI-CD133+ cell injection in end stage CLI patients. Clinical and instrumental data suggest a dramatic effect of CD133+ treatment, except in diabetic patients.

Effects of G-CSF On Acute Lymphoblastic Leukemia.

AbstractAbstract 2564G-CSF is commonly used to treat chemotherapy-induced neutropenia and for the mobilization of hematopoietic stem cells for transplantation in patients with leukemia. Administration of G-CSF has profound effects on the bone marrow microenvironment including the cleavage of molecules required for the maintenance of lymphopoiesis, including CXCL12 and VLA-4. We have recently reported that G-CSF results in the dramatic suppression of B-lymphopoiesis. This, together with previous reports by ourselves, and others, showing that disruption of CXCL12 or VLA-4 slow the progression of B-lineage ALL lead us to consider that G-CSF may similarly antagonize the progression of ALL.To explore this possibility, we examined the impact of G-CSF administration on six human ALL xenografts using a NOD/SCID mouse model. Mice were engrafted without radiation and G-CSF commenced when 1% of the bone marrow consisted of ALL cells. G-CSF was administered twice daily for 10 days, at which time all animals were culled and leukemia assessed in the blood, bone marrow and spleens. Surprisingly G-CSF was found to increase disease progression in two of xenografts investigated (1345 and 0398, referred to as G-CSF responsive xenografts hereafter), while the remainder demonstrated a small reduction in leukemia, with one showing a statistical significant decrease. No evidence for a direct mitogenic effect of G-CSF could be demonstrated in any of the xenografts using exogenous G-CSF in vitro cultures in the presence or absence of human or murine stromal support. Consistent with these findings, and previous reports, little to no G-CSF receptor was detected by flow cytometry or microarray analysis of xenografts.Microarray analysis of the xenografts revealed significant differences in gene expression between the G-CSF responsive xenografts and the remainder of the samples. A total of 83 genes were expressed at a higher level and 127 genes at a lower level in the G-CSF responsive xenografts. The more highly expressed genes included cell cycle regulators (eg cyclin A1), adhesion molecules (eg ALCAM), extracellular matrix components and surface receptors. Perhaps the most interesting was the exclusive expression of the acetylcholine receptor (cholinergic receptor, nicotinic, beta 4, nAChRb4) in the G-CSF responsive cases. Analysis of a large public dataset of childhood ALL samples revealed significantly higher expression of this gene in ALL samples with rearranged MLL (p<0.03). However, small numbers of cases in all ALL subgroups had greater than an 2 fold higher expression compared to normal B cell progenitors. The role of nAChR in the response of ALL cells to micro-environmental changes induced by G-CSF remains to be determined, however, nAChR has known roles in cell proliferation and inhibition of apoptosis. Furthermore G-CSF is known to induce acetylcholine production in other tissues.In summary, G-CSF inhibited leukemia progression in the majority of patient xenografts, however, in a subset of samples G-CSF accelerated disease progression. Clinically, G-CSF administration to ALL patients has not been associated with any major adverse outcomes. However our data suggest that a small subset of patients may experience accelerated disease. Identification of features associated with adverse responses to G-CSF will permit the identification of patients for whom G-CSF may present a risk for increased disease progression. Disclosures:No relevant conflicts of interest to declare.

2-O, 3-O Desulfated Heparin (ODSH) May Mitigate Chemotherapy-Induced Thrombocytopenia and Neutropenia in Patients Treated with Combination Gemcitabine (G)/Nab-Paclitaxel (A), a Myelosuppressive Chemotherapy Regimen

AbstractAbstract 4723 Introduction:Combination G/A has been shown to be apromising investigational therapy for patients with metastatic pancreatic cancer. A randomized phase II trial was initiated to determine if ODSH, a low anticoagulant heparin derivative that blocks several pancreatic cancer cell survival pathways, would enhance the efficacy of G/A. Preliminary data from the first 10 patients administered ODSH demonstrated an unexpected and profound preservation of peripheral blood counts, despite the highly myelosuppressive G/A regimen they also received. Methods:Patients with previously untreated, metastatic pancreatic cancer were treated with G (1000 mg/m2) and A (125 mg/m2) on days 1, 8, and 15 every 28 days until either disease progression or unacceptable toxicities ensued. ODSH (4 mg/kg bolus followed by a 48 hr. continuous IV infusion at a dose of 0.375 mg/kg/hr for 48 hrs) was administered with each treatment. The first 10 patients received A/G plusODSH with plans for a randomized comparison between A/G with and without ODSH involving 50 patients. Patients were required to have ECOG 0–1 as well as adequate hematological and bilirubin level <1.5 ULN. G-CSF administration was permitted per investigator discretion. Here we present hematological data on the 10 patients treated with A/G plus ODSH. Results:10 patients were administered up to 5 cycles of G/A plus ODSH. The patients had a median age of 66 years (range, 59 to 75 years) and 50% were male. The median platelet and neutrophil counts prior to each cycle are listed in Table 1. No patients had grade 3/4 thrombocytopenia during any of the cycles. Forty percent of patients had grade 3 neutropenia, but none had grade 4 during any of the cycles. Only 1 patient received 1 dose of G-CSF after day 15 in cycle #1. Conclusions:A previous trial of G/A in patients with previously untreated, metastatic pancreatic cancer reported grade 3/4 thrombocytopenia and neutropenia in 28% (19% grade 3 and 9% grade 4) and 75% (26% grade 3 and 49% grade 4) of patients, respectively (Von Hoff, J Clin Oncol, 2011). With the addition of ODSH to an equivalent chemotherapy regimen in a similar patient cohort, the current trial reported grade 3 thrombocytopenia and neutropenia in 0% and 40% of patients, respectively, with no patients having grade 4 thrombocytopenia or neutropenia. With successive cycles, median platelet counts were stable to improved and a trend existed for reduced neutropenia with ODSH treatment. An explanation for the apparent salutary effects of ODSH on marrow function may involve its interaction with platelet factor 4 (PF4). PF4 is released from the alpha-granules of injured megakaryocytes after chemotherapy delivery. Increased PF4 levels promote thrombocytopenia by inhibiting thrombopoietin production and megakaryopoiesis (Lambert, Blood, 2007; Lambert, Int J Radiation Oncology Biol Phys, 2011). The high molecular weight and strong negative charge of ODSH may result in avid binding to platelet factor 4 (PF4), possibly leading to decreased PF4 levels and more rapid post-chemotherapy platelet recovery (Joglekar, Thromb Haemost, 2012). PF4 may also facilitate neutrophil adhesion to endothelial cells, and reduced PF4 levels from ODSH binding may demarginate and preserve neutrophil values (Kasper, Blood, 2006). Further study of ODSH as a marrow protective agent in myelosuppressivechemotherapy regimens is warranted.Table 1Blood counts prior to the first 4 cycles of G/A plus ODSH therapyPlatelets (109/L)Neutrophils (109/L)Cycle #MedianRangeMedianRange1273120 to 4294.22.1 to 6.22418175 to 6144.11.6 to 133387123 to 6553.21.8 to 3.94404184 to 6673.72.2 to 5.95*408288 to 4992.651.6 to 5*This cycle has data on 4 patients Disclosures:Off Label Use: The use of nab-paclitaxel and ODSH in pancreatic cancer. Marcus:Paringenix: Employment, Equity Ownership. Quintana Diez:Paringenix, Inc.: Employment.

Use of Plerixafor in Predicted and Proven Poor Mobilizer (according to GITMO criteria). Experience of Policlinico Universitario Tor Vergata

AbstractAbstract 4415 BACKGROUND.Autologous stem cell transplantation (ASCT) of PBSCs has become a widely applied treatment for Multiple Mieloma (MM), non- Hodgking’s lymphoma (NHL) and Hodgking’s lymphoma (HL). Successful engraftment correlates with the number of CD34 hemopoietic progenitors cells infused. However, a part of MM or lymphoma patients (5% to 40%) fail to mobilize adequate numbers of PBSCs and thus cannot undergo to ASCT. The success of PBSCs mobilization is usually assessed by the total number of CD34+ stem cells collected, with a cutoff of 2.0–2.5 ×106 CD34+ cells/kg recipient body weight being considered as a minimum requirement for transplant. Poor mobilization of PBSCs is a major limitation to ASCT. Recently GITMO Working Group worked to define operational criteria for the identification/prediction of the poor mobilizer (PM) patients (Olivieri et al. 2011). Plerixafor, a CXCR4 chemochine antagonist, has been showed to improve significantly PBSC mobilization in PM patients. We present our experience using Plerixafor in PM patients classified according to GITMO criteria. METHODS.Between September 2009 and June 2012, a total of 17 patients (9F-8M) were enrolled. The diagnosis were: 10 MM (5F-5M), 1HL (1M), 6 NHL (4F-2M). The median age was 57 (range 15–66). 7 patients (3MM, 4NHL) were defined “Proven PM” and 10 patients (7MM, 2NHL, 1HL) “Predicted PM” according to GITMO criteria. The mobilization protocol included G-CSF, administered at a dose of 10μg/kg daily on 4 consecutive days. In the evening of the fourth day, patients received subcutaneous plerixafor at a dose of 0,24 mg/kg. Apheresis was initiated on the fifth day, 10–12 h after plerixafor and 1 h after G-CSF administration. Apheresis and daily administration of G-CSF and plerixafor continued until the patient collected enough CD34+ cells for auto- HSCT (> 2 ×106/kg; max 7 plerixafor injections if required). PBSC collection was initiated if peripheral CD34+ cells count was >10μl. A successful mobilization was defined as a total yeld of > 2×106/kg. RESULTS.13 patients (76,5%) collected the minimum number of CD34 cells > 2×106/kg. The diagnosis were: 8MM, 1HL,1 NHL. 7 patients (2NHL; 4 MM; 1 LH; 7 predicted) were able to collect > 5×106/Kg. Only 4 patients (3 MM; 1 LNH; 4 proven) failed the mobilization because the numbers of cells CD34 were < 10μL and these patients did not undergo to apheresis procedures. The collection target of 2×106/Kg was reached in a median of 2 apheresis session (range 1–3). The technical characteristics of the procedures were (median value): blood volume processed 12 L (range 9–14), total CD34+/Kg collected 3,06 × 106(range 2,21-8,62), procedure efficiency 47,5% (range 35,3–79), duration of the procedure 261 minutes (range 210–309). Plerixafor was well tolerated and mild side effects were: reactions in the injection site, gastrointestinal disturbs, muscle pain. During administration of plerixafor we did not observe any significant laboratory abnormalities of liver or renal function. CONCLUSION.Unsuccessful mobilization represents an important limitation to ASCT in lymphoma and MM. In our experience plerixafor allowed to collect an appropriate amount of CD34 also in patients defined “proven PM” significantly reducing the percentage of patients that could not undergo ASCT (target value obtained in 43% of “proven PM”). Confirming the recent literature plerixafor is well tolerated with minimal side effects. We retrospectively applied GITMO criteria for PM patients and our experience, although limited, confirm that the use of a correct definition of PM allows the appropriate use of new mobilizing agents like plerixafor increasing significantly the therapeutic options also in patients who had no possibilities to receive an ASCT with the traditional mobilizing therapy. Disclosures:No relevant conflicts of interest to declare.

Efficacy and Safety of Conditioning Treatment Using Bendamustine in Autologous Hematopoietic Cell Transplantation in Heavily Pretreated Patient with Lymphoma

Abstract 4537Most of lymphoma patients are chemo- and radiosensitive. Using a conventional therapy we can cure about 50% of patients (pts). Unfortunately the rests achieved only partial remission or have a rapid relapse after treatment. These pts are a real challenge for nowadays hematology. Till now there is not a recommended scheme of therapy. Using high dose chemotherapy with autologous hematopoietic cell transplantation (AHCT) is the most popular one. During the last year in our Department we have been started using BeEAM as the conditioning treatment, and the results of a single centre observation are presented now. Material and MethodsActually: between April, 2011 and February, 2012 we conducted AHCT using BeEAM as conditioning treatment in 23 pts with lymphoma (18 pts with Hodgkin Lymphoma (HLy), 2pts with diffuse large B cell lymphoma (DLBCL), 2 pts with T cell lymphoma (TCL), 1 with follicular lymphoma (FL). There were 14 male and 9 female, with a median age of 37,6 (range 21–62 yrs). Ann Arbor staging at diagnosis was as follows: II-33%, III-50%, IV-17%; 83% of patients manifested B-symptoms. Clinical manifestation at diagnosis included: enlargement of the lymph nodes (n=18), bone marrow infiltration (n=2), lung infiltrates (n=3). Initially, all patients received at least 2 cycles of chemotherapy; in HLy all pts received ABVD, in non HLy- RCHOP. None of them achieved complete remission (CR). Partial response (PR), defined as the reduction of measurable disease by ≥50% without the appearance of any new lesions, achieved 11pts. The rests underwent high dose chemotherapy (HDT) followed by AHCT without remission (NR).Stem cells were collected from peripheral blood after IVE chemotherapy (Ifosfamide 3g/m2 iv in 1–3d, Etoposide200mg/m2 iv in 1–3d, Epirubicine 50mg iv in1d) and subsequent administration of G-CSF at a dose of 10ug/kg/d, starting from +5 day after chemotherapy till the last day of collection. Collections were performed using Optia Spectra. All pts collected the sufficient number of CD34+ cells for AHCT procedure. Conditioning regimens before AHCT consisted of BeEAM (Bendamustine 200mg/m2 in -8-7d, Etoposide 200mg/m2 in -6-3d, Ara-C 400mg/m2 in -6-3d, Melphalan 140mg/m2 in -2d). A median number of transplanted CD34+ cells was 5,58 (2,05–17,8×10∧6/kg). All except one pt successfully engrafted. 1 pt with Hly died within 8 day after transplantation due to infection. Hematopoietic recovery was as following: WBC count > 1,0×10∧9/L after median of 12 days (range 9–15 days),ANC> 0,5×10∧9/L after median of 13 days (range 9–16 days) and platelet count >20×10∧9/L after median of 13days (range 7–20 days). Results3 pts died after AHCT. Two of them due to progression of the disease within 5 and 16 months after AHCT. Both were without remission before AHSCT. 1 pt died due to heart insufficient after 3 months after AHCT.The complications after transplantation procedure were rare and included mainly: bacterial infection in the upper respiratory tract (n=3), viral skin infection (n=1), oral mucositis (n=2). At the last contact, 19 pts are alive and 9 of them achieved CR, and 3 – stable disease. One pt underwent sibling alloHCT and 2 others will undergo alloHCT within 2 months. 4 pts with Hly were treated with anty CD 30+. ConclusionAutologous hematopoietic cell transplantation using IVE for mobilization and BeEAM as conditioning is effective treatment for lymphoma patients with refractory disease. Disclosures:No relevant conflicts of interest to declare.

Poor Graft Function in Recipients of T Cell Depleted (TCD) Allogeneic Hematopoietic Stem Cell Transplants (HSCT) Is Mostly Related to Viral Infections and Anti-Viral Therapy.

AbstractAbstract 3147 Introduction:Poor graft function (PGF) without immune rejection, defined as persistent cytopenias with hypocellular marrow and full donor myeloid chimerism, can be a life-threatening complication after allogeneic HSCT. It is commonly caused by viral infectious, myelosuppressive drugs like antivirals, and graft-vs-host disease (GvHD). Treatment options include supportive therapy with transfusions and growth factors and in severe cases administration of additional hematopoietic stem cells (HSCs) from the same donor without conditioning (stem cell boost). The incidence, natural history, and the indications for stem cell boost therapy are not well defined. Aims:To assess the incidence, etiologies, and indications for stem cell boost for PGF in a homogeneous group of patients with advanced MDS and AML who underwent TCD HSCT from matched or mismatched related or unrelated donors after conditioning with the same myeloablative regimen. Patients and methods:Poor graft function was defined as persistent neutropenia (ANC <1,000 μL and G-CSF administration x3 in 30 days), thrombocytopenia (platelets <50,000 μL or platelets transfusion × 4 in 30 days), and/or hemoglobin <8 g//dL after engraftment with hypocellular BM and full donor myeloid chimerism. Severe PGF was defined as ANC <500 μL, red cell transfusion-dependent anemia with reticulocytopenia of < 20,000 μL, and platelets <20,000 μL. The patient population in which this study was done included 42 patients enrolled between 09/2009 and 05/2012 in a phase 2 trial of palifermin peri-transplant to reduce transplant-related mortality. The median age was 57.5 years (1–65). All patients received the same myeloablative conditioning regimen with busulfan, melphalan, fludarabine, rabbit ATG and palifermin peri-transplant. G-CSF mobilized donor peripheral blood stem cells underwent CD34+ selection and depletion of T cells using CliniMACS immunomagnetic selection columns (Milteny Biotec). Donors were HLA matched (31; 13 related and 18 unrelated) or mismatched unrelated (11). Chimerism was determined in bone marrow as well as neutrophils, B cells, and T cells by short tandem repeat analysis on DNA extracted from bone marrow and peripheral blood cell subsets. Results:Forty-one patients were evaluable for this analysis; 1 patient was not included as he rejected the allograft shortly after engraftment. There were 8 cases of PGF with a cumulative incidence (CI) at 1 year of 18% (13% HLA matched, 33% HLA mismatch). The etiology was infection in 7 cases, and unknown in the 8th case. This patient presented with presumed autoimmune anemia and thrombocytopenia associated with a hypercellular marrow and did not respond to multiple lines of therapies. Her marrow became later hypocellular and met the criteria for PGF. None of the PGF cases in this series was associated with GvHD at the time of diagnosis of PGF. The infectious etiologies included: 6 viral infections and 1bacterial sepsis + myelosuppressive drugs. The most common viral etiology associated with PGF was CMV (50%). The 1-year CI of PGF in CMV seropositive patients was 25% and in CMV seronegative patients was 14%. Of note, HHV6 viremia was detected in patients with PGF. HHV6 is not routinely monitored, however, making it difficult to establish a causative role. All patients had moderate PGF at diagnosis and 3 cases had worsening of cytopenias and met the criteria for severe PGF. To date, 3 PGF patients have died from EBV-PTLD, adenovirus infection or GVHD (developed after CMV treatment with liposomal cidofovir), 3 continue to suffer from PGF and 2 patients are alive with recovered good blood counts after eradication of CMV. Of the 3 patients with persistent PGF, one received a TCD boost with no response, and 2 continued to be treated for CMV viremia. A stem cell boost was indicated if pancytopenia persisted despite eradication of cause of the PGF. In this small series, there were not enough events to evaluate association between PGF and CD34 cell dose, CD3 cell dose or day 100 T-cell chimerism. Conclusions:In this homogenous population of patients with MDS who underwent TCD allogeneic HSCT, the incidence of PGF is about 20%. The most common cause was viral infection with predominance of CMV. Therefore, strategies to prevent CMV reactivation in patients undergoing allogeneic HSCT has the potential to reduce the risk of PGF and avoid the need for infusion of additional stem cells. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Microrna-155 Promotes Hematopoietic Stem and Progenitor Cell Mobilization and Proliferation

Abstract 214MicroRNAs (miRNAs) are small non-coding RNAs involved in various physiological processes, including hematopoiesis. Although miRNAs are broadly studied with regards to normal and malignant leukocyte development, the role of miRNAs in hematopoietic stem and progenitor (HSPC) migration and mobilization is poorly understood. Currently, induction of HSPC mobilization from the bone marrow (BM) to the peripheral blood (PB) is the major mean to harvest HSPCs for clinical transplantation. Recently, several miRNAs were found to be upregulated in macaque G-CSF-mobilized CD34+ HSPCs, among them the oncogenic miRNA mir-155 (Donahue et al., Blood 2009).To study the involvement of mir-155 in HSPC regulation, we examined hematopoiesis in mir-155 knock out (KO) mice. Of interest, mir-155 KO mice had normal BM and PB levels of mature cells, but reduced levels of immature BM Lineage−/Sca-1+/c-Kit+ (LSK) and primitive BM CD34−LSK HSPCs. Profiling of mir-155 expression in murine hematopoietic BM populations, following G-CSF treatment, revealed differential expression patterns in wild type (WT) mice. G-CSF treatment upregulated mir-155 levels in immature LSK cells, in T-cells and in Mac-1+/Gr-1+ monocyte/macrophages. In contrast, G-CSF downregulated mir-155 levels in common lymphoid progenitors and in B-cells. Suggesting that mature hematopoietic cells may also participate in HSPC mobilization process. G-CSF administration to mir-155 KO mice resulted in reduced HSPC mobilization, as assessed by CFU-C and LSK cell counts in the PB. Surprisingly, G-CSF treatment increased BM LSK cell frequency in mir-155 KO mice to the same levels as in WT mice. On the contrary, G-CSF treatment reduced BM CD34−LSK cell frequency in WT mice and increased it in mir-155 KO mice showing an opposing effect on the more primitive HSPC population. Since mir-155 is involved also in mesenchymal development regulating osteoblast differentiation, we propose that BM HSPC pool reduction could be mediated by the stromal microenvironment. Additionally, osteoblasts and other BM residing cells undergo substantial changes in response to G-CSF that might be mediated by mir-155.To determine whether the mobilization defect is hematopoietic cell-autonomous or due to an abnormal microenvironment, we examined G-CSF-induced mobilization in chimeric mice. Mir-155 KO mice reconstituted with wild type (WT) BM cells had normal mobilization as WT mice reconstituted with WT BM cells. Of interest, WT mice reconstituted with mir-155 KO BM cells showed reduced mobilization as mir-155 KO mice reconstituted with mir-155 KO BM cells. These results indicate that the mobilization defect in mir-155 KO mice is also due to a defect in HSPC motility. Since the CXCL12/CXCR4 axis plays a major role in HSPC mobilization, we examined the ability of mir-155 KO cells to perform CXCL12-induced migration and found reduced migration capacity of HSPCs in vitro. Although having reduced migration potential, mir-155 KO LSK cells had normal CXCR4 expression levels, suggesting that an aberrant intracellular response to SDF-1 is responsible for the observed defect. In support, AMD3100 treatment to mir-155 KO mice resulted in reduced HSPC rapid mobilization. Since SHIP-1 phosphatase mRNA is targeted by mir-155 in hematopoietic cells (Costinean et al., Blood 2009) and SHIP-1 KO hematopoietic cells exhibit increased migration towards CXCL12 (Kim et al., JCI 1999), we examined intracellular SHIP-1 expression during HSPC mobilization. SHIP-1 levels were downregulated in WT BM LSK cells in response to G-CSF or AMD3100 mobilizing treatments. In contrast, mir-155 KO BM LSK cells upregulated SHIP-1 levels in response to the mobilizing treatments. These results suggest that mir-155 may promote HSPC mobilization and increased motility via SHIP-1 downregulation.In summary, our data indicates that mir-155 directly promotes HSPC motility and mobilization by SHIP-1-mediated regulation of intracellular response to CXCL12 signaling. We also propose the mechanism of indirect regulation of BM HSPC pool size during steady state and following G-CSF treatment by mir-155, via stromal BM microenvironment, which is currently under investigation. Deciphering the mechanisms of HSPC migration and maintenance in general and by mir-155 in particular, may potentially improve clinical mobilization protocols and contribute to increased donor BM engraftment following transplantation. Disclosures:No relevant conflicts of interest to declare.

Differential Distribution of Activated Innate and Adaptive Immune Subsets in G-CSF Mobilized Hematopoietic Stem Cell Allografts May Influence Incidence of Acute (aGVHD) and Chronic Graft-Versus-Host Disease (cGVHD)

AbstractAbstract 4192Donor T cells are known to be the most powerful effectors for GVHD. Recently, the dose of invariant NKT and B cells in the allograft have been associated with incidence of aGVHD in sibling transplants (Chaidos et al, Blood 2012; Michonneau et al, Brit.J.Hem 2009). Murine models suggest a role for donor B cells and CD4+ T cells in development of cGVHD (Young et al, J.Immunol 2012). The influence of NK cells, NKT cells, B cells in allografts from matched unrelated donor (MUD) transplants on incidence of cGVHD is unknown. Here we present results on influence of innate and adaptive immune subsets in the allograft and the incidence of aGVHD and cGVHD in 108 patients (pts). We analyzed the absolute numbers of T, NK, NKT and B cells along with extensive immunophenotypic characterization of their activation status in consecutive allografts obtained from both sibling and MUD between 2010– 2011. All allografts were peripheral blood stem cells mobilized after 5 days of G-CSF administration. Of the 108 pts, 67 pts received grafts from MUD; 71 pts had received a reduced-intensity conditioning regimen. All pts receiving allografts from MUD received anti-thymocyte globulin. Median age of pts at transplant was 55 years (21–73 years). Median CD34+ dose was 6.51 x106/kg and median CD3+ dose was 2.33 x108/kg. The incidence of aGVHD and cGVHD was 58% and 38% respectively. We compared in an unsupervised approach whether absolute numbers of resting and activated NK, NKT, B, CD4+ and CD8+ T cells were different in pts with vs. without aGVHD/cGVHD. Univariate logistic regression models and Wilcoxon rank sum tests were used to assess differential expression of absolute and percent immune subset markers between those who did vs. did not develop GVHD, analyzing separately for aGVHD and cGVHD. In addition, some markers had a number of absolute values of zero, where the parameter of interest was whether or not a donor had a non-zero value and its association with GVHD in pts. Results:Median numbers of activated NK cells (CD3−CD56+CD16+CD69+) cells were significantly higher in pts with vs. without aGVHD (586.4/μL vs. 270.5/μl, p=0.003). Higher median numbers of resting NK cells (CD3−CD56+CD16+) and activated B cells (CD19+CD80+) were associated with cGVHD but not aGVHD. Non-zero levels of CD4+ T cells expressing prostaglandin D2 (PGD2) receptor (CD4+CD294+), chemokine receptor CCR3 (CD4+CD193+), chemokine receptor 4 CCR4 (CD4+CD194+) and CXCR3 (CD4+CD183+) in the allograft were associated with lower incidence of cGVHD. Also CD4+ T cells expressing both CCR4 and PGD2 receptors were associated with lower incidence of cGVHD. Significant differences were seen in median numbers (Table 1) and % of non-zero values (Table 2) of NK cells, B cells, and activated CD4+ T cells between those who did vs. did not develop cGVHD. Immunophenotypic analyses of pts receiving these allografts at days 30 and 100 post transplant were also performed. Updated results about correlation of these subsets between allografts and post-transplant patient samples will be presented. Multivariate analyses of these subsets in conjunction with clinical factors are being performed. In conclusion, activated B cells in the allograft may influence the incidence of cGVHD; in particular B cells expressing CD80, a co-stimulatory molecule for T cells and their association with cGVHD confirms results from murine models. Interestingly, CD4+ T cells associated with lower incidence of cGVHD expressed receptors for chemokines, CXCR3 and prostaglandin D2. These homing receptors are up regulated selectively in CD4+ T cells in the absence of chronic GVHD, suggesting a protective role for these receptors in the pathogenesis of cGVHD. Activated and resting NK cells were also associated with acute and chronic GVHD respectively. These results showing an association of specific immune subsets in the allograft and GVHD may provide opportunities for therapeutic interventions for graft engineering.Table 1Median absolute numbers of NK and B cells by cGVHD statusSubsetcGVHDNo cGVHDp-valueCD3−/CD56+/CD16+/CD117−636148920.036CD19+/CD80+109.671.20.012Table 2Percent with non-zero absolute numbers of activated CD4+ T cells by cGVHD statusSubset% non-zero values in pts without cGVHD% non-zero values in pts with cGVHDp-valueCD4+/CD193+27.9%5.1%0.004CD4+/CD294+41.0%10.3%0.0013CD4+/CD183+41.0%10.3%0.0007CD4+/CD194+42.6%10.3%0.0006CD4+/CD294+/CD194+36.1%7.7%0.0017 Disclosures:Off Label Use: Lenalidomide in AML. Jaglowski:Pharmacyclics: Research Funding. Benson:Innate Pharma: Research Funding. Devine:Sanofi: Honoraria, Research Funding.

Bendamustine (Treanda®), Etoposide and Dexamethasone (BED) Followed by GCSF Effectively Mobilizes Autologous Peripheral Blood Hematopoietic Stem Cells

AbstractAbstract ▪4126▪This icon denotes a clinically relevant abstract Background:High dose chemotherapy followed by autologous stem cell transplantation (ASCT) is a standard of care for patients with advanced or treatment refractory multiple myeloma (MM) and non-Hodgkin lymphoma (NHL). Stem cell proliferation and mobilization can be enhanced though the addition of myelosuppressive chemotherapy to GCSF administration. Chemotherapeutic agents without cross resistance to prior therapies may support peripheral blood stem cell (PBSC) collection and improve patient outcomes by exacting a more potent direct anti-tumor effect prior to ASCT. Bendamustine (Treanda®) is a synthetic chemotherapeutic agent that shares structural similarities to both purine analog and alkylating agents without significant cross resistance to other compounds in either drug class. Bendamustine appears to have low stem cell toxicity in vitro, is well tolerated, and has activity in MM and NHL. We hypothesized that bendamustine’s activity in patients with disease resistant to first line therapies makes it a logical candidate for chemotherapy based PBSC mobilization. Methods:Patients were eligible if they had relapsed or refractory MM, B-cell NHL or T-cell NHL and were candidates for ASCT. Other criteria included: age >18 years, ANC >1,500/mm3, platelets >100,000/mm3, adequate renal and hepatic function, <3 prior myelotoxic regimens, <6 cycles of lenalidomide, no prior failed mobilization attempt, and no prior pelvic/spinal irradiation. Patients received 1 cycle of BED therapy [bendamustine (120 mg/m2 IV d 1, 2 - provided along with financial support for this study by Teva Pharmaceuticals), etoposide (200 mg/m2 IV d 1– 3), dexamethasone (40 mg PO d 1– 4), delivered as an outpatient, followed by filgrastim (10 mcg/kg/day; starting on d 5 through end of collection)]. Apheresis was initiated when peripheral blood CD34 cell counts were >5/μL. The primary endpoint was successful mobilization, defined as collection of >2.0 × 106CD34 cells/kg. Adverse events (AEs) were graded using the CTCAE v4.0. Results:Twenty patients (16 MM, 3 B-cell NHL, 1 NK/T-cell NHL) were treated. The median age was 59 years (range 43–70), and the median number of prior therapies was 1 (range 1–3) for MM and 2 (range 2–3) for NHL patients. All patients (20/20) were successfully mobilized. The median number of CD34+ cells collected was 19.11 × 106/kg (Mean 22.49; range 4.35 to 55.51 × 106). All MM patients collected >10 × 106 CD34+cells/kg. The median time from BED mobilization therapy to the first day of CD34 stem cell collection was 12 days (mean 12.05; range 10 to 20 days). The median number of days of apheresis was 1 (mean 1.45; range 1 to 4). A predictable pattern of leucocyte nadir and recovery was demonstrated (88% of patients started apheresis between days 10–12). One patient (5%) was given plerixafor and for 2 patients (10%) the dose of GCSF was increased to 16 mcg/kg twice daily. Among the 20 patients mobilized and collected, 12 have thus far undergone ASCT and 100% (12/12) have achieved an unsupported neutrophil count >500/μL at an average of 14.3 days after PBSC infusion and a platelet count >20K/μL at an average of 10 days. Serious AEs (SAEs) were observed in 5 patients and 1 patient died due to disease progression. No unexpected grade 3 or greater treatment related SAEs were seen. Disease response assessments are ongoing. The original protocol design involved 3 agents (bendamustine, dexamethasone and GCSF [BDG]). After the first 3 patients enrolled, the mobilization regimen was modified to include etoposide because BDG did not yield a predictable pattern of leucocyte nadir and recovery, thus complicating timing for apheresis (median time to collection 22 days). The first 3 patients were censored from the analysis, however all 3 patients were successfully mobilized and collected. Conclusions:The initial experience with PBSC mobilization after BED in this phase II study suggests the regimen is safe and effective, while the use of BDG does not yield predictable CD34 kinetics. Time to neutrophil and platelet engraftment after ASCT appears unimpaired when compared with other chemotherapy based mobilization regimens. Large numbers of stem cells were rapidly mobilized and resulted in short durations of apheresis. No patient with MM collected <10 × 106 CD34+ cells/kg (sufficient for 2 ASCTs). The regimen was very well tolerated and these findings suggest that the role of bendamustine in PBSC mobilization should be further explored. Disclosures:Green:Teva Pharmaceuticals: Research Funding. Holmberg:Millenium: Research Funding; Otsuka: Research Funding; Merck: Research Funding; Seattle Genetics: Research Funding; Sanofi: Research Funding. Budde:Teva Pharmaceuticals: Research Funding. Gopal:Teva Pharmaceuticals: Research Funding.

G-CSF Stimulation and Coronary Reinfusion of Mobilized Circulating Mononuclear Proangiogenic Cells in Patients with Chronic Ischemic Heart Disease: Five-Year Results of the TOPCARE-G-CSF Trial

Prognosis of patients with heart failure remains poor despite improved conventional and interventional treatment regimens. The improvement of neovascularization and repair processes by administration of bone marrow-derived cells modestly improved the recovery after acute myocardial infarction. However, circulating patient-derived cells are reduced in number and function particularly in chronic heart failure. Therefore, we tested the hypothesis whether the mobilization of circulating mononuclear proangiogenic cells (CPCs) by G-CSF may overcome some of these limitations. In the present pilot study, 32 patients with at least 3-month-old myocardial infarction were randomized to G-CSF alone (G-CSF group) or intracoronary infusion of G-CSF-mobilized and cultured CPCs into the infarct-related artery (G-CSF/CPC group). Primary endpoint of the study was safety. Efficacy parameters included serial assessment of LV function, NT-proBNP levels, and cardiopulmonary exercise testing. G-CSF effectively mobilized circulating CD34(+)CD45(+) cells after 5 days in all patients (408 ± 64%) without serious adverse events. At 3 months, NYHA class and global LV function did not show significant improvements in both treatment groups (G-CSF: ΔLVEF 1.6 ± 2.4%; p = 0.10; G-CSF/CPC: ΔLVEF 1.4 ± 4.1%; p = 0.16). In contrast, target area contractility improved significantly in the G-CSF/CPC group. During 5-year follow-up, one patient died after rehospitalization for worsening heart failure. Eleven patients underwent further revascularization procedures. NT-proBNP levels, cardiopulmonary exercise capacity, and NYHA class remained stable in both treatment groups. The results from our pilot trial indicate that administration of G-CSF alone or G-CSF-mobilized and cultured CPCs can be performed safely in patients with chronic ischemic heart disease. However, only minor effects on LV function, NT-proBNP levels, and NYHA classification were observed during follow-up, suggesting that the enhancement of CPCs by G-CSF alone does not substantially improve intracoronary cell therapy effects in patients with chronic ischemic heart failure.

Granulocyte colony stimulating factor exacerbates antineutrophil cytoplasmic antibody vasculitis

ObjectivesGranulocyte colony stimulating factor (GCSF) is important in mobilising neutrophils from the bone marrow but also has a range of proinflammatory effects. We therefore decided to investigate the role of...

Phase I Study of Intravenous Low-dose Granulocyte Colony-stimulating Factor in Acute and Subacute Ischemic Stroke

Granulocyte colony-stimulating factor (G-CSF; filgrastim) may be useful for the treatment of acute ischemic stroke because of its neuroprotective and neurogenesis-promoting properties, but an excessive increase of neutrophils may lead to brain injury. We examined the safety and tolerability of low-dose G-CSF and investigated the effectiveness of G-CSF given intravenously in the acute phase (at 24 hours) or subacute phase (at 7 days) of ischemic stroke. Three intravenous dose regimens (150, 300, or 450 μg/body/day, divided into 2 doses for 5 days) of G-CSF were examined in 18 patients with magnetic resonance imaging (MRI)-confirmed infarct in the territory of the middle cerebral artery. Nine patients received the first dose at 24 hours poststroke (acute group) and 9 patients received the first dose on day 7 poststroke (subacute group; n = 3 at each dose in each group). A scheduled administration of G-CSF was skipped if the patient's leukocyte count exceeded 40,000/μL. Patients received neurologic and MRI examinations. We found neither serious adverse event, drug-related platelet reduction nor splenomegaly. Leukocyte levels remained below 40,000/μL at 150 and 300 μg G-CSF/body/day, but rose above 40,000/μL at 450 μg G-CSF/body/day. Neurologic function improvement between baseline and day 90 was more marked after treatment in the acute phase versus the subacute phase (Barthel index 49.4 ± 28.1 v 15.0 ± 22.0; P < .01). Low-dose G-CSF (150 and 300 μg/body/day) was safe and well tolerated in ischemic stroke patients, and leukocyte levels remained below 40,000/μL.

1547PD - G-CSF as Secondary Prophylaxis of Chemotherapy-Induced Neutropenia in Patients with Solid Tumors: Results of a Prospective, Observational Study

ABSTRACT Background There are little available data on secondary prophylaxis of chemo-induced neutropenia. We designed this multicentre, prospective and observational study to identify predictive factors of occurrence of neutropenic events (NE) subsequent to a previous episode, in patients with solid tumors (primary objective). Secondary objectives were to determine the incidence of NE, describe prophylactic strategy: cycle delay, dose reduction, G-CSF prescription, and its impact on the recurrence of NE. Patients and methods Patients ≥ 18 years were included if they experienced a NE during any previous cycle (reference cycle A), with no prior G-CSF administration. Neutropenic events were defined as any neutropenia grade 1-4, febrile or not, that impacts on the subsequent cycles (cycle delay and/or dose reduction and/or use of G-CSF). Patients were followed for a total of 5 cycles. Risk factors of febrile neutropenia (FN), and prophylactic strategies (with or without G-CSF) were included in univariate and multivariate analyses to assess their predictive value on recurrence of NE. Results 625 patients included, 548 (87.7%) evaluable.378 (69%) female, mean (SD) age (years) 61.7 (12.3), WHO PS 0-1 88.3%, breast: 40%, colorectal: 15.7%, lung: 11.9%. Metastatic disease: 53.3%. During cycle A, 88 patients (16.1%) experienced FN, 42 (7.7%) neutropenic fever and 418 (76.3%) neutropenia (any grade w/o fever). 44.5% had cycle delay, 22.3% dose reduction and 466 (85%) received prophylactic G-CSF (pegfilgrastim 59.7%, lenograstim 27.3%, filgrastim 10.3% and biosimilar 2.1%). Incidence of NE in subsequent cycles and prophylactic strategies are shown in the table below. In multivariate analysis, G-CSF use (HR:0.32 (0.24; 0.43; p Conclusion Prophylactic strategy with G-CSF has significant efficacy in reducing the incidence of NE, and should be considered as the best option in the secondary prophyalxis setting. Cycle A (no G-CSF) N = 548 Cycle B N = 548 Cycle C N = 548 CycleD N = 442 Cycle E N = 344 Febrile neutropenia 88 (16.1%) 3 (0.5%) 4 (0.7%) 0 1 (0.3%) Neutropenic fever 42 (7.7%) 2 (0.4%) 4 (0.7%) 1 (0.2%) 0 Neutropenia w/o fever 418 (76.3%) 111 (20.3)% 95 (17.3%) 50 (11.3%) 47 (13.7%) Cycle delay - 244 (44.5%) 44 (8.0%) 23 (5.2%) 18 (5.2%) Dose reduction - 122 (22.3%) 27 (4.9%) 17 (3.8%) 12 (3.5%) use of Prophylactic G-CSF - 466 (85.0%) 413 (75.4%) 332 (75.0%) 247 (71.8%) Disclosure G. Freyer: Consultant for Amgen All other authors have declared no conflicts of interest.