Dose Of G-CSF Research Articles

Genistein Protects Hematopoietic Stem Cells Against G-CSF Induced DNA Damage

Abstract 3486Granulocyte colony-stimulating factor (G-CSF) is widely utilized in multiple clinical settings to lessen the effects of neutropenia. Although clearly beneficial, there are concerns about the long term effects of G-CSF. A particular concern is that G-CSF therapy may increase the risk of MDS and or AML. The most striking example is that of Severe Congenital Neutropenia (SCN). While G-CSF clearly improves survival, there are several lines of evidence to suggest that G-CSF treatment contributes to development of leukemia in these patients. First, the risk of leukemia appears to correlate with the cumulative dose of G-CSF. Second, of all the congenital marrow failure syndromes predisposed to AML, SCN alone does not appear to be a hematopoietic stem cell disorder. Since AML appears to rise from sequential mutations in hematopoietic stem cells, this would suggest that therapy, not the intrinsic cell defect is causal. It has been demonstrated that G-CSF does initiate signaling pathways in hematopoietic stem cell (HSC). We hypothesize that G-CSF induced excessive HSC proliferation can lead to DNA damage and genome instability. To test our premise, mice were treated with G-CSF for 4 months and bone marrow cells were analyzed. Our results demonstrated a 3 fold increase in linage negative, Sca positive and cKit positive (LSK) population and a 2 fold increase in the amount of DNA double strand breaks via the presence of nuclear pH2AX in the LSK population. To determine if the G-CSF induced proliferation lead to chromosome alterations, we performed array-comparative genomic hybridization analyses (CGH). DNA from lineage negative bone marrow cells from animals treated with G-CSF for 4 months were compared to untreated mice. Our results demonstrate variations in gains and losses of several chromosome regions. Fluorescence in situ hybridization (FISH) of Lin-Sca+ bone marrow cells confirmed loss on regions of chromosome 2 (6%) and 17 (30%). Since prolonged G-CSF exposure promotes genomic instability in HSCs we hypothesize that an alternative strategy would be to co-administer a drug that selectively blocks the effect of G-CSF on HSCs. Previous studies suggested genistein as an attractive compound. Genistein is a natural soy isoflavone with excellent bioavalibity that has anti-oxidant and anti-proliferative properties. In this study, we utilized a dose of genistein that can easily be obtained through oral supplementation. Mice were concomitant treated with G-CSF and genistein 3 times a week. Genistein partially blocked the G-CSF induced expansion of LSK cells and reduced pH2AX levels in this population by 40%. This was also accompanied by a reduction in LSK cells with an abnormal FISH signal (50% reduction). Importantly, genistein did not block the G-CSF driven expansion of mature neutrophils as total number of neutrophils in mice treated with G-CSF and genistein are the same as those treated with G-CSF alone. Our results suggest that genistein’s effects are mediated primarily through inhibition of HSC proliferation. We demonstrate that G-CSF treatment induces GSK3β phosphorylation and Cyclin D1 and D3 expression. Genistein blocked GSK3β phosphorylation and Cyclin D1 and D3 induction. Inhibition of GSKβ3 has been demonstrated to delay HSC entry into cell cycle by promoting degradation of β-catenin, while HSCs from the triple cyclin knock out mouse (Cyclins D1, D2, and D3) display delayed cell cycle entry. Collectively, our results imply that prolonged G-CSF treatment induces DNA damage in HSCs by initiating cell cycle progression. HSCs are long lived, quiescent cells that preferentially utilize non-homologus end joining for DNA repair when progressing from G0 to G1. NHEJ is a relatively error prone DNA repair mechanism. Its preferential use by HSCs has been postulated as reason chromosomal deletions and translocations are often seen and many times are causal in the development of acute leukemia. Importantly, we demonstrate, that genistein, at levels obtainable through oral supplementation, is able to reduce DNA damage by attenuating G-CSF induced HSC proliferation without compromising G-CSFs ability to accelerate terminal neutrophilic differentiation. These results suggest that genistein may be an effective therapeutic agent in patients with SCN who require prolonged G-CSF support. Disclosures:No relevant conflicts of interest to declare.

The Natural History of Cyclic Neutropenia: Long-Term Prospective Observations and Current Perspectives.

AbstractAbstract 2141Cyclic neutropenia (CyN) is an autosomal dominant and sporadically occurring hematological disease first described in 1910. Cyclic neutropenia is usually diagnosed in children before age 2 years based of regularly occurring fever, mouth ulcers, and recurrent skin or respiratory infections. In affected families, recognition is generally earlier than for the sporadic cases. Serial neutrophil counts usually show periods of very severe neutropenia (ANC< 0.2 × 109/L) at three week intervals and an intervening peak ANC less than 2.0 × 109/L. A reciprocal monocytosis often occurs during the neutropenic period. Mild anemia is common, particularly in patients with recurrent infections. Cycling of blood neutrophils is more apparent in young children than in adults, who often report severe childhood symptoms ameliorating near puberty or soon thereafter.Through the Severe Chronic Neutropenia International Registry and Repository- Seattle (SCNIR-Seattle) we have prospectively studied the natural history of 348 patients with a clinical diagnosis of CyN, 181 sporadic cases and 167 patients from 36 families whose pedigrees illustrate autosomal dominant inheritance. Sequencing results for the gene for neutrophil elastase, ELANE, are available for 119 of these patients including at least one member of 18 families. We have prospectively followed all of these patients for up to 25 years.Patients with typical, 21 day oscillations of blood neutrophils almost always have mutations of ELANE, most frequently mutation in exons 4 or 5. Neutropenic relatives (ANC < 1.5 × 109/L) of an index case of CyN with an ELANE mutation uniformly have the same ELANE mutation, even if they have few symptoms and the neutropenia is not severe. Patients with severe congenital neutropenia and CyN may also have the same or nearly identical mutations; thus the diagnosis of cyclic neutropenia can not be made solely from mutation analysis.Since 1987 we have treated cyclic neutropenic patients with G-CSF; some patients have been on G-CSF on a daily or alternate-day basis for 25 years. For the population of 209 treated patients enrolled in the SCNIR and treated for a median of 14.8 years, the median daily G-CSF dose is 2.1 mcg/kg/day. We have observed that the dose established during the first year of treatment can be adjusted for body weight and usually maintained indefinitely. Adverse effects are infrequent, particularly with daily or alternate-day treatment, although development of osteopenia remains a concern. A few patients who have transiently interrupted G-CSF; almost all have resumed G-CSF because of recurrence of fever, mouth ulcers, infections or fatigue. We are currently aware of only two CyN patients who have discontinued G-CSF treatment for greater than 2 months without recurrence of symptoms or returning to G-CSF therapy.In this population, there has been no hematopoietic stem cell transplantation for treatment of CyN and no G-CSF treatment failures. 15 deaths have occurred: 1 from ovarian cancer, 2 related to discontinuation of G-CSF, 2 from cardiac causes, 3 from homicide or accidents, 5 from complicated illnesses with multi-system problems. One closely followed patient never treated with G-CSF developed CML and one patient treated long term with immunosuppressive drugs and G-CSF developed AML. Other than this case, there have been not CyN patients developing AML in more than 5000 years of patient observation. Clostridial sepsis, the frequent cause of death in CyN patients in the pre-G-CSF era, has not been observed in CyN patients on G-CSF.These long-term observations serve to characterize cyclic neutropenia is a distinctive hematological disorder, very responsive to treatment with G-CSF and without recognized risk of evolution to AML. Disclosures:Dale:AMGEN: Consultancy. Boxer:Amgen: Equity Ownership.

Profound Signaling Abnormalities Due to Cytoplasmic Variants of CSF3R in Patients with Myelodysplastic/Myeloproliferative Syndromes Associated with CSF3R Mutations or Mutations of Splicing Factor U2AF1

AbstractAbstract 3792Myelodysplastic Syndromes (MDS) are a group of bone marrow disorders closely related to acute myeloid leukemia (AML). Even though a number of genetic mutations have been recently identified in patients with MDS, their contributions to MDS pathogenesis remains poorly understood. Some of these genetic mutations involve transcription factors, but they are also found in AML: TET2, EZH2, and ASXL1. One group of mutations distinct to MDS are those encoding proteins involved in RNA splicing (e.g. U2AF1/U2AF35, ZRSR2, SRSF2, SF3B1). Based on RNA-Seq of MDS/AML patients, we report exon skipping in the 3′ end of the CSF3R transcript, which encodes the granulocyte colony-stimulating factor receptor (GCSFR), in a patient carrying the S34F mutation in the U2AF1 gene. U2AF1 is one of the more recurrent genes affected by mutation in MDS, and it is associated with progression to secondary AML. The S34F mutation in U2AF1 is a gain of function mutation that promotes excess splicing and exon skipping. Alternative splicing of CSF3R results in 7 transcripts, of which the two most common are Class I and Class IV. There are putative splicing sequences within exon 17 of the CSF3R locus; GT (GU) at the 5′ site and AG at the 3′ site - a recognition sequence for U2AF1. In addition, we also identified mutations affecting CSF3R in two patients with chronic myelomonocytic leukemia (GCSFR T595I or Q726X), one patient with Refractory Cytopenias with Multilineage Dysplasia and Ring Sideroblasts (GCSFR W650L), and one patient with primary AML (GCSFR G659fs). In the last case, this mutation affects the Class III transcript of CSF3R, an alternatively spliced form expressed highly in the placenta. The Class IV isoform lacks much of the C-terminal domain, similar to the protein produced by nonsense mutations found in patients with severe congenital neutropenia who develop MDS/AML or the patient we identified.Little is known about the signaling-phenotype relationship of a mutant or alternatively-spliced GCSFR. To address these questions, we first studied the expression patterns of Class I and Class IV GCSFR in the human NB4 promyelocytic leukemia cell line and primary human hematopoietic stem (CD34+) cells induced to differentiate into neutrophils. Quantitative PCR of Class I and Class IV transcripts showed a positive feedback loop for Class I. Expression of the Class IV transcript was downregulated during hematopoietic cell differentiation. Scatchard analysis showed no differences between the two receptors in high-affinity Kd (∼ 500 nM) for the GCSF-GCSFR. Because Class IV lacks the C-terminal di-leucyl motif that facilitates internalization, we measured internalization rates and found that indeed the Class IV internalized more slowly and less completely. Using an MTT assay to measure proliferation we observed Class IV isoform had lower proliferative capacity at lower GCSF concentrations (0.1 – 2 nM GCSF); however at higher GCSF dose (>100 nM) its proliferative response was greater than Class I. Using western blotting we observed that the Class IV isoform showed weaker signaling via the JAK/STAT and ERK1/2 pathways, but had higher Lyn activity when treated with 100 ng/ml GCSF. To determine effects on differentiation, we made chimeric human growth hormone receptor-GCSFR for transfection into murine 32D cells. 32D cells express low levels of murine GCSFR, thus we made the chimeric receptor. Expression of Class IV Receptor impaired their differentiation (as demonstrated by morphology and Gr-1 expression). We are now developing a mouse model of perturbed hematopoiesis due to dysregulated expression of Class IV GCSFR. Altogether, our studies show that S34F mutation of U2AF1 splicing gene is associated with exon skipping of CSF3R. This would result in expression of a C-terminal truncated GCSFR, similar to that observed in patients with nonsense mutations or alternative splicing. A C-terminal truncated GCSFR causes aberrant hematopoietic cell proliferation, altered post-receptor signaling events, and impaired myeloid differentiation.Our findings and those involving GCSFR E785K in high-risk MDS (Wolfler et al, Blood 105:3731, 2005) strongly suggest that aberrant signaling by alterations in the C-terminus of the GCSFR contributes to the pathogenesis of MDS. Disclosures:Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

B-lymphopoiesis is stopped by mobilizing doses of G-CSF and is rescued by overexpression of the anti-apoptotic protein Bcl2

Osteoblasts are necessary to B lymphopoiesis and mobilizing doses of G-CSF or cyclophosphamide inhibit osteoblasts, whereas AMD3100/Plerixafor does not. However, the effect of these mobilizing agents on B lymphopoiesis has not been reported. Mice (wild-type, knocked-out for TNF-α and TRAIL, or over-expressing Bcl-2) were mobilized with G-CSF, cyclophosphamide, or AMD3100. Bone marrow, blood, spleen and lymph node content in B cells was measured. G-CSF stopped medullar B lymphopoiesis with concomitant loss of B-cell colony-forming units, pre-pro-B, pro-B, pre-B and mature B cells and increased B-cell apoptosis by an indirect mechanism. Overexpression of the anti-apoptotic protein Bcl2 in transgenic mice rescued B-cell colony forming units and pre-pro-B cells in the marrow, and prevented loss of all B cells in marrow, blood and spleen. Blockade of endogenous soluble TNF-α with Etanercept, or combined deletion of the TNF-α and TRAIL genes did not prevent B lymphopoiesis arrest in response to G-CSF. Unlike G-CSF, treatments with cyclophosphamide or AMD3100 did not suppress B lymphopoiesis but caused instead robust B-cell mobilization. G-CSF, cyclophosphamide and AMD3100 have distinct effects on B lymphopoiesis and B-cell mobilization with: 1) G-CSF inhibiting medullar B lymphopoiesis without mobilizing B cells in a mechanism distinct from the TNF-α-mediated loss of B lymphopoiesis observed during inflammation or viral infections; 2) CYP mobilizing B cells but blocking their maturation; and 3) AMD3100 mobilizing B cells without affecting B lymphopoiesis. These results suggest that blood mobilized with these three agents may have distinct immune properties.

Safety and efficacy of healthy volunteer stem cell mobilization with filgrastim G‐CSF and mobilized stem cell apheresis: results of a prospective longitudinal 5‐year follow‐up study

G-CSF-mobilized peripheral blood stem cells have long replaced marrow as the major source for allogeneic transplants. Conclusive evidence questioning the long-term safety of G-CSF for donors has not been provided, but the cumulative number of followed donors remains insufficient to rule out rare adverse events. A long-term active follow-up study of G-CSF-mobilized healthy volunteer donors was therefore performed. Two hundred and three successive donors were evaluated pre-apheresis, subjected to G-CSF-mobilization/apheresis, and actively followed for 5 years by the same physicians and laboratories. Follow-up laboratory work included standard biochemical/haematological tests and T-cell phenotyping. Donor epidemiology was typical for reported stem cell donor cohorts. Acute adverse effects of G-CSF and apheresis were mild and transient, consistent with the previous reports. Mean circulating CD34(+) cells after nine doses of G-CSF were 124 per μl. Other biochemical/haematological parameters were also altered, consistent with G-CSF treatment. Spleen enlargement was modest. At first follow-up, all clinical and laboratory parameters had normalized. Leucocyte/lymphocyte counts and CD4/CD8 ratios were the same as during premobilization work-up and remained unchanged throughout. A single severe but likely unrelated adverse event, a case of papillary thyroid carcinoma, was reported. The studies add an observation time of almost 500 donor years to the growing body of evidence of the long-term safety of G-CSF for allogeneic donor stem cell mobilization.

Use of granulocytes concentrates from a single high yield apheresis to support more than one patient.

6622 Background: Allogeneic granulocyte transfusion therapy is sometimes the only therapeutic option in immunocompromised neutropenic leukemic/HSCT patients with life threatening bacterial and fungal infections. Collection of larger cell doses of granulocyte concentrates (GC) by automated apheresis following G-CSF/steroids administration has renewed interest and its use has grown steadily. Methods: Donors were recruited from family/friends of the patients and informed consent was obtained. GCs were collected via donor stimulation with a single subcutaneous dose of G-CSF (600 mcg). First time donors received an oral dose of dexamethasone (8 mg). GCs were harvested by Hetastarch-assisted leukapheresis 12 hours later via peripheral venous access with the continuous flow cell separator Spectra (COBE Caridian BCT, Lakewood, CO). Results: GCs were collected from 93 donors (67M: 26F; age median 40 (19-73 years) and 1 ½ x blood volume processed median 7048 (3212-8085 mL). The pre/post wbc were median 8.1 (4.1-22.8)/ 42.7 (22.4-77.5). The volume collected was based on the post G-CSF wbc count (&gt; 35 K/UL - 800 mL; &lt;35 K/UL between 600-700 mL). The original yield (10 x10e) was median 9.7 (5.1-17.2 units), volume median 718 (538-860 mL) and WBC median 132.7 (84.2 -241.6 K/UL per bag). The Split GCs were median 4.48 (2.70-7.72 units), with neutrophils median 87% (54-97% per bag). GCs were transfused to 51 patients (31M: 20F; age median 58 [19-83 years]) who received median 3.5 (1-18 split units), containing wbcs median 10734.69 [5778-42 -162698.09 (x10^3/uL)/unit) and achieved a WBC increment median 0.2 (0.0-8.3K/UL). Conclusions: The GCs were split due to the primary care team's reluctance to transfuse the entire volume of the product due to volume restrictions or for some other medical reason. The same rationale to split platelets from high yield single apheresis was used to split our GC products. We found that GCs from each donor was utilized maximally and also brought partial relief to other patients who lacked donors. We would like to make the case that when adequate numbers of GCs are collected it may be feasible to split the unit. This may offer a glimmer of hope to other patients in need of GCs who lack a donor network support.

GM-CSF is not able to induce granulopoiesis in CN patients due to absence of LEF-1 transcription factor

G-CSF is successfully used to treat patients with severe congenital neutropenia (CN). However, daily treatment with high doses of G-CSF (up to 50µg/kg/d) are required to induce granulocyte numbers of above 1000/µl in CN patients. Intriguingly, GM-CSF failed even at high dosages (30µg/kg/day) to increase the neutrophil numbers, but led to a dramatic increase of eosinophils and monocytes in those patients. We aimed to investigate the mechanisms of abrogated GM-CSF-triggered granulopoiesis in CN patients. Recently, we found that in CN patients “steady-state” granulopoiesis could not be activated due to a lack of LEF-1 and C/EBPα transcription factors and that G-CSF induces C/EBPß-triggered “emergency” granulocytic differentiation. In the present study, we demonstrated that GM-CSF only slightly induces C/EBPß expression, in comparison to G-CSF. Moreover, in CFU assays, inhibition of LEF-1 in CD34+ hematopoietic cells using LEF-1 shRNA led to completely abolished CFU-G, but unaffected CFU-M production upon stimulation with GM-CSF. Therefore, we concluded, that GM-CSF is not able to induce granulopoiesis in CN patients due to a lack of LEF-1/C/EBPα and its inability to induce C/EBPß.

A role of GADD45b protein in myeloid differentiation and stress response of human hematopoietic cells

Growth arrest and DNA-damage-inducible, beta (GADD45b) protein modulates stress responses in mouse myeloid cells. Recently, we demonstrated, that in patients with severe congenital neutropenia (CN) daily treatment with high doses of G-CSF stimulated C/EBPß-dependent emergency stress-induced granulopoiesis.

Evaluation of Human and Rhesus CD34+CXCR4+ and CD34+CXCR4- Subpopulations

Abstract 1007CXCR4 is an α-chemokine receptor specific for the ligand stromal-derived-factor-1 (SDF-1). CXCR4 is expressed on several cell types, including CD34+ cells. In this study, we sought to evaluate the difference between subpopulations of human and rhesus CD34+ cells that express CXCR4. Rhesus CD34+ cells were mobilized using a five day course of G-CSF (10mcg/kg SQ) and a single dose (1mg/kg SQ) of the CXCR4 antagonist, AMD3100, on the 5th day. Leukapheresis was performed two hours following the last dose of G-CSF and AMD3100 and CD34+ cells were isolated by immunoselection from the product. Human CD34+ cells were collected by leukapheresis and immunoselection following a 5 day course of 480 mcg G-CSF SQ. CXCR4+ and CXCR4- fractions in the CD34+ cells were separated by cell sorting. Rhesus and human CD34+CXCR4+ and CD34+CXCR4- cells were used for microarray analysis as well as cultured and transduced with an EGFP-expressing lentiviral vector. On average (SD) 21.6 ± 4.4% of AMD3100+G-CSF mobilized rhesus (n=6) and 39.3 ± 4.3% of G-CSF mobilized human (n=3) CD34+ cells express CXCR4. Two days following in vitro culture, rhesus CD34+CXCR4+ cells showed greater numbers of adherent spindle-shaped cells (56.4 ± 7.4%) when compared to CXCR4- cells (1.3 ± 0.4%, p<0.01) and bulk CD34+ cells (21.0 ± 2.0%, p<0.01). Following transduction, the rhesus CD34+CXCR4+ derived cells also had significantly lower EGFP expression (7.0 ± 0.3%) when compared to their CD34+CXCR4- counterpart (25.4 ± 1.2%, p<0.01) and bulk CD34+ cells (22.6 ± 2.2%, p<0.01). In CFU assay, human CD34+CXCR4+ cells showed fewer colony numbers (Erythroid (E): 6.2 ± 1.7/1000 cells, Myeloid (M): 7.0 ± 0.0/1000 cells) when compared to CD34+CXCR4- derived cells (E: 21.5 ± 7.9/1000 cells, ns, M: 24.7 ± 4.8/1000 cells, p<0.05) and bulk CD34+ cells without lentiviral transduction (E: 37.3 ± 1.2/1000 cells, p<0.05, M: 24.7 ± 4.8/1000 cells, p<0.01). To determine whether the CD34+CXCR4+ or CD34+CXCR4- subpopulation contained hematopoietic repopulating cells, human CD34+CXCR4+ cells (4×10e4 cells/mouse) and CD34+CXCR4- cells (4×10e4 cells/mouse or 2×10e6 cells/mouse) with lentiviral gene-marking were transplanted into NOD/SCID/IL2Rγ null mice. Human cell chimerism and %EGFP in the peripheral blood cells were analyzed every 4 weeks for over 6 months (Figure). CD34+CXCR4+ cell-transplanted mice showed no engraftment of human cells (less than 1% of human CD45+ cells) at all time points, while human cells were detected in the peripheral blood of the CD34+CXCR4- cell-transplanted mice (4×10e4 cells/mouse) until 12 weeks after transplantation. The CD34+CXCR4- cell-transplanted mice (2×10e6 cells/mouse) showed human cell engraftment for over 6 months. These data suggest that the CD34+CXCR4- cell population contain hematopoietic repopulating cells. To evaluate the nature of the CD34+CXCR4+ cell population, global microarray gene expression analysis was performed both on human and non-human primate CD34+CXCR4+ cells. Microarray signatures showed different gene profiles between CD34+CXCR4+ and CD34+CXCR4- populations in both human and rhesus CD34+ cells. Both human and rhesus CD34+CXCR4+ populations demonstrated strong monocyte- and neutrophil-mediated inflammatory response gene expression profiles, suggesting that the CD34+CXCR4+ subpopulation in both human and non-human primates contain progenitors instrumental in the innate immune response. These results indicate that while the addition of AMD3100 to G-CSF mobilization protocols may increase the number of CD34+ cells mobilized, some of these cells may not be as potent for gene therapy and transplantation applications and support the design of immunoselection protocols which target specific subpopulations of CD34+ cells for therapeutic applications in the future. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Phase 2 Trial of G-CSF Priming, Clofarabine, and High Dose Cytarabine (GCLAC) for the Treatment of Newly Diagnosed Acute Myeloid Leukemia (AML) or Advanced Myelodysplastic Syndrome or Advanced Myeloproliferative Neoplasm,

Abstract 3617Clofarabine and cytarabine combinations have been effective in the treatment of adult acute myeloid leukemia (AML) in both the relapsed/refractory and upfront settings. Based on our results with GCLAC (G-CSF priming, clofarabine, and high dose cytarabine) in a trial for relapsed/refractory AML (Becker et al. Br J Haematol in press), we are currently testing this regimen in newly diagnosed patients age < 65. The G-CSF dose is 5 mcg/kg/day by subcutaneous injection beginning the day prior to chemotherapy, clofarabine 30 mg/m2/day × 5 and cytarabine 2 gm/m2/day × 5. Second induction with the same regimen was permissible if marrow blasts over 5% persisted on day 21 or thereafter. Consolidation courses were administered for up to 3 cycles, with clofarabine at a dose of 25 mg/m2/day × 4 days and cytarabine 2 gm/m2/day × 4 days. The primary objectives of this trial are to estimate rates of CR (complete remission) and EFS (event free survival). A stopping rule would be imposed if there was reasonable evidence that the CR rate was inferior to that obtained with standard induction 7+3, 70% (Fernandez et al. NEJM 2009; 361:1249–59). Absent early stopping, 50 patients will be treated. Twenty-five patients with non-APL AML, RAEB2, CMML2, or myelofibrosis with >10% blasts have been treated thus far; their median age is 52, range 22–63. Eleven patients had antecedent hematologic disorders(AHD). Four patients had poor risk cytogenetics, four patients had normal cytogenetics with Flt3+, and 5 patients had good risk cytogenetics. The most significant grade 3/4 toxicity occurring in 2 patients, was a constellation of pulmonary infiltrates, hypoxia, and diffuse alveolar hemorrhage that responded to steroids. This incidence is not dissimilar to the pulmonary toxicity described with single-agent high-dose cytarabine (Andersson et al. Cancer 1990; 65:1079–84). Pulmonary toxicity has not occurred in 8 subsequent patients given steroid premedication. The other grade 3 adverse events (AEs) included pneumonia (8), viral respiratory infection (6), abscess (4), bacteremia (13), and one additional grade 4 AE was septic shock. There has been no treatment related mortality. Fifteen of 17 currently evaluable patients have achieved CR, all but one with a single course, and 1 additional patient attained CRp (complete remission with incomplete platelet recovery). Using a model that accounts for cytogenetics, age, AHD, and organ function, the observed CR rate of 88% (95% CI 64%to 95%) exceeds the expected rate of 61% had the same patients received other high-dose cytarabine containing regimens but without clofarabine. Given the recent shortage of daunorubicin and the lack of assurance that an idarubicin dose (18mg/m2) that would be the nominal equivalent of 90mg/m2 daunorubicin is safe (Garcia-Manero et al. Haematologica 2002; 87:804–7), GCLAC may be a suitable alternative induction regimen for newly diagnosed AML and advanced myelodysplastic syndrome or myeloproliferative neoplasm. Disclosures:Becker:Sanofi-Oncology: Research Funding. Off Label Use: Clofarabine is FDA approved for treatment of relapsed pediatric acute lymphoblastic leukemia.

ELANE Mutations in Cyclic and Congenital Neutropenia: Genotype-Phenotype Relationships,

Abstract 3398Mutations in ELANE are the most common cause of cyclic and congenital neutropenia. We have detected 65 distinctive and presumably causal mutations in 212 patients with cyclic (91) and congenital neutropenia (121), including 28 families with a total of 101 affected members, followed longitudinally through the Severe Chronic Neutropenia International Registry and Repository. The diagnosis of cyclic neutropenia was associated with 13 different mutations. Congenital neutropenia occurred with 61 different mutations and 9 mutations were found in both cyclic and congenital neutropenia patients.ELANE mutations causing cyclic neutropenia cluster at the junction of exons 4 and 5 and include mutations in intron 4 (83/91). In a majority of the patients (64/91), the mutations are predicated to alter the binding pocket for the enzyme with its substrates based on application of the tertiary structure of neutrophil elastase using ICM-Browser-Pro software (Molsoft, San Diego, CA). However, the proportion of specific mutations affecting the binding pocket was similar for cyclic neutropenia 6/13 versus congenital neutropenia 29/61 (P=1, Fisher exact test). For the unique mutations associated with cyclic neutropenia, 2/4 mutations were predicted to affect the binding pocket.ELANE mutations causing autosomal dominant severe congenital neutropenia have been detected in exons 1, 2,3,4,5, and introns 3 and 4, with a wide diversity of predicted effects on the structure of mutant neutrophil elastase. An equivalent proportion of cyclic and congenital neutropenia patients affect the two glycosylation sites (congenital 55/121 versus cyclic 49/91, p=0.26, Fisher exact test). There was also an equivalent proportion of specific mutations in the two groups (cyclic 7/13, congenital 29/61, p=0.77, Fisher exact test).Seven of 7 mutations predicted to affect the structure of the active site of the enzyme caused congenital neutropenia in 10 patients (mutations exon 2: C26S, C42F, C42S; exon 4: Del 145–152, Del 161–170 2fs; exon 5: Del G172 fs182ter, C194ter). A significantly higher proportion of patients with active site mutations evolved to develop leukemia or myelodysplasia (AML/MDS) (4/10 active site mutations vs. other ELANE mutations 16/111, p<0.05, Fisher exact test), There was no difference in the age, age at the initiation of G-CSF therapy or the daily G-CSF dose for this cluster of 10 patients.As previously reported (Rosenberg et al Br J Haematol. 2008; 140: 210 and 2010; 150: 196) patients with congenital neutropenia who require higher doses of G-CSF are at greater risk of AML/MDS, whether or not they have ELANE mutations. We have not identified specific mutations associated with a higher risk of leukemia. There were no cases of AML/MDS in the 91 patients with cyclic neutropenia, but there were 20 cases of AML/MDS in the population of 121 congenital neutropenia patients. Two congenital patients with G185R mutations evolved to develop AML/MDS; 5 others with this same mutation have not shown signs of evolution. There are 18 different mutations in the other 18 patients. In the congenital neutropenia patients, we have found no apparent difference in the risk of AML/MDS for mutations affecting the binding pocket or glycosylation sites of the enzyme. In this group, we also cannot associate a requirement for higher G-CSF doses or failure to respond with specific mutations or categories of mutations. The distinctive features of ELANE mutations causing cyclic neutropenia, refractoriness to G-CSF and the risk of leukemia are still unknown. Disclosures:Dale:Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Boxer:Amgen: Equity Ownership.

FACS Analysis of Stat3/5 Signaling Reveals Ligand Sensitivity As a Significant Prognostic Factor in Pediatric AML: A Children's Oncology Group Report

Abstract 938Pediatric acute myeloid leukemia (AML) remains a devastating disease with a relapse rate near 50%. Kinase inhibitors are being incorporated into treatments, yet the signaling abnormalities targeted by these drugs remain poorly understood. Pathways involving Stat3 and Stat5 are known to be aberrantly active in AML, and increased activity has been associated with chemoresistant disease and poor outcome. Our hypothesis is that analysis of Stat3/5 activation patterns will delineate signaling abnormalities, and thereby guide the development of targeted therapies. We performed FACS analysis of the Stat3/5 signaling pathways in 140 diagnostic bone marrow samples from patients on the AML study CCG 2961. After thawing, cells were rested in serum-free medium for 2 hr. We measured tyrosine-phosphorylated Stat3 (pY-Stat3), serine-phosphorylated Stat3 (pS-Stat3) and pY-Stat5 in unstimulated cells, cells stimulated with low (1 ng/ml), intermediate (10 ng/ml), or high (100 ng/ml) dose G-CSF, and cells stimulated with low (0.5 + 1), intermediate (5 + 10), or high (50 + 100) dose IL-6 + soluble IL-6 receptor alpha. Constitutively phosphorylated proteins were quantified as the percent of events in the positive gate in unstimulated cells. Constitutive pY-Stat3 and pY-Stat5 were found to be common and highly variable. The median percent of pY-Stat3+ events was 37.3% (range 0.1–91.8%); the median %pY-Stat5+ was 18.6% (range 0.5–86.5%). In contrast, pS-Stat3 was generally low (median 3.63%, range 0.1–78.2%). There was non-significant correlation between %pY-Stat3+ and %pY-Stat5+. There was a significant correlation between %pY-Stat3+ and %total Stat3+, with Pearson correlation coefficient R=0.523 (p<0.001). Constitutively activated Src (pY-Src) was generally low (median 4.2%), although there was a population with 20–40 %pY-Src+, which also had high constitutive %pY-Stat3, suggesting that aberrant Src activation may account for high basal pY-Stat3 in some cases. Ligand responses were expressed as the fold increase in mean fluorescence intensity over unstimulated cells (δMFI). There was a strong correlation between pY-Stat3 and pY-Stat5 δMFIs to G-CSF (R=0.91, p<0.001), suggesting overlap of the pathways from G-CSF to each STAT. Likewise, the pY-Stat3 δMFI to G-CSF significantly correlated with the pY-Stat3 δMFI to IL-6 (R=0.343, p<0.001), suggesting convergence of the pathways from each receptor to Stat3. G-CSF receptor expression positively correlated with the GCSF-induced pY-Stat3 δMFI (R=0.396, p<0.001), whereas gp130 (IL-6 receptor) expression negatively correlated with the IL-6-induced pY-Stat3 δMFI (R=-0.262, p<0.005). None of the constitutive or ligand-induced pStat measurements, pY-Src, total Stat3, or receptor expression alone were significant predictors of survival. However, we did identify patterns of ligand sensitivity that predicted outcome. Patients with G-CSF-sensitive AML cells, defined as a pY-Stat3 δMFI > 2 in response to the low or intermediate G-CSF dose (33% of patients in the cohort), had a superior 5 year event-free survival (EFS; 57.8 ± 15% v. 37.2 ± 10%, p=0.025). A similar trend was observed for IL-6 sensitivity (p=0.1). When pY-Stat3 sensitivities to G-CSF and IL-6 were considered together, patients whose blasts were equally sensitive to both ligands had the best 5 yr EFS (69 ± 20%). Patients whose blasts responded to G-CSF only, or were unresponsive to both, had intermediate outcomes (5 yr EFS 38.5 ± 16% and 41.8 ± 14%, respectively). Surprisingly, patients whose blasts responded to IL-6 only had the worst outcome, with a 5 yr EFS of 25 ± 22% (p=0.047). These ligand sensitivity results highlight the importance of understanding not only molecular changes in AML, but also functional aberrancies. For example, cases with low constitutive and ligand-induced Stat3 activation may be resistant to Stat3 pathway inhibitors, whereas cases with high basal pY-Src or aberrant IL-6 sensitivity may be effectively treated with inhibitors of these pathways. In summary, multiparameter FACS analysis of Stat3 and Stat5 activities provides biologic and prognostic insight into the role of signaling aberrancies in AML, and this knowledge will promote rational implementation of new therapies. Disclosures:No relevant conflicts of interest to declare.

Cell Population Data As a Predictor of Stem Cell Aphaeresis Efficiency

Abstract 2991 Introduction:Autologous Hematopoietic Stem Cell Transplantation (AHSCT) is a necessary component of treatment for many oncohaemathological diseases. For success of AHSCT a sufficient quantity of Hematopoietic Stem Cells (HSC) are needed. The estimation of the quantity of HSC post aphaeresis is vital. The procedure of aphaeresis is time consuming and expensive but the CD34+ count (equivalent to HSC) measured by traditional flow cytometry, in peripheral blood can predict the quality of the aphaeresis product but is much more expensive than a complete blood count. It has been proposed that morphological changes of myeloid cells detected by some hematological analyzers could reflect the processes of bone marrow stimulation and may provide useful information for the prediction of the efficiency of stimulation and expected outcome of CD34+ stem cells.Materials and methods: Nine patients, 7 with multiple myeloma and 2 with Hodgkins disease were studied after informed consent: 8 female, 1 male, age range 21 to 59. Patients received the standard dose G-CSF, protocol-driven chemotherapy +G-CSF (dose=5 mcg/kg/day). Initial mobilization typically processed 10–12 liters of blood. The Beckman Coulter Cellular Analysis System DxH800 performs Flow Cytometric Digital Morphology analysis of leukocytes with measurement of cell volume (impedance), internal complexity and nucleo/cytoplasm ratio (cell conductivity in the radio-frequency current) and granularity (measurement of 5 angles of light scatter). All these measurements (Mean and Standard Deviations (SD)) are reported as numerical values, called Cell Population Data (CPD). Analysis of CD34+ stem cells was performed on FC500 Flow Cytometer with Stem Kit (Beckman Coulter) using the single-platform ISHAGE protocol. All patients were followed-up daily, starting from the day before G-CSF administration. Results:For patients, responding to therapy (8 from 9 included in the study) an increase in Ne CPD - Neutrophil Mean Volume (NeMV), and Neutrophil SD Volume (NeSDV) was seen from 2 to 4 days before the increase in CD34+ count in peripheral blood. The calculated parameter, Ne Immaturity Index (ImmNeIndex), using the formula (NeMV × NeSDV)/100 was introduced for the analysis. Patients that responded to the stimulation had an increase in NeMV greater than 15%, increase in NeSDV more than 60% and increase in ImmNeIndex more that 85% compared to the values pre- treatment. There was significant correlation between CD34+ and NeMV (correlation coefficient = 0.491, significance level P=0.0011, n=41), CD34+ and NeSDV (correlation coefficient = 0.418, significance level P=0.0065, n=41) and CD34+ and Imm Ne Index (correlation coefficient = 0.489, significance level P=0.0012, n=41). Patients were classified into 3 groups according to %CD34+ cells in peripheral blood at the end of one cycle of stimulation with NeMV, NeSDV and ImmNeIndex (Table 1), and this estimation can give information for the best time for harvest of CD34+ cells after aphaeresis.Table 1Ratio before treatment/after treatment%CD34+ cells in PB at the end of one cycle of stimulationImm Ne IndexNeMVNeSDV>0.52.28–2.921.26–1.471.87–2.370.2–0.51.9–2.031.14–1.241.64–1.77<0.21.131.041.09ROC curve analysis with MedCalc Software (Mariakerke, Belgium) showed that patients with % CD34+ cells higher than 0.2% in peripheral blood can be detected with NeMV (AUC 0. 903, cut-off 162, sensitivity 93%, specificity 89%) and Imm Ne Index (AUC 0.847 cut-off >41.43, sensitivity 93%, specificity 70%). Discussion:Cell Population Data available from Beckman Coulter Cellular Analysis System DxH800 are able to detect morphological changes in the cell size of neutrophils and anisocytosis of neutrophil population. These morphological features follow the stimulation of the bone marrow activity and the production of immature cells, including CD34+ cells. Changes in CPD were detectable 2–4 days before the observed increase in CD34+ count in the peripheral blood and can improve the management of patients. These parameters available on the routine haematological analyser without any additional cost or time can be used as additional criteria in the laboratory to make the decision about timing of aphaeresis for patients after the G-CSF stimulation. Disclosures:No relevant conflicts of interest to declare.

Remobilization with High Dose Methotrexate and Cytarabine in Patients with Non-Hodgkin Lymphoma and Multiple Myeloma Previously Failing Mobilization with Chemotherapy and Cytokine Treatment

Abstract 4402 BackgroundHigh-dose chemotherapy supported by autologous stem cell transplantation is a widely used therapeutic modality especially in patients with non-Hodgkin lymphomas (NHL) and multiple myeloma (MM). However, mobilization failure rates following first attempt are estimated to be between 5% and 30% and remobilization is required in those patients. Although plerixafor, a selective CXCR4 antagonist, is a promising agent for these purpose, mobilization with chemotherapeutic agents would be a hands-on option.°° Therefore we investigated the feasibility of high dose methotrexate and cytarabine with G-CSF as a remobilization regimen in those who failed a prior mobilization and collection with chemotherapy and G-CSF. Patients and methodsMobilization failure was defined as a collection of less than 5 × 106 CD34+ cells after 3–5 apheresis procedures. Methotrexate (3,500 mg/m2 in a 120 min infusion) on day 1 and cytarabine (3,000 mg/m2 infusion for 120 min) on day 4 and day 5 were followed by G-CSF. Supportive care and leucovorine rescue were done as standard protocol. Peripheral blood (PB) hematopoietic progenitor cells (HPCs) were counted with the Sysmex SE9000. PB progenitor cell harvest was initiated when HPC levels reached at least 5 per mm3. ResultsTotal 8 patients (6 NHL and 2 MM, median age: 55 years) who have failed in previous mobilization with conventional chemotherapy and G-CSF were tried again with high dose methotrexate, cytarabine and G-CSF. (table 1) Successful collection of CD34+ cells (> 5 × 106/kg) was achieved in six patients (75%). The total yield of CD34+ cells per kg of body weight harvested by 3 to 5 apheresis procedures was 6.28 × 106/kg (median; range, 1.53– 10.09 × 106/kg). (table 2) ConclusionHigh dose-MTX and cytarabine plus G-CSF may be a feasible chemotherapeutic regimen for stem cell mobilization in NHL and MM patients who have failed in previous mobilization with other type of chemotherapy and G-CSF.Table 1Patients characteristics and mobilization outcome (1st & 2nd °°mobilization)NAgeDiagnosisPrevious Treatmentprevious Mobilization regimen(1st)No. Of apheresis in 1st mobilizationTotal CD34+ yield (x106/kg ) in 1st mobilizationNo. Of apheresis in 2nd mobilizationTotal CD34+ yield (x106/kg ) in 2nd mobilizationCumulative CD34+ yield (x106/kg ) in 1st & 2nd mobilization148PTCLCHOP SMILESMILE44.0333.77.73257MZLRCVP ESHAPESHAP41.8845.337.21359MCLCHOP ESHAPESHAPHD Ara-C3,40.67, 1.5331.533.73454MMVAD PADCYC44.73410.0914.82564PTCLCHOP ESHAPESHAP20.647.017.61645DLBCLRCHOP ESHAPESHAP31.1257.738.85762MMVAD PADCYC43.7639.2112.97842EN NK/T cell lymphomaVIPDCYC53.1645.558.71Abbreviations: PTCL=Peripheral T-cell Lymphoma; MZL=Marginal Zone Lymphoma; MCL=Mantle Cell Lymphoma; MM=Multiple Myeloma; EN=extra nodal; DLBCL=Diffuse Large B-cell Lymphoma; CHOP=cyclophosphamide, doxorubicin, vincristine, prednisolone; SMILE=dexamethasone, methotrexate, ifosfamide, L-asparaginase, etoposide; RCVP=rituximab, cyclophosphamide, vincristine, prednisolone; ESHAP=etoposide, methylprednisolone, cytarabine, cisplatin; VAD=vincristine, doxorubicin, dexamethasone; PAD=bortezomib, doxorubicin, dexamethasone; RCHOP=rituximab+CHOP; VIPD=etoposide, ifosfamide, cisplatin, dexamethasone; HD Ara-C=high dose cytarabine; CYC=cyclophosphamide; No.=number;Table 2Remobilization of CD34+ cells after HD-MTX and cytarabine plus G-CSFNApheresis 1Apheresis 2Apheresis 3Apheresis 4Apheresis 5Cumulative CD34+ yield (x106/kg)10.121.542.043.720.671.890.891.885.3330.290.760.481.5341.831.293.693.1810.0950.410.953.262.397.0160.390.471.871.543.467.7371.353.094.779.2181.121.272.021.145.55Median (range)0.54 (0.12−0.83)1.28 (0.47−3.09)2.03 (0.48−4.77)1.88 (1.14−3.18)3.46 (3.46)6.28 (1.53 – 10.09) Disclosures:No relevant conflicts of interest to declare.

Impact of High Dose Cyclophosphamide on the Outcome of Autologous Stem Cell Transplant in Patients with Newly Diagnosed Multiple Myeloma,

Abstract 4127In the age of novel targeted agents, autologous stem cell transplant (ASCT) remains the standard of care for younger patients with newly diagnosed multiple myeloma (MM), offering similar treatment responses and overall survivals as standard chemotherapeutic agents but with the added benefit of a prolonged treatment-free period. Nevertheless, a standard of care for stem cell mobilization for ASCT has yet to be determined. Even in the era of new mobilization agents such as Plerixafor, Cyclophosphamide (Cy) and G-CSF combination remains the preferred mobilizing approach for patients with MM. Several studies have shown that Cy improves the stem cell yield at the expense of increased toxicity, but whether the administration of this chemotherapeutic agent pre-transplant has any impact on the long-term event-free and/or overall survival of myeloma patients remains controversial.In this study, we present a retrospective analysis of 186 patients with newly diagnosed MM who underwent ASCT with high-dose melphalan 200 mg/m2 (HDM) between December of 2000 and 2008 at our Institution. Eighty-three patients were mobilized with single agent G-CSF and 103 patients received high dose Cy (4 gm/m2) and G-CSF combination. Patient characteristics were similar between the treatment groups, including: age, gender, disease stage, and disease status prior to transplant. However, toxicity post-mobilization with Cy/G-CSF was significantly higher compared with G-SCF alone, including: febrile neutropenia (23%), hemorrhagic cystitis (8%), GI toxicity (57%), re-hospitalization due to complications and transplant delay (14%).The overall post-transplant toxicity was similar in the 2 groups, though the treatment related mortality was slightly higher in the Cy/G-CSF arm (4% versus 2%). Post transplant responses were not significantly different in the 2 groups, with 60% of patients achieving a VGPR or better after ASCT in the G-CSF group and 49% in the Cy/G-CSF group (p = 0.33). The median event-free survivals (EFS) for the Cy/G-CSF and G-CSF cohorts were 21.6 and 22.6 months, respectively, (p = 0.62) yielding no significant difference (Figure 1). Similarly, with a median follow up for surviving patients of 34.3 and 32.7 months, the median overall survivals were 68.2 and 62.3 months (p = 0.23) for the Cy/G-CSF and G-CSF cohorts, respectively (Figure 2). This retrospective analysis confirms that the addition of high dose Cy as part of the mobilizing regimen offers no improvement on the transplant outcome for patients with newly diagnosed myeloma and should therefore only be used in cases of difficult stem cell mobilization. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Renal Function Measures Improve on Lenalidomide and Dexamethasone and Compete with Patient Characteristics to Predict Lenalidomide Dose Density and Hematologic Toxicity: An E4A03 Analysis

Abstract 1882 Introduction:Lenalidomide (L) with dexamethasone (D) has demonstrated activity in untreated symptomatic multiple myeloma (MM) (Rajkumar et al. Lancet Oncol 2010; 11: 29–37). Neutropenia may occur, requiring dose reductions, interruptions, or G-CSF support in up to 20% of patients. Renal clearance accounts for 67% of L elimination, and renal insufficiency is associated with an increased risk of cytopenias. Product information recommends the use of the Cockcroft-Gault (C-G) equation for creatinine clearance (CrCL) estimation for initial dosing; however, other estimates including the Modification of Diet in Renal Disease (MDRD) method may be more accurate. We evaluated the impact of renal function on L dosing, cytopenias, and response in patients on the Eastern Cooperative Group (ECOG) E4A03 study. Methods:All patients (n=445) enrolled on E4A03 were included in this analysis. Clinical data at baseline and after 4 cycles was reviewed. Renal function over the first 4 treatment cycles was estimated using both last reported on cycle 4 and last ever reported serum creatinine (SCr) as the former method resulted in a loss of 70 more patients. Patients were classified by CrCL (mL/min) stage: (1) ≥90 (2) 60–89 (3) 30–59 (4) 15–29 (5) <15. Patients were further categorized into CrCL groups collapsing stages 3–5 while maintaining division of normal status. Demographic and disease data of age, ECOG performance status (PS), sex, race, ISS stage, and complete blood counts were included. Percent of planned L dose and dose interruptions were calculated. Descriptive statistics were used to assess measures at each timepoint. Associations between baseline MDRD, C-G, SCr and patient characteristics were assessed by chi-square testing, and logistic regression models of L dose modification, cytopenias, and response status with renal function measures were created. Results:Mean SCr improved in 361/445 (81%) by 0.23 mg/dL following treatment initiation. Mean MDRD CrCL improved in 264/445 (59%) by 24.3 mL/min. Because only initial weight was captured, C-G estimates over time were not available. ISS stage II/III was associated with baseline elevated SCr (> 1.5 mg/dL) and low CrCL (p<0.001). Dose modification of L occurred in 44% of patients over 4 cycles (Table 1). In adjusted analyses, C-G predicted L dose modifications, p<0.001 [stage 3–5 vs 1 OR 2.09 (1.23-3.55)] along with ECOG PS. Similarly, in adjusted analyses, grade 2–5 cytopenias were better predicted by C-G stage, p<0.001 [stage 3–5 vs 1 OR 5.08 (2.18-11.84)], over MDRD stage, p=0.025 [stage 3–5 vs 1 OR 2.66 (1.07-6.61)]. No association between baseline or change in renal function and ≥ VGPR after 4 cycles was noted.Table 1Baseline Characteristics Associated with Lenalidomide Dose ModificationsVariableOR (95% CI)p-valueAge (≥ 65 vs < 65y)1.57 (1.08, 2.29)0.019SCr (> 1.5 vs ≤ 1.5 mg/dL)1.83 (1.06, 3.15)0.031ISS Stage II/III vs I2.04 (1.33, 3.12)0.004ECOG PS 1/2 vs 01.80 (1.23, 2.64)0.002MDRD (Stage 3–5 vs Stage 1)1.97 (1.10, 3.51)0.041C-G (Stage 3–5 vs Stage 1)2.59 (1.59, 4.21)< 0.001 Conclusion:Renal function improvement in patients receiving LD warrants regular CrCL monitoring and L dose increases in patients on reduced doses without cytopenias. Baseline MDRD is not superior to C-G for CrCL estimation for predicting L dosing and cytopenia development. Optimizing L exposure through pharmacokinetic modeling or other methods may further refine dosing. Disclosures:Lonial:Millenium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Onyx: Consultancy; Merck: Consultancy.

UPDATE On the RISK of SECONDARY LEUKEMIA In GENETIC SUBGROUPS (ELANE, HAX1, WAS, G6PC3, p14) of CONGENITAL NEUTROPENIA In EUROPE

Abstract 1106▪▪This icon denotes a clinically relevant abstractCongenital neutropenia (CN) is well known as one of the premalignant bone marrow failure syndromes with an overall incidence of secondary leukemia of more than 10 percent. With the identification of new causative gene mutations the number of genetic subgroups is still increasing. Due to the limited patient numbers for each subgroup we re-assessed the incidence and potential risk factors of leukemic transformation in CN patients with known gene mutations in ELANE, HAX1, G6PT, G6PC3, WAS, SBDS, TAZ1 and p14 or no identified mutation, respectively, by analyzing all available data from the European Branch of the Severe Chronic Neutropenia Registry (SCNIR). For comparison we also analyzed patients with cyclic neutropenia (CyN) with or without ELANE mutations.Results from genetic testing were available for 255 of 336 CN patients. Of the 255 CN patients 71 patients revealed ELANE mutations, 31 HAX1 mutations, 47 SBDS, 18 WAS, 21 G6PT, 9 G6PC3, 4 p14 and 5 TAZ1 mutations. In addition, in 36 patients neither ELANE nor HAX1 mutation was detectable. 81 patients were not tested to date, but further genetic evaluation is not yet completed. Results from genetic testing were also available in 28 of 66 patients with CyN of whom 22 revealed ELANE mutations and 6 were negative for ELANE mutations.Secondary malignancies occurred in 36 of the 336 CN patients and 1 of the 66 patients with CyN. Leukemia distribution is unequal in genetic subtypes of CN: Patients with ELANE-CN and HAX1-CN and ELANEneg/HAX1neg-CN have the highest risks followed by patients with WAS-CN and Shwachman-Diamond syndrome. Progression to MDS or leukemia has so far not been reported in G6PT, G6PC3, TAZ1 or p14 CN cases in our registry.All subgroups benefit from G-CSF treatment. However, patients requiring higher maintenance doses of G-CSF are at greater risk of leukemic transformation (Rosenberg, Zeidler et al., 2010).G-CSF receptor mutations usually occur without additional clinical features, e.g. change in response to G-CSF treatment in different CN subtypes. Mutations can be used as early indicators for the process of malignant transformation. Conclusion:The incidence of secondary leukemia reflects the heterogeneity of congenital neutropenia ranging between no leukemia and more than 10 percent in patients with an underlying mutations in ELANE, HAX1, WAS or ELANE/HAX1 negative patients. However, patient numbers within each genetic subgroup are still limited.Despite mutations in the ELANE gene patients with cyclic neutropenia exhibit no increased risk for malignant transformation. Mutational analysis is helpful to identify the genetic cause of severe congenital or cyclic neutropenia but with limited numbers in genetic subgroups still does not serve to identify patients at risk of leukemic transformation. Detection of G-CSF receptor mutations should result in close observation of the patient with consideration of stem cell transplantation as soon as additional aberrations occur. Disclosures:No relevant conflicts of interest to declare.

Oxygen-Independent Stabilization of the Oxygen-Labile Transcription Factor HIF-1α with Dimethyloxalyl Glycine or FG-4497 Increases Hematopoietic Stem Cell Quiescence In Vivo and Mobilization in Response to G-CSF

Abstract 2334The endosteal region of the bone marrow (BM) is hypoxic in steady-state and most quiescent hematopoietic stem cells (HSC) reside in hypoxic, poorly perfused niches. Mobilizing doses of G-CSF renders most of the BM space hypoxic. Most cellular effects of hypoxia are mediated by O2-labile hypoxia-inducible transcription factors (HIF). At O2 concentration above 2%, HIF-α is rapidly hydroxylated on Pro residues by the prolyl hydroxylases PHD1, PHD2, and PHD3. HIF-α prolyl hydroxylation recruits the E3 ubiquitin ligase VHL, which targets HIF-α to the proteasome. In hypoxia (O2 < 2%), HIF-α proteins are stable, associate with the β subunit ARNT, and translocate to the nucleus to activate transcription. It has emerged that HIF-1α regulates HSC proliferation and is critical to maintain long-term HSC self-renewal in vivo.In this study, we investigated the effect of pharmacological stabilization of HIF-1α protein on HSC cycling and mobilization in mice using two different HIF prolyl hydroxylase (PHD) inhibitors, dimethyloxalyl glycine (DMOG) and FG-4497. We first assessed whether DMOG and FG-4497 stabilized HIF-1α protein in BM leukocytes in vivo by western-blot. Following a single injection of 400mg/kg DMOG, HIF-1α protein was stabilized for up to 6 hrs in BM leukocytes. With 20mg/kg FG-4497, HIF-1α protein persisted over 12 hours. HIF-1α protein was below detection in the BM from saline injected animals.C57BL/6 mice were injected daily with 400mg/kg DMOG to measure effect on HSC cycling and BrdU incorporation by flow cytometry. Using Hoecht33342 and FITC-conjugated anti-Ki67 antibody, a 18 day DMOG treatment increased the proportion of Lin-negative Kit+ Sca1+ CD48- (L-K+S+48-) HSC in phase G0 from 61±11% to 84±6% (p<0.001, 5 mice/group). Conversely, DMOG decreased the proportion of HSC in phase G1 from 31±5% to 14±5% (p<0.001). Similar enhancement of quiescence was observed in less primitive cells such as L-K+S+48+ cells and L-K+S- myeloid progenitors. In mice given BrdU in their drinking water for the last 3 days of the experiment, a 18 day DMOG treatment reduced 3-fold the proportion of L-K+S+48- HSC that incorporated BrdU and halved the proportion of BrdU+ L-K+S+48+ progenitors. Shorter DMOG treatments (6 days or 12 days) did not alter hematopoietic stem and progenitor cell (HSPC) cycling or BrdU incorporation. In contrast, mice treated with 20mg/kg/day FG-4497 had significantly increased proportion of HSPC in G0 phase after only 6 days of treatment, likely due to the more lasting effect of FG-4497 on HIF-1α stabilization. Proportion of HSPC that incorporated BrdU was also significantly decreased. As HIF-PHD inhibitors also increase erythropoietin (EPO) expression by stabilizing HIF in the kidney, we injected a parallel cohort of mice with EPO daily for 18 days. Despite a very strong increase in red cells and hemoglobin in the blood, EPO had no effect of HSPC cycling and BrdU incorporation. Therefore the effects of HIF-PHD inhibitors on HSPC cycling are not an indirect effect of increased EPO.Since HIF-PHD inhibitors slow HSC cycling in vivo, we tested whether they could protect HSC from sublethal irradiation. Mice were treated with 400mg/kg DMOG or saline for 22 days and then irradiated with 9.0Gy. Both cohorts were leukopenic betweens days 7 and 14 post-irradiation but DMOG treated mice had significantly higher blood leukocytes at days 22 and 30, and higher platelet numbers day 22 and all subsequent time-points suggesting that DMOG enhances HSC survival with higher blood recovery.Finally mice were injected with DMOG for 4 days and pegylated rhuG-CSF 3 and 1.5 days before harvest. DMOG doubled mobilization in response to G-CSF with 1,620±530 CFC / mL blood in response G-CSF alone, and 3,250±830 CFC / mL in the G-CSF + DMOG group (p<0.05). Without G-CSF, circulating CFC were less than 10 / mL blood. Similarly, FG-4497 pre-treatment increased the number of CFC mobilized into the spleen 4-fold compared to G-CSF alone (p<0.01).In conclusion, these data highlight the importance of the hypoxia pathway and HIF in the regulation of HPSC cycling and trafficking in vivo. Furthermore HIF-PHD inhibitors may provide therapeutic opportunities to protect HSC from deleterious effects of irradiation as well as increasing mobilization efficiency for transplantation. Disclosures:Walkinshaw:Fibrogen Inc.: Employment, Equity Ownership.

Impact of G-CSF on Outcomes of Pregnancy in Women with Severe Chronic Neutropenia

Abstract 4786Pregnancy in patients with severe chronic neutropenia is associated with high risk of spontaneous abortions. Patients and physicians often ask about benefits and safety of G-CSF administration during pregnancy. We therefore conducted a telephone survey of women of child-bearing potential with the diagnoses of congenital, cyclic, idiopathic or autoimmune neutropenia who are enrolled in the North American branch of the Severe Chronic Neutropenia International Registry (SCNIR).We identified 99 women who had 214 pregnancies before and on G-CSF for this cross sectional study, expanding our previous preliminary investigation, (Boxer et al, ASH abstract 2010). In this population, 31 women had pregnancies before availability of G-CSF, 35 were on long term G-CSF at the time of their pregnancies and 33 were taking G-CSF for some, but not all, of their pregnancies. For those on G-CSF during their pregnancies, the median G-CSF dose was 1.30 mcg/kg/day (mean dose 2.2 ± 0.4 [SEM] mcg/kg/day, dose range 0.1 to 28 mcg/kg/day). Treatment was administered throughout the pregnancy in 74% (49/65) of the pregnancies. Five percent (3/65) were treated only in the 1st trimester, 11% (7/65) were treated only in the last 2 trimesters, 5% (3/65) treated for the last trimester only, and 5% (3/65) treated in 2 of 3 trimesters (2 treated in 1st and 3rd trimesters, 1 treated in the 1st and 2nd trimesters). Sixty-two women with 137 pregnancies while not on G-CSF [congenital neutropenia (CN) N=3, cyclic neutropenia (CyN) N=15, idiopathic neutropenia (IN) N=43, autoimmune neutropenia (AN) N=1] were compared with 47 women having 77 pregnancies while on G-CSF therapy [CN=9, CyN=14, IN=21, AN=3].Pregnancies occurring in neutropenic women without G-CSF therapy had the following complications: premature labors (8), premature rupture of membranes (1), life-threatening infections (2), and minor infections (5). Patients on G-CSF during pregnancy reported no premature labors (0), no life threatening infections (0), minor infections (5), thrombocytopenia (1), placenta previa (1), abruptio placenta (1), and death following incomplete elective termination (1). By diagnostic category complications in the no G-CSF mothers occurred in 16 patients (CN=1, CyN=5, IN=9, AN=1), compared with nine in the G-CSF treated group (CN=1, CyN=4, IN=4). There were 31 spontaneous miscarriages in the women not on G-CSF (CyN=8, and IN=23) compared with 5 in the women (CyN=4, IN=1) on G-CSF.There were 93 live births from 137 pregnancies in the non G-CSF group. Thirteen of these infants had neonatal neutropenia. Other major neonatal complications in the no G-CSF group included significant infections (3) [meningitis 1, septicemias 2], minor infection (1), cerebral palsy associated with prematurity (1), respiratory distress syndrome (1), and collapsed lung (1). In the G-CSF treated group there were 65 live births from 77 pregnancies. Fifteen of these infants were neutropenic. Other complications in the G-CSF treated group included minor infections (2), abruptio placenta associated with neonatal apnea (1), and premature infants with associated complications (2). There were 2 congenital abnormalities: tracheal esophageal fistula (1) and hydronephrosis (1) in 65 live births, a frequency similar to the general population.This study showed that women with chronic neutropenia who were on G-CSF during pregnancy had a higher percentage of live births, 84% (65/77) than those not on G-CSF, 68% (93/137) (p=0.011, two tailed Fisher exact test). G-CSF therapy was associated with a lower number of spontaneous abortions, with a 6% (5/77) spontaneous abortion rate in the G-CSF treated group versus 23% (31/137) in the non G-CSF group (p=0.003, two tailed Fisher exact test). G-CSF therapy was also associated with a lower rate of serious maternal complications. Two common congenital anomalies (2/65) were observed in the offspring of the G-CSF treated mothers and no anomalies were observed in the offspring of the mothers not receiving G-CSF. Overall the newborns from the G-CSF treated mothers appeared to have fewer neonatal complications. Based on this analysis, we recommend that G-CSF therapy should be offered and continued during pregnancy in women with severe chronic neutropenia. The available data indicates that administration throughout pregnancy is well tolerated and protective of maternal health, including reducing the risk of infections and spontaneous abortions. Disclosures:Boxer:Amgen: Equity Ownership. Dale:Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Hematopoietic Cell Mobilization for Gene Therapy of Adult Patients with Severe Beta-Thalassemia: The Challenge of Splenectomy and the Role of Plerixafor

Abstract 166▪FN2▪This icon denotes a clinically relevant abstractIn preparation for a trial for gene therapy of thalassemia, we investigated the safety and efficacy of stem cell mobilization in adult patients with severe thalassemia using either GCSF or Plerixafor. We first assessed mobilization with G-CSF or G-CSF following pretreatment with Hydroxyurea (HU). HU was used in non splenectomized patients in order to reduce extramedullary hemopoiesis in spleen and the splenic size and in splenectomized patients in order to decrease the high number of platelets and mainly WBCs before G-CSF, so as to reduce the chance of complicated leukocytosis during mobilization. We present data on 23 patients from the G-CSF study (EudraCT number 2005-000315-10, NCT00336362) and on 14 patients from the Plerixafor study (EudraCT number 2009–014136-37, NCT01206075). The bulk of the CD34+cell enriched leukaphereses product in either study was cryopreserved for the future gene therapy trial. Mobilization with G-CSF was safe and effective in non-splenectomized patients (CD34+cellsX106/kg/2aphereses: 6.67±2.87, n=6). HU+G-CSF-treated subjects (n=4), mobilized successfully only when an optimal wash out period of approximately two weeks was maintained before G-CSF administration (CD34+cellsX106/kg/2aphereses: 7.34±1.19 vs 1.86±0.76, respectively, p=0.03). HU reduced the spleen size before G-CSF (376±96cm3 vs 556±218cm3) and resulted in less splenic enlargement during mobilization as compared to non-HU pretreated subjects (26.8% vs 60%, p=0.1), although this difference did not reach statistical significance. Splenectomized patients responded excessively to G-CSF by developing early hyperleukocytosis (day 3 mean WBCs: 80.0±7.5×103/μl) without a corresponding rise in blood CD34+cells. This necessitated a significant G-CSF dose reduction or hold resulting in poor yields in the majority of cases (CD34+cells/kg/2aphereses: 2.0±1.7, n=4). One-month HU-pretreatment (n=9), prevented the G-CSF-associated hyperleukocytosis during mobilization (mean WBCs: 60.4±20.8×103/μl) allowing for safe and successful CD34+cell collections (CD34+cells/kg/2aphereses: 6.6±2.3, p=0.02 vs G-CSF-alone), but again, only when an optimal 2-week wash out period was maintained. Despite the safe and effective mobilization with the optimal HU+G-CSF combination of splenectomized patients, HU pretreatment significantly prolonged the mobilization procedure. Plerixafor induced a rapid and effective mobilization in both the splenectomized and non-splenectomized patients (CD34+celsX106/kg/1 or 2 aphereses: 7.29±1.82, n=8 and 5.38±1.99, n=6, respectively) and it was very well tolerated. In SPL subjects, the mean yield per apheresis with plerixafor was higher, with a trend to significance, over its G-CSF counterpart and similar to the mean yield obtained by the optimally HU+G-CSF-treated patients in the G-CSF study (CD34+cells/kgX106/apheresis: 4.19±3.13 vs 1.01±0.85 vs 3.32±1.16, p=0.06 and p=0.6 respectively). One patient who was remobilized with the combination of G-CSF+plerixafor due to previous failure to collect 32×106/kg/2aphereses with G-CSF-alone, yielded 6.5χ10^6 CD34+cells/kg by one apheresis. Importantly also for the non-SPL patients treated with plerixafor, a mean increase of splenic volume of 10.9±14.3% was encountered which was significantly lower than the 60% mean spleen volume increase during G-CSF mobilization (p=0.03). There was no significant difference in the clonogenic capacity (CFU-GM, BFU-E) of CD34+cells mobilized by G-CSF, HU+G-CSF or Plerixafor. However, there was a trend for Plerixafor to mobilize more primitive HSC subpopulations (CD34+/CD38−, CD34+/CD38−/HLADR−) as compared to HU+G-CSF- or G-CSF-alone-mobilized cells.These results suggest that either G-CSF or Plerixafor could be used for mobilizing non-splenectomized patients. Plerixafor seems to represent the agent of choice for mobilization of splenectomized patients with thalassemia. Disclosures:Off Label Use: Plerixafor is used off-label for mobilization of thalassemic subjects.