High-dose Granulocyte Colony-stimulating Factor Research Articles

Comparing Phenotyping, Gene Expression, and Function of T Lymphocytes before Mobilization Chemotherapy Versus after Stem Cell Harvesting in Patients with B Cell Lymphoma and Multiple Myeloma

Chimeric Antigen Receptor T cell (CAR-T) therapy is an effective treatment for relapsed and refractory hematological diseases. However, a significant medical unmet need persists, particularly for patients at the relapse and refractory (R/R) status, who often experience leukopenia or lymphopenia during lymphocyte collection. This limitation poses considerable challenges to the successful implementation of CAR-T treatment, primarily due to the impaired function of T cells resulting from multiple lines of treatment and disease progression. As a consequence, the quality of the CAR-T cell product may be compromised. Before introducing CAR-T therapy, most patients with B cell lymphoma or multiple myeloma (MM) experienced mobilization chemotherapy and high-dose granulocyte colony-stimulating factor (G-CSF) administration for stem cell harvesting, followed by autologous stem cell transplantation as a salvage or consolidation treatment. Given that the procedure of lymphocyte collection for CAR T cell manufacturing is quite similar to stem cell harvesting, it becomes reasonable to explore the possibility of collecting lymphocytes immediately after the stem cell harvesting process. The advantage of this strategy is that patients are typically in a healthier condition with better T cell function compared to their status during R/R status. However, the effects of mobilization chemotherapy and high-dose G-CSF on T lymphocytes remain inadequately understood. To assess the feasibility of lymphocyte collection, it is essential to delve into the function of T lymphocytes at two critical time points: before mobilization chemotherapy compared to after stem cell harvesting. In this study, we recruited 19 patients with B cell lymphoma or MM. Paired peripheral blood samples were obtained before administering mobilization chemotherapy and after stem cell harvesting. Flow cytometry analysis using CCR7 and CD45RA markers was applied to classify T cell subsets, including T naive or T stem cell-like (TN/SCM, CCR7+/CD45RA+), T central memory (TCM, CCR7+/CD45RA-), T effector memory (TEM, CCR7-/C5RA-), and T effector (Teff, CCR7-/CD45RA+) (Figure A). The data indicated no significant difference in T cell phenotype between pre-mobilization chemotherapy and after stem cell harvesting. CD4and PD-1 expression were also analyzed and showed no significant difference between the two time points. To gain further insights, we performed RNA sequencing on 3 paired samples from patients with large B cell lymphoma (UPN#3, UPN#11, UPN#16). Through our observations, we noticed that these 3 paired samples could be clustered into 2 groups based on differential gene expression (Figure B), which corresponds to pre-mobilization chemotherapy versus post-stem cell harvesting status. Our findings strongly indicate that T cells at the pre-mobilization chemotherapy status exhibit significant differences in gene expression compared to those at the post-stem cell harvesting status. Specifically, T cells from post-stem cell harvesting show significantly higher expression of NIBANI, ALOX5AP, and GLUL compared to those from pre-mobilization. NIBANI plays a vital role in down-regulating apoptosis by interacting with the MDM2/p53/Bcl2/Bax pathway, promoting cell survival under stress-dependent conditions. GLUL is responsible for glutamine synthetase, and glutamine is an important source of biosynthetic precursors in active T cells. On the other hand, T cells from pre-mobilization show higher expression of TOX, DUSP4, and TGFBR3 compared to those from post-stem cell harvesting. TOX is a transcription factor involved in T cell exhaustion and senescence, while DUSP4 expression is associated with defective TCR signaling and accelerated T cell senescence. Our data indicate that the proportion of T naive or T stem cell-like phenotypes remains unchanged after mobilization chemotherapy and G-CSF injections during stem cell harvesting. RNA sequencing data further reveal that T cells at the post-stem cell harvesting status are associated with down-regulated apoptosis, favorable survival and activation, and reduced exhaustion and senescence compared to those at the pre-mobilization stage. This study suggests that instead of collecting lymphocytes during relapse or refractory status, collecting them right after stem cell harvesting may be a viable alternative.

Direct Potential Modulation of Neurogenic Differentiation Markers by Granulocyte-Colony Stimulating Factor (G-CSF) in the Rodent Brain.

The hematopoietic granulocyte-colony stimulating growth factor (G-CSF, filgrastim) is an approved drug in hematology and oncology. Filgrastim’s potential in neurodegenerative disorders is gaining increasingly more attention, as preclinical and early clinical studies suggest it could be a promising treatment option. G-CSF has had a tremendous record as a safe drug for more than three decades; however, its effects upon the central nervous system (CNS) are still not fully understood. In contrast to conceptual long-term clinical application with lower dosing, our present pilot study intends to give a first insight into the molecular effects of a single subcutaneous (s.c.) high-dose G-CSF application upon different regions of the rodent brain. We analyzed mRNA—and in some instances—protein data of neurogenic and non-neurogenic differentiation markers in different regions of rat brains five days after G-CSF (1.3 mg/kg) or physiological saline. We found a continuous downregulation of several markers in most brain regions. Remarkably, cerebellum and hypothalamus showed an upregulation of different markers. In conclusion, our study reveals minor suppressive or stimulatory effects of a single exceptional high G-CSF dose upon neurogenic and non-neurogenic differentiation markers in relevant brain regions, excluding unregulated responses or unexpected patterns of marker expression.

Mobilization with high-dose granulocyte colony-stimulating factor alone at 12 μg/kg twice a day in high-risk pediatric patients: A retrospective analysis of the experience in a single center.

Mobilization regimes in pediatric patients at high risk for poor mobilization are not standardized across different institutions. We present a retrospective analysis of our experience with a high-dose granulocyte colony-stimulating factor (G-CSF) regime of 12μg/Kg per body weight (BW) twice a day for 4 days used in high-risk patients. We report the results of all pediatric patients mobilized with high-dose G-CSF between January 1999 and February 2021 in our center. A successful mobilization was defined as a peripheral blood (PB) CD34+ cell count of ≥10 CD34+ cells/μl on the fifth day of mobilization immediately before leukapheresis. A minimum cell yield of ≥2× 106 CD34+ cells/Kg of BW was required for a successful collection. Of the 262 patients included in the analysis, mobilization failure was found in 27 (10.3%). In a univariate analysis, this was associated with age, weight, baseline diagnosis, and having undergone a previous mobilization cycle, the latter being the only factor that remained significantly associated in a multivariate analysis (P= 0.03). The 54 patients (20.6%) did not reach the minimum required CD34+ cell yield. 50.4% of the patients reported adverse events (AEs) during the mobilization period, and 23 (9.1%) reported 3 or more concomitant AEs. However, all of them were mild and did not affect the mobilization schedule. Although most high-risk pediatric patients are successfully mobilized with the high-dose G-CSF regime, this approach does not salvage all of them and significantly increases the presence of AEs in comparison to standard-dose regimes.

Granulocyte colony-stimulating factor protected against brain injury in a rat cerebral hemorrhage model by modulating inflammation.

Granulocyte colony-stimulating factor (G-CSF) has been reported to exert a protective effect against secondary brain damage, but the underlying mechanisms remain unknown. We explored the ability of G-CSF to protect the brain from injury in a rat autologous blood-induced model of intracerebral hemorrhage (ICH), with a special focus on the anti-inflammation effect. An ICH was induced in 8-week-old male rats by an infusion of autologous blood, and the rats were then randomly assigned to five treatment groups: sham, ICH, and ICH+ low-dose (25 µg/kg), middle-dose (50 µg/kg), and high-dose (75 µg/kg) G-CSF. We then evaluated the levels of brain inflammation-related genes and proteins. The levels of tumor-necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) mRNA increased between days 1 and 14 post-ICH, with the highest expression on day 3. These changes were rectified by G-CSF in a dose-dependent manner. At day 3 post-injury, an elevation of the nuclear factor-kappa B (NF-κB) p65 protein level and a reduction of the inhibitor of NF-κB alpha (IκBα) protein level were observed; G-CSF treatment exerted a beneficial effect on both protein expressions. The expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins were increased; these changes were rectified by the highest dose of G-CSF. The brain-protecting effects of G-CSF are likely to be attributable, at least in part, to attenuation of the TNF-α, IL-6, iNOS, and COX-2 expressions induced by NF-κB activation in the brain tissues of this autologous blood-induced ICH rat model.

SERF1 Is Required for G-CSF Resistance of Start-Codon Mutant ELANE Granulocytic Precursors

▪Severe congenital neutropenia (SCN) is frequently associated with mutations in the ELANE gene encoding neutrophil elastase (NE), an azurophilic granule protein which represents a major fraction of all protein synthesized by neutrophilic promyelocytes and myelocytes. The reduction of mutant ELANE translation inversely correlates with neutrophil differentiation. Chronic administration of granulocyte colony-stimulating-factor (G-CSF) has been successfully used to treat some of these patients, especially those ones with mutations in C-terminus translating exons-4 and -5. Patients with proximal mutations of ELANE tend to be resistant to G-CSF therapy. The SCN pathobiology is poorly understood due to the lack of efficient disease animal models, and difficulty in obtaining relevant primary cell populations from patients. We have demonstrated that ELANE exon-3 mutations, sensitive to higher doses of G-CSF administration, cause maturation arrest and apoptosis of promyelocyte and myelocyte stages due to mutant NE inducing unfolded protein response (UPR) and endoplasmic reticulum (ER) stress. We have also shown that SCN patients with mutations in ELANE at its translation initiation codon (ELANE-ATG mut) result in synthesis of alternate truncated neutrophil elastase peptides from downstream in-frame ATG due to the presence of internal ribosomal entry sites. However, the mechanisms of SCN pathogenesis associated with mutations at the translation initiation codon of ELANE gene associated with G-CSF resistance remain unclear. We employed a disease-in-a-dish approach by directed hematopoietic and granulopoietic differentiation of gene edited (CRISPR/Cas9) isogenic patient-derived iPSC lines. We used four iPSC lines (healthy donor 28L and its start codon knock-in isogenic 28L-GTG; and patient-derived ATG mut SCN110 and its isogenic corrected line SCN110C) to evaluate if ELANE start codon mutations are both necessary and sufficient to cause disease pathogenesis, and to unravel the underlying molecular mechanisms. We found that myeloid precursors derived from 28-GTG and SCN110 iPSC lines generate low molecular weight truncated NE that is detected by a C-terminal region (C225-H238) specific anti-NE antibody (Ab). The survival, expansion and differentiation of ELANE start codon mutant hematopoietic progenitors were significantly reduced in cytokine supplemented in vitro myelopoiesis cultures, and the correction of the mutation (SCN110C) restored the granulocytic precursor expansion and differentiation. The reduced expansion in 28L-GTG and SCN110 lines was associated with both increased apoptosis and expression of proapoptotic BH3-only proteins in comparison to their isogenic 28L and SCN110C lines. However, this apoptosis was not associated with UPR/ER-stress in ELANE-ATG mut myeloid precursors. Granulopoietic differentiation of the 28L-GTG and SCN110 iPSC derived hematopoietic progenitors was significantly reduced, while it was rescued in corrected isogenic lines. Correlating with their clinical behavior, high-dose G-CSF in vitro did not restore the granulopoietic differentiation of ELANE ATG mutant hematopoietic progenitors. Mechanistically, 28L-GTG and SCN110 myeloid precursors contained high-molecular weight, sodium dodecyl sulfate (SDS) resistant NE bands that were detected by anti-NE Ab specific for the C-terminal (C225-238), but not the N-terminal region (C19-38), suggesting that truncated mutant NE generates insoluble NE aggregates. The association of truncated NE with classical autophagy marker LC3B strongly suggested the activation of an aggrephagy process in these cells. We found that SERF1 (small EDRK-rich factor), an RNA-chaperoning protein, known to localize in misfolded protein aggregates of neurodegenerative diseases, was highly upregulated in GTG knock-in and SCN patient myeloid precursors. SERF1 was distributed in both nucleus and cytoplasm, co-localized and physically interacted with mutant truncated NE in the cytoplasm of ELANE-ATG mut cells as shown by proximity ligation assays. Silencing of SERF1 made the survival and differentiation of 28L-GTG and SCN110 derived myeloid precursors sensitive to G-CSF. ELANE translation initiation mutant was associated with truncated NE aggregates and induced apoptosis mediated by SERF1. This data opens a path for therapeutic intervention of G-CSF resistant ELANEmut SCN. DisclosuresKalfa: Agios Pharmaceuticals, Inc.: Other: Steering Committee, Research Funding; FORMA Therapeutics, Inc: Research Funding. Lutzko: Aruvant Sciences: Patents & Royalties: preclinical vector development. Cancelas: Hemerus LLC: Research Funding; Vascular Solutions Inc.: Research Funding; Westat Inc: Research Funding; Fresenius-Kabi LLC: Research Funding; Cytosorbents Inc: Research Funding; Hemanext: Membership on an entity's Board of Directors or advisory committees, Research Funding; TerumoBCT: Research Funding; Cerus Co: Research Funding; University of South Florida/MEQU Inc: Research Funding.

Optimal timing of apheresis for the efficient mobilization of peripheral blood progenitor cells recruited by high-dose granulocyte colony-stimulating factor in healthy donors

Predictors of peripheral blood stem cell (PBSC) yield can potentially improve the comfort, safety, and efficacy of CD34+ cell collection from donors treated with recombinant human granulocyte colony-stimulating factor (G-CSF). We investigated 181 apheresis procedures on 109 healthy allogeneic donors to identify factors correlating with efficient PBSC collection. Apheresis started on Day 4 or 5 and continued up to Day 6 of G-CSF administration. CD34+ cell yields on Days 4 and 5 were comparable, and significantly higher than on Day 6. This suggests that starting apheresis on Day 4 rather than Day 5 may be preferable, to reduce G-CSF exposure and optimize yield, even if multi-day collection is required. More CD34+ cells were collected from male and cytomegalovirus (CMV)-seronegative donors than from female and CMV-seropositive donors, respectively. The yields of CD34+ cells were similarly high in both male and female donors aged 20–29 years; yields decreased in female donors in their thirties, and were comparably low in both male and female donors in their forties and thereafter. These findings should guide decision-making about when to begin apheresis, and encourage careful consideration of donor factors such as gender, age, and CMV serostatus when collecting PBSCs.

Effects of continuous high-dose G-CSF administration on hematopoietic stem cell mobilization and telomere length in patients with amyotrophic lateral sclerosis – a pilot study

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of complex and still poorly understood etiology. Loss of upper and lower motoneurons results in death within few years after diagnosis. Recent studies have proposed neuroprotective and disease-slowing effects of granulocyte-colony stimulating factor (G-CSF) treatment in ALS mouse models as well as humans. In this study, six ALS patients were monitored up to 3.5 years during continuous high-dose G-CSF administration. Repetitive analyses were performed including blood count parameters, CD34+ hematopoietic stem and progenitor cell (HSPC) and colony forming cell (CFC) counts, serum cytokine levels and leukocyte telomere length. We demonstrate that continuous G-CSF therapy was well tolerated and safe resulting in only mild adverse events during the observation period. However, no mobilization of CD34+ HSPC was detected as compared to baseline values. CFC mobilization was equally low and even a decrease of myeloid precursors was observed in some patients. Assessment of telomere length within ALS patients’ leukocytes revealed that G-CSF did not significantly shorten telomeres, while those of ALS patients were shorter compared to age-matched healthy controls, irrespective of G-CSF treatment. During G-CSF stimulation, TNF-alpha, CRP, IL-16, sVCAM-1, sICAM-1, Tie-2 and VEGF were significantly increased in serum whereas MCP-1 levels decreased. In conclusion, our data show that continuous G-CSF treatment fails to increase circulating CD34+ HSPC in ALS patients. Cytokine profiles revealed G-CSF-mediated immunomodulatory and proteolytic effects. Interestingly, despite intense G-CSF stimulation, telomere length was not significantly shortened.

Granulocyte colony stimulating factor treatment in non-alcoholic fatty liver disease: beyond marrow cell mobilization.

Protective effects of granulocyte colony stimulating factor (G-CSF) in acute liver injury via marrow cell mobilization have been reported in several studies. But exact mode of action and optimal protocol of G-CSF has been still doubt in chronic disease. Here we investigated mode of action and optimization of G-CSF as a treatment for non-alcoholic fatty liver disease (NAFLD). Various doses of conventional G-CSF (30 μg/kg once weekly, once daily for 5 days, twice weekly) and long acting G-CSF (30 μg/kg once a month) were evaluated in two kinds of NAFLD animal models to optimize the G-CSF protocol. G-CSF receptor expression highest increased in NAFLD model among various liver diseases compare to control (NAFLD: 14.7 times, alcohol hepatitis: 7.1 times, cirrhosis: 2.4 times, and ischemia reperfusion: 6.8 times). G-CSF treatment reduced intrahepatic fat accumulation, and inflammation in two kinds of NAFLD animal models. G-CSF increased PI3K/Akt expression in hepatocyte as well as decreased apoptotic drive (increased Bcl-2 expression and decreased Bax expression) in animal model. Five day consecutive G-CSF treatment and once a month long acting G-CSF increased marrow derived stem cell marker in peripheral blood. But twice a week conventional G-CSF treatment did not increased CD34+ cell in peripheral blood and liver neither. Not only high dose G-CSF (once daily for 5 days) but also hepatotropic dose G-CSF (twice a week) significantly reduced hepatocyte apoptosis via PI3K and Akt pathway activation without marrow cell mobilization in NAFLD animal model.

G-CSF Plus Preemptive Plerixafor Versus Cyclophosphamide Plus G-CSF for Autologous Stem Cell Mobilization in Multiple Myeloma: Effectiveness, Safety and Cost Analysis

Introduction: The optimal stem cell mobilization regimen for patients with multiple myeloma (MM) remains undefined. Most transplant centers use either a chemo-mobilization strategy using cyclophosphamide (CY) and granulocyte-colony stimulating factor (G-CSF) or a steady state strategy using G-CSF alone or with plerixafor in case of mobilization failure. However, very few studies compared efficacy, toxicity and cost-effectiveness of stem cell mobilization with cyclophosphamide (CY) and G-CSF versus G-CSF with preemptive plerixafor. In this study, we retrospectively compared our single center experience at the American University of Beirut in 89 MM patients using fractionated high-dose CY and G-CSF as our past preferred chemo-mobilization strategy in MM patients with our new mobilization strategy using G-CSF plus preemptive plerixafor. The change in practice was implemented when plerixafor became available, in order to avoid CY associated toxicity.Patients and methods: Patients in the CY group (n=62) (Table 1) received either fractionated high-dose CY (n=56) (5g/m2 divided in 5 doses of 1g/m2 every 3 hours) or CY at 50mg/kg/day for 2 doses (n=6). G-CSF was started on day +6 of chemotherapy at a fixed dose of 300 µg subcutaneously every 12 hours. All patients in the plerixafor group (n=27) (Table 1) received G-CSF at a fixed dose of 300 µg subcutaneously every 12 hours daily for 4 days. On day 5, if peripheral blood CD34+ was ≥ 20/µl, apheresis was started immediately. Plerixafor (240 µg/kg) was given 7-11 hours before the first apheresis if CD34+ cell count on peripheral blood on day 5 was <20/µl and before the second apheresis if CD34+ cells on the first collect were <3х106/kg. The median number of prior therapies was 1 (range: 1-3) in both groups.Results: Compared with plerixafor, CY use was associated with higher median peak peripheral blood CD34+ counts (35 vs 111 cells/µl, P= 0.000003), and total CD34+ cell yield (7.5 х 106 vs 15.9 х 106 cells/kg, P= 0.003). All patients in both groups collected ≥4x106 CD34+ cells/Kg. Moreover, 60 (96.7%) and 46 (74.2%) patients in the CY group vs 24 (88.8%) and 6 (22%) patients in the plerixafor group collected >6х106 and >10x106 CD34+ cells/kg, respectively (P=0.16; P<0.00001). Only 4 (6.4%) patients required two apheresis sessions in the CY group compared to 11 (40%) in the plerixafor group (P=0.0001). Conversely, CY use was associated with higher frequency of febrile neutropenia (60% vs 0%; P<0.00001), blood transfusions (27% vs 0%; P<0.00001), platelets transfusion (25% vs 0%; P<0.00001) and hospitalizations (64% vs 0%; P<0.00001). No one required intensive level of care and all recovered. Autografting was successfully performed in all patients using high-dose melphalan with a median time from mobilization to the first transplant of 31 days (range: 16-156) in the CY group compared to 13 days (range: 8-40) in the plerixafor group (P=0.027); and median infused CD34+ cells were 7х106/kg (range: 3.1-15.3) versus 5.27 (2.6-7.45), respectively (P=0.002). The average total cost of mobilization using the adjusted costs based on National Social Security Fund (NSSF) prices in Lebanon in the plerixafor group was slightly higher compared with the CY group ($7964 vs $7536; P=0.16).Conclusions: Our data indicate robust stem cell mobilization in MM patients with either fractionated high-dose CY and G-CSF or G-CSF alone with preemptive plerixafor. The chemo-mobilization approach was associated with two-fold stem cell yield, slightly lower cost (including cost of hospitalization) but significantly increased toxicity. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Bladder cancer: ESMO Practice Guidelines for diagnosis, treatment and follow-up

Bladder cancer: ESMO Practice Guidelines for diagnosis, treatment and follow-up

Allometric Dose Retranslation Unveiled Substantial Immunological Side Effects of Granulocyte Colony–Stimulating Factor After Stroke

Granulocyte colony-stimulating factor (GCSF) showed robust neuroprotective and neuroregenerative properties after stroke in rodents but failed to meet study end points in patients. Because immunologic side effects of GCSF may have escaped preclinical testing because of nonallometric dose translation, we hypothesized those as possible reasons. Stroke was induced in C57BL/6 mice by 45-minute filament middle cerebral artery occlusion. GCSF was administered at 50 and 832.5 μg/kg body weight. Treatment was controlled by vehicle injection, sham surgery, and naive animals. Immune cell counts were assessed in blood, spleen, and brain by multidimensional flow cytometry 1 day after stroke. High-dose GCSF significantly altered myeloid and T-cell subpopulations in blood and spleen and caused a tremendous increase of monocytes/macrophages infiltrating the ischemic brain. Dose-dependent immunomodulation superimposes central nervous system-specific effects of GCSF after stroke. Adaption of dose or treatment time may overcome this drawback.

Fractionated High-Dose Cyclophosphamide and G-CSF: An Extremely Effective Chemo-Mobilization Modality In Patients With Multiple Myeloma

Background The optimal stem cell mobilization regimen for multiple myeloma (MM) is undefined. Most centers use either granulocyte-colony stimulating factor (G-CSF) alone (steady state strategy) or cyclophosphamide (CY) followed by G-CSF (chemo-mobilizing strategy). However, the impact of CY dose on stem cell yield and subsequent engraftment, and toxicity is unknown. We retrospectively analyzed our experience using fractionated high-dose CY and G-CSF as our preferred chemo-mobilization strategy in MM patients (pts) and its impact on the mobilization outcomes, engraftment and the observed toxicity. Methods Between 01/2000 and 12/2012, 220 chemo-mobilization attempts were undertaken. Among these, 62 pts (M=37, F=25) had MM (1st-line=54, relapsed=8) and all received high-dose CY and G-CSF. Median age was 56 (37-75) yrs. ISS stage was I (n=34), II (n=16), and III (n=12). Pre-transplant induction consisted of VAD or VAD-like chemotherapy (n=26), bortezomib(bor)/dexamethasone (dex) (n=15), thalidomide (thal) /dex (n=10), bor/thal/dex (n=10), and 1 received bor/lenalidomide/dex. Fifty-six received fractionated high-dose CY (5g/m2 divided in 5 doses of 1g/m2 q 3 hrs) whereas 6 received CY 50 mg/kg for 2 doses. G-CSF was given at a fixed dose of 300 µg SQ q 12 hrs. Results All 62 (100%) pts achieved a circulating CD34 count ≥20/µl which is the cut-off level at our center to proceed with apheresis. The median peak peripheral blood CD34+ cell count was 111.5 (21-575) cells/μL. Success rate of stem-cell mobilization defined as collection of more than 2x106 CD34+ cells/kg was 100%. Median stem cell collection yield was 15.9x106 CD34+ cells/kg. Moreover, 61 (98.4%) pts and 46 (74%) pts collected &gt;5x106 and &gt;10x106 CD34+ cells/kg, respectively. Only 4 (6.4%) pts required 2 apheresis sessions. Conversely, 40 (64.5%) pts required hospitalization for febrile neutropenia (n=38) or transfusion support (n=2) for a median of 4 (1-8) days. No one required intensive level of care and all recovered. Also, 17 (27.4%) pts required blood transfusions and 16 (25.8%) required platelets transfusion. Autografting was successfully performed in all pts using high-dose melphalan with a median time from mobilization to the first transplant of 31 (16-156) days and median infused CD34+ cells of 7x106/kg (3.1-15.3). All pts achieved successful hematologic engraftment with a median time for neutrophil engraftment (ANC ≥500/µL) of 11 days and platelet engraftment (platelet ≥20000/microliter) of 12 days. Conclusion Fractionated high-dose CY and G-CSF is a highly effective chemo-mobilization strategy in MM in terms of successful rate of mobilization (100%), efficiency of stem cell collection (high yield), and timely hematologic engraftment (100%). However, the relatively high-rate of hospitalizations for febrile neutropenia requires an assessment of its cost-efficiency as compared to new mobilization strategies using G-CSF and preemptive plerixafor. Disclosures: No relevant conflicts of interest to declare.

Just‐in‐time rescue plerixafor in combination with chemotherapy and granulocyte‐colony stimulating factor for peripheral blood progenitor cell mobilization

Plerixafor, a recently approved peripheral blood progenitor cell mobilizing agent, is often added to granulocyte-colony stimulating factor (G-CSF) to mobilize peripheral blood progenitor cells in patients with lymphoma or myeloma who cannot mobilize enough CD34+ cells with G-CSF alone to undergo autologous stem cell transplantation. However, data are lacking regarding the feasibility and efficacy of just-in-time plerixafor in combination with chemotherapy and G-CSF. We reviewed the peripheral blood stem cell collection data of 38 consecutive patients with lymphoma (Hodgkin's and non-Hodgkin's) and multiple myeloma who underwent chemomobilization and high-dose G-CSF and just-in-time plerixafor to evaluate the efficacy of this treatment combination. All patients with multiple myeloma and all but one patient with lymphoma collected the minimum required number of CD34+ cells to proceed with autologous stem cell transplantation (>2 × 10(6) /kg of body weight). The median CD34+ cell dose collected in patients with non-Hodgkin lymphoma was 4.93 × 10(6) /kg of body weight. The median CD34+ cell dose collected for patients with multiple myeloma was 8.81 × 10(6) /kg of body weight. Plerixafor was well tolerated; no grade 2 or higher non-hematologic toxic effects were observed.

Peripheral blood CD34+ cell monitoring after cyclophosphamide and granulocyte-colony-stimulating factor: an algorithm for the pre-emptive use of plerixafor

Plerixafor “on demand” after chemotherapy plus granulocyte-colony-stimulating factor (G-CSF) is efficient in peripheral stem cell mobilization, but the timing of administration and criteria for patient selection are under investigation. To devise an algorithm for the “on demand” use of plerixafor at the first mobilization attempt, we analyzed the kinetics of hematopoietic recovery and peripheral blood CD34+ cells in 107 patients treated with high-dose cyclophosphamide plus G-CSF. Fifty-one patients with myeloma were treated with cyclophosphamide 3–4 g/m2 on day 0 followed by G-CSF 10 μg/kg from day + 6, and 56 patients with lymphoma received cyclophosphamide 6–7 g/m2 followed by G-CSF 5 μg/kg from day + 1. Peripheral blood CD34+ cell monitoring was started on day + 8 in patients with myeloma and day + 10 in patients with lymphoma. The outcome of interest was a collection of ≤ 2 × 106 CD34+/kg. By a multivariate logistic regression model, CD34+ cell count < 10/μL at leukocyte recovery (> 1000/μL) or leukocyte count < 1000/μL after day + 12 in myeloma and day + 14 in lymphoma predicted the failure of mobilization by 2.7 and 2.8 times (p = 0.001 and p = 0.02) with a sensitivity of 89% and specificity of 88%, respectively. Plerixafor “on demand” may be considered in patients with myeloma and lymphoma with delayed hematopoietic recovery and < 10/μL CD34+ cells, as a first-line mobilization strategy.

Defining “poor mobilizer” in pediatric patients who need an autologous peripheral blood progenitor cell transplantation

The definition of poor mobilizers is not clear in pediatric patients undergoing autologous peripheral blood hematopoietic progenitor cell (HPC) mobilization. Most studies conducted in children define those variables related to the collection of HPC after leukapheresis, but the information regarding exclusively the mobilization process is scarce. In our experience, most children (92.2%) reach the target CD34(+) cell dose for autologous peripheral blood progenitor cell transplantation ifCD34(+) cell count was higher than 10/μL. No differences were observed between those with >20 CD34(+) cells/μL and 11-20 CD34(+) cells/μL. In this study, we analyzed the variables that influence CD34(+) cell count; we found that prior use of radiotherapy was the main variable related to poor mobilization. Patients diagnosed with Ewing sarcoma, treated with radiotherapy and mobilized with standard doses of granulocyte colony-stimulating factor (G-CSF) were also at a high risk of mobilization failure. In these patients, we should consider mobilization with high dose G-CSF and be prepared with new mobilization agents to avoid delay on their course of chemotherapy.

Successful Treatment of Childhood Hypocellular Acute Myeloid Leukemia

Hypocellular acute myeloid leukemia (AML) is extremely rare in childhood. We report on a 7-year-old girl with hypocellular AML who was treated successfully with granulocyte-colony stimulating factor (G-CSF) and combined chemotherapy. High-dose G-CSF induced complete remission and she subsequently received reduced intensity conditioning and unrelated cord blood transplantation; however, this resulted in early rejection. After a complete hematological recovery, she received 3 courses of combination chemotherapy oriented toward AML. She has remained in complete remission for over 1 year after the completion of the therapy. G-CSF effectively induced remission, and combination chemotherapy has been proven to be feasible for patients with childhood hypocellular AML.

Successful collection and engraftment of autologous peripheral blood progenitor cells in poorly mobilized patients receiving high‐dose granulocyte colony‐stimulating factor

Granulocyte Colony-Stimulating Factor (G-CSF) alone or in conjunction with chemotherapy is commonly used to mobilize hematopoietic progenitor cells (HPC) into peripheral blood for progenitor cell harvest for autologous HPC transplantation. However, in up to 30% of patients, HPC are not effectively mobilized. In this study, we report the efficacy and safety profiles of a mobilization strategy using high-dose (up to 36 μg/kg) G-CSF in poorly mobilized patients. Retrospective medical record reviews were performed for 392 patients who underwent autologous peripheral blood progenitor cell collection. A total of 56 patients were given high-dose G-CSF due to very ineffective mobilization and 35 of these patients underwent autologous HPC transplantation. The efficacy of mobilization, apheresis collection, and infusion were reviewed and analyzed. More than 2.5 × 10(6) CD34/Kg were collected in 88% of patients (49 of 56) who were placed on high-dose G-CSF due to very ineffective mobilization. Of the 35 patients who underwent HPC transplantation using the progenitor cells that were mobilized with high-dose G-CSF due to very ineffective mobilization, all had rapid and complete neutrophil and platelet engraftment comparable with good mobilizers. We conclude that collection of HPC using hyperstimulation with G-CSF is an effective alternative approach for HPC harvest for poorly mobilized patients.

Hepatic Hemangioendothelioma in an Infant With Severe Congenital Neutropenia

Severe congenital neutropenia (SCN) is a rare disorder caused by heterogeneous genetic mutations. We describe here a rare association of SCN caused by a novel ELANE mutation and infantile hepatic hemangioendothelioma. In a 2-month-old infant, an abdominal ultrasound performed for omphalitis revealed a hepatic tumor, which was resected. Histopathology confirmed the diagnosis of hemangioendothelioma. Postoperatively, severe neutropenia was noted. Bone marrow examination showed myeloid maturation arrest, diagnostic of SCN. Mutation analysis for the neutrophil elastase gene identified a novel heterozygous de novo ELANE missense mutation in exon 2 (c.215T>A, p.Val72Glu). He was managed successfully with broad-spectrum antibiotics and high-dose granulocyte colony-stimulating factor.

Secondary acute myeloid leukemia following severe congenital neutropenia: Case presentation.

e20001 Background: Therapy with granulocyte colony stimulating factor (G-CSF) is standard of care to prevent sepsis in severe congenital neutropenia (SCN). This successful approach has unmasked the predisposition to myelodysplastic syndrome and acute myeloid leukaemia (MDS/AML). This appears greatest in patients who require higher doses of G-CSF to achieve an adequate neutrophil response. Methods: 9 year old boy started severe infections since early infancy, neutropenia consistently below 500. Severe congenital neutropenia (SCN) was diagnosed at age 3 months, after marrow assessment. On high dose G-CSF since then, 10 to 15 mcg/Kg/d, to keep adequate ANC. Asymptomatic since then, normal development. No physical stigmata. Blood smears always dysplastic on myeloid line. At age 8, on check-up, had mild bicytopenia (anemia and thrombocytopenia). Was followed with bone marrow aspirates and biopsies. Normocellular, myeloid blasts 10 to 12%, by morphology and immunophenotype. Normal karyotype. Peripheral cytopenias advanced, until becoming transfusion-dependent for RBC and later platelets. WBC remained stable after discontinuing G-CSF. Viral and other infections were rulled out. Samples were analyzed for ELA-2 mutation, which was positive. G-CSF receptor mutation was negative. Course was progressive, transfusions given frequently. Splenomegaly stable at 3cm. Bone marrow performed monthly. Started iron chelation after 15 RBC transfusions and ferritin over 1000mcg/dl. No sibling available for HSCT. Results: Transformed to overt AML, with a rapid disease progression, despite giving AML chemotherapy. He ultimately died of refractory disease, after 2 blocks of chemotherapy. Conclusions: It is imperative that all patients with severe congenital neutropenia be closely monitored for leukaemic transformation. Risks and benefits of early haematopoietic stem cell transplantation should be evaluated for patients who respond poorly to a high dose of G-CSF.

Stem Cell Mobilization and Collection for Induction of Mixed Chimerism and Renal Allograft Tolerance in Cynomolgus Monkeys

We have previously observed that donor bone marrow hematopoietic stem cells successfully induce transient mixed chimerism and renal allograft tolerance following non-myeloablative conditioning of the recipient. Stem cells isolated from the peripheral blood (PBSC) may provide similar benefits. We sought to determine the most effective method of mobilizing PBSC for this approach and the effects of differing conditioning regimens on their engraftment. A standard dose (10 μg/kg) or high dose (100 μg/kg) of granulocyte colony-stimulating factor (GCSF) with or without stem cell factor (SCF) was administered to the donor, and PBSC were collected by leukapheresis. Cynomolgus monkey recipients underwent a nonmyeloablative conditioning regimen (total body irradiation, thymic irradiation, and ATG) with splenectomy (splenectomy group) or a short course of anti-CD154 antibody (aCD154) (aCD154 group). Recipients then received combined kidney and PBSC transplantation and a 1-mo post-transplant course of cyclosporine. Treatments with either two cytokines (GCSF+SCF) or high dose GCSF provided significantly more hematopoietic progenitor cells than standard dose GCSF alone. Recipients in the aCD154 group developed significantly higher myeloid and lymphoid chimerism (P < 0.0001 and P = 0.0002, respectively) than those in the splenectomy group. Longer term renal allograft survival without immunosuppression was also observed in the aCD154 group, while two of three recipients in the splenectomy group rejected their allografts soon after discontinuation of immunosuppression. Protocols including administration of two cytokines (GCSF + SCF) or high dose GCSF alone significantly mobilized more PBSC than standard dose GCSF alone. The recipients of PBSC consistently developed excellent chimerism and survived long-term without immunosuppression, when treated with CD154 blockade.