Course Of G-CSF Research Articles

Targeting CXCR4, VLA4, and CXCR2 for Hematopoietic Stem Cell Mobilization

Background: Hematopoietic stem cell (HSC) transplant is an essential treatment for a variety of blood disorders and malignancies. A key step in this procedure is the mobilization of donor stem cells. The most commonly used regimen for donor mobilization is a 5-day course of G-CSF. The length of this regimen coupled with the associated side effects emphasizes a need for superior alternatives. In recent years, there has been a growing understanding of mechanisms governing stem cell retention within the bone marrow niche. This has led to the development of new mobilization drugs that specifically target these processes. Two examples of previously described drugs that target mechanisms of stem cell retention are Plerixafor (a CXCR4 inhibitor already in clinical use), and truncated Gro-Beta (tGroβ; a CXCR2 agonist). Another potential target for inducing mobilization is disruption of the interaction between the VLA-4 integrin and its ligand VCAM-1. In this study, we evaluate the efficacy of novel VLA-4 inhibitors (VLA4i) alone and in combination with Plerixafor and/or tGroβ for the purposes of hematopoietic stem cell mobilization. Methods: We synthesized over 15 novel VLA-4 inhibitor molecules and tested their potency using soluble VCAM-1 binding assays. The 5 inhibitors determined to be most potent were then tested in vivo in DBA mice for their ability to mobilize HSCs alone and in combination with tGroβ and/or Plerixafor (n=5). HSC mobilization was measured in wild-type and splenectomized mice via flow cytometry to quantify the proportion of LSK (Lineage- Sca+ cKit+) cells as well as via Colony Forming Unit (CFU) assays. For competitive transplant, mobilized CD45.1+ BALB/c mouse blood (10 uL) was injected into lethally irradiated CD45.2+ BALB/c recipients alongside 2.5x105 CD45.2+ BALB/c bone marrow cells (n=10 / cohort). HSC engraftment was monitored monthly via flow cytometry for ratio of 45.1+ vs. 45.2+ cells in peripheral blood. Results: Firetagrast and BIO5192 are previously characterized VLA4i that have been administered to humans for indications unrelated to HSC mobilization. Our best VLA4i to date, LGB-2019, exhibited similar potency as BIO5192 in preventing the binding of sVCAM-1 to VLA-4 (IC50: 1.7nM) and was >200-fold more potent than firategrast. LGB-2019 showed increased aqueous solubility and mobilized 1.5-fold more murine LSK cells for a longer time period (peak HSC mobilization maintained for 4 hours) than BIO5192 when administered alone. Simultaneous injection of C57BL/6 mice with LGB-2019 (VLA4i), Plerixafor (CXCR4i) and tGro-β (CXCR2a) resulted in a synergistic increase in circulating CFUs (Fig. 1A; 9.8 x 103 CFUs/mL) and LSKs (Fig. 1B; 12.8 LSKs/uL) at 4 hours post-injection. In contrast, 5 days of G-CSF treatment mobilized approximately 3-fold and 8-fold less CFUs and LSKs, respectively (Fig. 1A-B). We saw no significant difference in mobilization for splenectomized vs. wildtype mice (23.4 x 103 CFUs/mL vs. 23.0 x 103 CFUs/mL) when mobilizing DBA/2 mice via VLA4i+CXCR4i+CXCR2a. Three months after competitive transplantation, blood obtained from BALB/c mice mobilized with the triple combination engrafted significantly better than blood obtained from mice treated with G-CSF or the dual combinations (Fig. 1C). Summary: New insights about the stem cell niche have allowed for the development of targeted drugs for the purposes of mobilization. Here, we show that a novel VLA-4 receptor inhibitor in combination with two other known mobilizers induces mobilization of hematopoietic stem and progenitor cells (CFU/LSK) at levels superior to the standard of care G-CSF and in a dramatically shortened time frame. Mouse transplant data also show superior engraftment in lethally irradiated recipients when using the triple cocktail regimen compared to the G-CSF mobilized graft. Secondary transplants are ongoing and will provide a more complete picture of primitive HSC mobilization and serial engraftment properties of the cells. Disclosures Rettig: WashU: Patents & Royalties: Patent Application 16/401,950. Karpova:WashU: Patents & Royalties: Patent Application 16/401,950. Ruminski:WahU: Patents & Royalties: Patent Application 16/401,950. Morrow:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. DiPersio:Cellworks Group, Inc.: Membership on an entity's Board of Directors or advisory committees; RiverVest Venture Partners Arch Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Equity Ownership; Incyte: Consultancy, Research Funding; Bioline Rx: Research Funding, Speakers Bureau; Macrogenics: Research Funding, Speakers Bureau; Karyopharm Therapeutics: Consultancy; Celgene: Consultancy; Amphivena Therapeutics: Consultancy, Research Funding; WUGEN: Equity Ownership, Patents & Royalties, Research Funding; NeoImmune Tech: Research Funding.

Cost effectiveness of G-CSF after allogeneic peripheral blood stem cell transplant.

e18540 Background: In spite of the fact G-CSF has been used post stem cell transplant (SCT) to accelerate neutrophil engraftment its use post allogeneic SCT remains controversial. ASCO does not recommend its use after allogeneic SCT. To further evaluate the effectiveness of its use, we compared outcomes in pts who underwent related and unrelated peripheral blood SCT(PBSCT) either with or without the use of G-CSF post SCT. Methods: This is a retrospective study comparing early outcomes in pts who received G-CSF starting on day + 6 post SCT until engraftment with pts who did not receive a planned course of G-CSF. Pts who underwent Allogeneic PBSCT between 2012-2014 at our institution were included. Pts who received marrow, haploidentical or cord blood transplants were excluded. Associations with survival outcomes were assessed by univariable and multivariable Cox proportional regression models. Results: A total of 162 patients were evaluated. Sixty-five pts received G-CSF post SCT and 97 did not. The only difference between the two groups was that more pts in the G-CSF group received myeloablative-conditioning (MAC) regimen (78% vs. 55%, p = 0.008). Other pt characteristics were not significantly different. Length of hospital stay was significantly lower in the G-CSF group (24 vs. 27 days P = 0.002). Pts who received G-CSF had earlier neutrophil engraftment (median, days 11 vs. 14 p = < 0.001). The median day of platelet engraftment was 15 days in both groups. There was no significant difference between the 2 groups in re-admissions in the first 100 days, and the incidence of acute or chronic GVHD. In multivariate analysis use of G-CSF did not significantly impact non- relapse mortality, relapse free survival and overall survival. However, relapse rate was significantly lower in G-CSF group in multivariable analysis (hazards ratio = 0.44, p = 0.03). Conclusions: Use of G-CSF post allogeneic PBSCT is associated with earlier neutrophil engraftment, shorter hospital stay and a suggestion of a reduced relapse rate after PBSCT. Our experience suggests that use of G-CSF (on average for approximately 5 days) in this setting is cost effective as it reduces hospitalization duration without adversely impacting post-transplant outcomes.

Combined Targeting of CXCR2 and VLA4 Results in Rapid and Synergistic Mobilization of Hematopoietic Stem and Progenitor Cells in Mice

Introduction:Since the first description of hematopoietic stem and progenitor cell (HSPC) mobilization over forty years ago, it has become the standard of care for both autologous and allogeneic transplantation. A five-day course of G-CSF represents the most commonly used mobilization regimen today. The CXCR4 inhibitor, plerixafor, is a more rapid but weak mobilizer when used as a single agent, thus emphasizing the need for faster acting agents with more predictable mobilization responses and fewer side effects.Methods:Given the critical role of VLA4/VCAM1 signaling for migration and retention of HSPC, we were seeking to identify small molecule antagonists of VLA4 with improved potency and bioavailability. Relative to previously described comparators Bio5192 (□4□1-specific) and firategrast (□4□1 and □4□7 dual-specific), our lead candidate, CWHM-823, exhibited increased aqueous solubility and ~10-100 fold better activity in blocking VLA4 and mobilizing HSPC in mice. CWHM-823 pharmacokinetics and mobilization were assessed in BALB/c and DBA/2 mice at different doses (3 to 15 mg/kg) and time points (15 to 240 min) when administered alone or in combination with the truncated isoform of the CXCR2 agonist Gro-beta (tGro-β, 2.5 mg/kg, generously provided by GlaxoSmithKline). HSPC mobilization was monitored using flow cytometry and clonogenic in vitro assays. "True" stem cells were measured in a serial competitive transplantation assay. The combination of tGro-β and VLA4 antagonist was further tested in diabetic mice in comparison to G-CSF (9 x 100μg/kg, q12h). RNA profiling of flow-sorted HSPC was performed via microarray analysis.Results:The combination of tGro-β with each VLA4 antagonist resulted in a dramatic synergistic increase in circulating HSPC numbers when compared to steady state (50-70-fold) or treatment with single agents (3-10 fold) including tGroβ. Mobilization with tGro-β + CWHM-823 was rapid, peaking at 15-30 minutes after injection. In a model of streptozotocin-induced diabetes, the mobilopathy (reduction in stem cell mobilization compared to wild type mice) was considerably less pronounced with the combination tGro-β + CWHM-823 (~1.5-fold lower CFU mobilization in diabetic mice) versus the 5-day course of G-CSF (~3-fold reduction).Despite the superior progenitor cell mobilization achieved with G-CSF (~2-fold more CFU and LSK/ml), the concentration of serially repopulating units (RU) was equally high in the tGro-β + CWHM-823 and G-CSF mobilized grafts suggesting a higher HSC frequency (1 RU out of 200 vs. 1 RU out of 400 LSK/CFU) in the tGro-β + CWHM-823 mobilized grafts (Figure 1). RNA profiling demonstrated close similarity between the expression profile of tGro-β + CWHM-823 mobilized, BM resident, and G-CSF mobilized LSK, with less than 0.5% of genes found to be significantly up- or downregulated.CXCR2 chemokine receptor stimulation was critical for the observed synergistic response, as pretreatment ("priming") or simultaneous treatment with tGro-β resulted in subsequent enhanced mobilization using VLA4 inhibitors, whereas reversed administration (VLA4 antagonist followed by tGro-β) had no effect on potency of either agent. Lack of surface CXCR2 expression on HSPC suggested that a rapidly acting effector molecule released from tGro-β-stimulated mature myeloid cells may subsequently influence VLA4-mediated HSPC adhesion/retention. Consistent with this theory, we observed increased protease MMP-9 in plasma within minutes after treatment with tGro-β + CWHM-823.Conclusions:We describe a novel strategy for rapid, reliable, and potent mobilization of HSPC in mice using a combination of VLA4 blockade (via novel and potent □4□1 inhibitors) and CXCR2 activation (via tGro-β). The combination of tGro-β + VLA4 inhibitors or tGro-β followed by VLA4 inhibitors results in synergistic and rapid HSPC mobilization with quantity and quality of repopulating units similar to optimal mobilization with G-CSF. These data suggest further development of tGro-β + VLA4 inhibitor combinations for clinical testing is warranted. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Expansion and Maintenance of Hematopoietic Stem and Progenitor Cells in Course of Long-Term Inhibition of CXCR4/CXCL12 Signaling

During the past two decades peripheral blood stem cells have become the favored graft source for HSCT with 80 % of allogeneic and almost 100 % of autologous HSCT performed with mobilized blood. The critical role of the interaction between the chemokine receptor CXCR4 and its chief ligand CXCL12 for retention and migration of hematopoietic stem and progenitor cells (HSPC) has been well established. Interference with CXCR4/CXCL12 signalling iscurrentlybeing exploited as a strategy to mobilize HSPC indirectly with the most clinically relevant mobilizing agent to date, G-CSF as well as directly with the bicyclam CXCR4 antagonist Plerixafor (AMD3100).In this study, qualitative and quantitative effects of long-term pharmacologic inhibition of CXCR4/CXCL12 axis within the HSPC compartment were investigated in healthy C57BL/6 mice using the non-peptidic small molecule CXCR4 antagonists Plerixafor and ALT1188 along with the Protein-EpitopeMimeticsInhibitor POL5551.Up to 12-14 fold higher mobilization efficiency was achieved by applying the antagonists via two weeks of continuous infusion (up to 8-10x104 CFU-C and LSK/ml) as compared to bolus treatment (4-6x103 CFU-C and LSK/ml) or 5-day course of G-CSF (3-6x103 CFU-C/ml).Despite dramatic increase in numbers of circulating HSPC, the BM HSPC pool dis not decrease; in fact it expanded up to 2-4-fold compared to steady state reservoir (sham-operated control mice) as measured by immunophenotypical (LSK SLAM) and functional (e.g. serial competitive transplantation) properties of the cells. Thus, in contrast to genetically CXCR4 ablatedHSPC, the reversible long-term blockade of the receptor did not diminish the long-term repopulating capacity of HSPC.Cell cycle analysis showed a 2-3-fold increase in cycling activity of BM HSPC: only 10-20% of LSK and 30-40 % of LSK SLAM cells were found to be quiescent (in G0 phase of the cell cycle) after two weeks of CXCR4 antagonist infusion versus 50-60 % of LSK and 70 % of LSK SLAM found in G0 under homeostatic conditions. This increased proliferation was very similar to the one induced transiently at day 3 G-CSF treatmentand would conceivably explain the sustained mobilization without concomitant depletion of the BM HSPC pool. Profiling of differentially treated BM HSC (LSK SLAM) via microarray analysis did not reveal substantial effects of CXCR4 inhibitor infusion on the expression signature.Ofnote, major cytological changes typically associated with G-CSF induced mobilization, e.g. depletion of bone lining osteoblast lineage cells and macrophages, were not detected in continuous infusion of POL5551 exposed BM suggesting limitedeffects within the BM niche compartment. Moreover analysis of the BM HSPC after different washout periods at the end of continuous infusion treatment revealed a rapid (within 1-3 days after discontinuation of infusion) reestablishment of steady state HSPC numbers in the BM.Our data suggest that prolonged pharmacologic blockade of the CXCR4/CXCL12 axis using multiple small molecule inhibitorsrepresents an approach thatreleasesHSPCwith efficiency superiorto any other knownmobilization strategybut also may serve as an effective method induce cell cycling and thus expand BM HSPCs. [Display omitted] DisclosuresLevesque:GlycoMimetics: Equity Ownership.

Potent Stem Cell Mobilization with the Novel CXCR4 Antagonist POL6326 - Results of a Phase IIa Dose Escalation Study in Comparison to G-CSF

Background:Stem cell mobilization (SCM) with G-CSF is efficient but - although overall safe - inconvenient because of the five-day injection regime and certain contraindications. Side effects, sometimes severe, are frequent. These disadvantages fuel the quest for alternative mobilizing agents. Mobilization with the CXCR4-inhibitor plerixafor is rapid, albeit insufficiently efficacious on its own. POL6326, a potent 2nd generation macrocycle CXCR4 antagonist, has demonstrated rapid mobilization kinetics and efficacy in mice. We herein report the results of a Phase IIa dose escalation trial where SCM in response to POL6326 was compared with G-CSF in healthy volunteer stem cell donors.Methods:In this Phase IIa open label trial, healthy volunteer stem cell donors with average mobilization (121±7 CD34+ cells/μL, MW±SEM)after a five-day course of G-CSF, and a wash-out period of at least 6 weeks, received POL6326 at 500-2500 µg/kg as a single 2-hour i.v. infusion. Safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) were assessed in 3-10 subjects/dose group. Subgroups received two doses of POL6326, 1000 and 2500 µg/kg or 1500 and 2500 µg/kg, at least 2 weeks apart (paired intra-individual analysis). For PK and PD blood samples were collected before (0) and at 2, 3, 4, 6, 8 and 24 hrs after infusion start. Complete blood count, CD34+, CFU-C count and PK were assessed at all time points. At 0, 4, 8 and 24 hrs extensive phenotyping of mobilized mature and immature leukocyte subsets was performed. Eight to 14 days after treatment volunteers underwent extensive clinical and laboratory follow-up.Results:POL6326 was very well tolerated. Several volunteers experienced a mild urticarial or itchy macular rash which responded well to H1/H2 blockade. Rating of tolerability/adverse events by volunteers (questionnaire) compared favourably with G-CSF administration. Exposure (Cmax, AUC) was dose-linear. At all doses tested POL6326 mobilized CD34+ progenitor cells and colony-forming cells (CFU-C, Figure 1) exceeding reported peak mobilization with plerixafor in donors at all except the lowest dose levels. In this dataset mobilization after doses of 2000 or 2500 µg/kg did not appear meaningfully stronger than after 1500 µg/kg. The SCM response for CD34+ cells to doses ≥1500 µg/kg was 36.9±2.4/µL (mean±SEM), or 1/3 that of G-CSF (y=0.324x). Good SCM with G-CSF was predictive of good SCM with POL6326 (r=0.63). One/5.7 POL6326-mobilized CD34+ cells was clonogenic (G-CSF: 1 CFU-C/3.4 CD34+ cells) possibly indicating a more immature phenotype of CD34+ cells mobilized by POL6326. POL6326 caused mixed leucocytosis with peak values in the mid-20K/µL. B-lymphocytosis was more and neutrophilia and monocytosis were less pronounced after POL6326 than G-CSF. Compared to G-CSF the subset of plasmocytoid dendritic cell progenitors (pDC) was enriched to a distinct population within the CD34+ cells following SCM with POL6326 as previously described for plerixafor. At the 24 h time point, blood values were well on their way towards normal, and at follow-up all laboratory values had normalized.Summary/Conclusions:The novel CXCR4-antagonist POL6326 is safe, well tolerated, and provides efficient mobilization of HSPCs. Based on the number of mobilized CD34+ cells at higher doses in this study, we conclude that a standard dose of 4x10E6 CD34+ cells/kg can be extracted with a single apheresis for most recipients unless their body weight significantly exceeds the donor weight. However, exploration of alternative dosing regimens may provide even higher mobilization responses. POL6326 can be an effective mobilizing agent for allogeneic donors, including subjects with contra-indications to G-CSF. [Display omitted] [Display omitted] DisclosuresEscot:Polyphor Ltd.: Employment. Douglas:Polyphor Ltd.: Employment. Romagnoli:Polyphor Ltd.: Employment. Chevalier:Polyphor Ltd.: Employment. Dembowsky:Polyphor Ltd.: Consultancy. Hooftman:Polyphor Ltd.: Employment. Bonig:Polyphor Ltd.: Research Funding.

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.

Mobilizing Agents G-CSF, Cyclophosphamide or AMD3100 (Plerixafor) Have Distinct Effects on Osteoblasts, Hematopoietic Stem Cell Niches, and B-Lymphopoiesis,

Abstract 4005The CXCR4 antagonist AMD3100 is progressively replacing cyclophosphamide as adjuvant to G-CSF to mobilize hematopoietic stem cells (HSC) for autologous transplants in patients who failed prior mobilization with G-CSF alone. We and others have recently demonstrated that G-CSF-induced mobilization and the associated response of HSC niches and bone formation depend on bone marrow (BM) macrophages1–3. Moreover medullar B lymphopoiesis is dependent on bone-forming osteoblasts in vivo. We therefore compared the effects of these three mobilizing agents (6 day course of G-CSF, versus a single injection of cyclophosphamide, versus 6 day course of AMD3100) on endosteal osteoblasts, bone formation, BM macrophages, expression of HSC-supportive cytokines and B lymphopoiesis in the mouse.G-CSF administration significantly reduced the number of endosteal osteoblasts and niche-supporting macrophages. G-CSF also inhibited expression of chemokines and cytokines such as CXCL12, Kit-ligand, angiopoietin-1 and IL-7 in the endosteal region resulting in a profound inhibition of medullar B lymphopoiesis with suppression of pre-pro-B, pro-B, pre-B cells and mature sIgM+ B cells as long as G-CSF was administered and HSC mobilization persisted.Cyclophosphamide also suppressed osteoblasts, niche-supportive macrophages and inhibited endosteal expression of CXCL12 and angiopoietin-1 but did not inhibit KIT-ligand expression. Of note, the down-regulation of CXL12 was more pronounced in response to cyclophosphamide than G-CSF. This could explain the higher mobilizing effect of cyclophosphamide. In contrast to G-CSF, medullar B lymphopoiesis rebounded in synchrony with endosteal IL-7 expression during cyclophosphamide-induced mobilization despite persistent suppression of osteoblasts and bone formation. Despite a marked rebound in the number of immature B cell progenitors in cyclophosphamide-mobilized BM, B cell progenitors could not mature into sIgM+ B cells which remained very depressed in BM, spleen and blood even 10 days after a single cyclophosphamide injection.Finally, while AMD3100 was effective at mobilizing HSC, it did not suppress osteoblasts, cytokine expression at the endosteum or B lymphopoiesis.In conclusion, although G-CSF, cyclophosphamide and AMD3100 efficiently mobilize HSC into the blood, their effects on HSC niches, bone formation and B lymphopoiesis are distinct. Furthermore the contrasted effects of G-CSF, cyclophosphamide and AMD3100 on medullar B lymphopoiesis indicates that grafts mobilized with these three agents may have different immune properties.(1) Winkler IG, Sims NA, Pettit AR, et al. Bone marrow macrophages maintain hematopoietic stem cell (HSC) niches and their depletion mobilizes HSCs. Blood. 2010;116(23):4815–4828. (2) Christopher MJ, Rao M, Liu F, Woloszynek JR, Link DC. Expression of the G-CSF receptor in monocytic cells is sufficient to mediate hematopoietic progenitor mobilization by G-CSF in mice. J Exp Med 2011;208(2):251–260. (3) Chow A, Lucas D, Hidalgo A, et al. Bone marrow CD169+ macrophages promote the retention of hematopoietic stem and progenitor cells in the mesenchymal stem cell niche. J Exp Med 2011;208(2):261–271. Disclosures:No relevant conflicts of interest to declare.

Dose Dependent Effects of G-CSF on Ca27.29 in Early Stage Breast Cancer Patients.

Abstract Background:13% of 2556 patients (pts) examined in the SUCCESS trial showed elevated levels of Ca27.29 only after chemotherapy (SABCS2008). Early data indicate a possible relationship between the administration of G-CSF and a rise in the tumor marker. This analysis focuses on the dose dependency of this effect.Methods:The SUCCESS Trial is a phase III trial comparing FEC-Docetaxel vs. FEC-Doc-Gemcitabine regime and 2 vs. 5 years of treatment with zoledronate in patients with primary breast cancer in 3754 pts (BC) (N+ or high risk). Blood samples for this analysis are drawn before and after chemotherapy (CHT). Ca27.29 has been measured with ST AIA-PACK Ca27.29 reagent using MUC-1 for AIA-600II (Tosoh Bioscience, Tessenderlo, Belgium). The cutoff for Ca27.29 is &amp;gt;=32 U/ml. For chi² analysis patients were grouped to increasing or not increasing values of Ca27.29 and 1 to 6 cycles with G-CSF or no G-CSF at all. The absolute difference of Ca27.29 values before and after CHT was correlated with the number of G-CSF cycles administered.Results:The analysis of Ca27.29 is based on the data of 2556 pts. 1252 pts (49%) received at least one course of G-CSF. 338 pts (13%) exceeded the threshold for CA27.29 only after CHT. In this group 209 pts (62%) received G-CSF and 129 (38%) did not. 1043 pts with stable or decreased CA27.29 received G-CSF (47%) and 1175 did not (53%). This difference was highly significant (p&amp;lt;0.0001). Correlating the number of G-CSF cycles received during CHT with the absolute difference in Ca27.29 levels showed a highly significant positive correlation of 0.13 (Spearman-Rho; p&amp;lt;0.0001[two-sided]).Discussion and Conclusion:This analysis gives strong evidence that there is a dose dependent correlation between elevated levels of CA27.29 post CHT and the application of G-CSF. This might be attributed to an illegitimate expression of MUC-1 in leucocytes during leucopoiesis. Whether this effect is also related to the destruction of remaining disseminated tumor cells needs to be further evaluated. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6030.

Effects of G-CSF on circulating tumor cells (CTC) and CA 27.29 in breast cancer patients

11027 Background: Some recent publications indicated that the use of G-CSF could be connected to an increase in CTC as well as elevated levels of tumor markers such as CA 27.29. In the SUCCESS Trial CTC and CA27.29 are examined before and after adjuvant chemotherapy (CHT) in 3754 breast cancer patients (pts). Methods: The SUCCESS Trial is a phase III trial comparing FEC-Docetaxel vs. FEC-Doc-Gemcitabine regime and 2 vs. 5 years of treatment with zoledronate in patients with primary breast cancer (BC) (N+ or high risk). Blood samples are taken before and after CHT. CTC were assessed with the CellSearchSystem (Veridex, Warren, USA). After immunomagnetic enrichment with an anti-Epcam-antibody, cells were labeled with anti-cytokeratin (8,18,19) and anti-CD45 antibodies to distinguish epithelial cells and leukocytes. CA27.29 has been measured with ST AIA-PACK Ca27.29 reagent using MUC-1 for AIA-600II (Tosoh Bioscience, Tessenderlo, Belgium). The cutoff for CA27.29 is 32 U/ml and &gt;1 cell for the CTC analysis. Patients were grouped to CTC/CA27.29 raise or no raise and 1 to 6 cycles with G-CSF or no G-CSF at all. Results: Data on 1510 pts are available for CTC analysis. 745 pts (49%) received at least one course of G-CSF. 117 pts (8%) showed an increase in CTC after CHT. In this group 52 (3%) pts received G-CSF and 65 (4%) did not. 693 pts with stable or decreased CTC received G-CSF (46%) and 700 did not (46%). There was no significant difference (p=0.29). The analysis of CA27.29 is based on the data of 2556 pts. 1252 pts (49%) received at least one course of G-CSF. 338 pts (13%) exceeded the threshold for CA27.29 only after CHT. In this group 209 pts (8%) received G-CSF and 129 (5%) did not. 1043 pts with stable or decreased CA27.29 received G-CSF (41%) and 1175 did not (46%). This difference was highly significant (p&lt;0.0001). Conclusions: No evidence can be provided for a significant correlation between an increase in the number of CTC and the application of G-CSF over CHT. Nevertheless the results on CA27.29 showed a highly significant correlation between the administration of G-CSF and elevated CA27.29 levels directly after CHT. This could be a possible explanation for the often observed increase of tumor markers after CHT. [Table: see text]

Chimerism and Tolerance after Combined Human Kidney and Hematopoietic Cell Transplantation Using Conditioning with Total Lymphoid Irradiation and Anti-Thymocyte Globulin.

Although transplantation tolerance to organ allografts has been achieved using a wide variety of immunologic interventions in laboratory animals, few tolerance induction protocols with complete immunosuppressive drug withdrawal have been tested in humans. In preclinical rodent models our group showed that long-term tolerance to vascularized organ allografts and donor specific unresponsiveness was achieved when hosts developed persistent mixed hematopoietic chimerism following conditioning with nonmyeloablative fractionated total lymphoid irradiation (TLI) combined with depletive anti-T cell antibodies (ATG) and infusions of donor bone marrow cells. We now report the translation of the preclinical model to human transplantation and have studied 8 patients enrolled in a tolerance induction protocol with a goal of complete immunosuppressive drug withdrawal after combined HLA- matched kidney and hematopoietic cell transplantation. Donors received a 5 day course of G-CSF 4-6 weeks before kidney transplant surgery, and purified CD34+ hematopoietic progenitor cells from the apheresis products were mixed with 1x10^6 donor CD3+ T cells/kg recipient weight (n = 7) or in the case of the patient with life-threatening lupus 10 x10^6 donor CD3+ T cells/kg before cryopreservation. After kidney transplantation surgery, recipients were conditioned with 10 equal doses of TLI administered once daily to a total dose of 800 (n=3) or 1200 (n=5) cGy combined with 5 intravenous infusions of rabbit anti-thymocyte globulin (ATG) at 1.5mg/kg/ dose, total dose of 7.5 mg/kg. Immediately after conditioning (day 10 after kidney transplantation), recipients were given an intravenous injection of the thawed donor cells in the outpatient clinic. Mycophenolate mofetil was given until day 30, and a tapering course of cyclosporine until day 180. Follow up for the first enrolled patient is 1230 days and the last enrolled patient is 105 days. The mean duration of hospitalization for the transplant was 5 days. The mean nadir WBC was 1100 cells/mm3 and no patient developed significant thrombocytopenia. Of the 8 patients transplanted, five have developed persistent chimerism; four of the five have persistent mixed chimerism and the one patient with lupus has complete chimerism. Among the patients with persistent chimerism two have had complete immunosuppressive drug withdrawal and three are in the midst of taper; none of these five patients have had clinical rejection episodes or have histologic evidence of rejection on protocol kidney biopsies. The three patients with transient hematopoietic cell chimerism are on maintenance low doses of immune suppression medications. All patients have excellent graft function with serum creatinine levels between 0.8 to 1.3 mg/dL. None of the patients developed acute or chronic GVHD. Two patients developed 1–2 dermatomal zoster and one patient had CMV reactivation in the blood; all were treated to resolution without hospitalization. Analysis of the gene array profiles of PBMC from the two patients off immune suppressive drugs showed the development of a “ tolerance” pattern after transplantation, and donor specific unresponsivesness in the MLR after drug withdrawal. In conclusion, using a CD34 selected G-CSF mobilized graft with a defined T cell dose and 1200 cGy of TLI we can establish persistent mixed hematopoietic cell chimerism for tolerance induction in patients undergoing kidney transplantation. Tolerance does not develop as soon as chimerism is established, and drug withdrawal without allograft rejection was feasible several months after persistent chimerism. The results suggest that tolerance induction in this protocol is an active and dynamic process.

Acute myeloid leukemia in a healthy hematopoietic stem cell donor following past exposure to a short course of G-CSF

Acute myeloid leukemia in a healthy hematopoietic stem cell donor following past exposure to a short course of G-CSF

Factors Predicting PBPC Collections in Healthy Adolescents and Children with Solid Tumours.

G-CSF is used for mobilization of hematopoietic cells in healthy adolescent donors and pediatric patients. In both groups it is important to ascertain factors predicting the success of apheresis as well as the number of procedures to achieve the target CD34+ cell number for hematologic recovery. We retrospectively analyzed factors predicting the yield for a target CD34+ cell dose in10 healthy adolescent donors and 43 children with solid tumours who were enrolled in the transplantation program at our center from January 1996 to July 2007. The healthy adolescents (n=10) had a median age of 15 years (range 13–16) and body weight (bw) of 45 kg (range 20–74) and the children with solid tumours (n=42) (Neuroblastoma n=28, Ewing sarcoma n=7 and other solid tumours n=7) had a median age of 4 years (range 1–15) and bw of 17 kg (range 9 – 63). Mobilization regimen consisted of G-CSF in a dosage of 10 μg/kg per day, applied subcutaneously for 5 or 6 days. A pre-collection peripheral blood (PB) CD34+ count was performed in all children, by flow cytometric analysis. Standard volume apheresis was carried out using a Cobe Spectra blood cell separator, through peripheral veins in donors and central lines in patients. We estimated a minimum of 4 x106 and 2x106 CD34+ cells/kg bw receptor for successful engraftment for allogeneic and autologous transplant, respectively. The healthy adolescent donors had high number of PB CD34+ cells (median 62/μl, range 42 – 75) and the majority (7/10) only required a single apheresis to collect sufficient cells for their family related recipients. The neuroblastoma patients with >10/μl PB CD34+ cells collected in one day the target cell dose (median 4.6x106 CD34+ cells/kg, range 2.6 – 27.0). Those with <10/μl PB CD34+ cells underwent 2 apheresis and the majority reached the cell target. The majority of children with Ewing sarcoma and other solid tumours (9/14) also collected >2.0 x106 CD34+ cells/kg in 2 apheresis. In summary all the children with solid tumours received a single course of G-CSF and presented on the 5th day of mobilization a median PB CD34+ cell count of 9/μl (range 1–216). The majority (75%) collected >2.0 x106 CD34+ cells/kg and the remaining (25%) had <10/μl PB CD34+ cells and did not achieve this minimum requirement. We found a correlation between the PB CD34+cell count and the number of apheresis and CD34+ cells yielded both for the adolescent donors and children with solid tumours. Based on these results, a PB CD34+ cell count >10/μl was a good indicator for performing PBPC collections in paediatric patients from our institution.

G-CSF Disrupts the Stem Cell Niche by Increasing Turnover of Bone Marrow Osteoblasts.

Accumulating evidence suggests that osteoblast-lineage cells play a key role in supporting hematopoiesis, providing signals that maintain the normal function of hematopoietic stem cells (HSC). We and others previously showed that treatment with G-CSF reduces the number and activity of osteoblasts in the bone marrow. Here we confirm these findings using transgenic mice expressing GFP in osteoblast lineage cells under control of a 2.3 kb fragment of the col1a1 promoter. Analysis of these mice confirmed that G-CSF treatment reduced the number of osteoblasts 2.9-fold compared to control mice (n=4, p<.01). Consistent with these findings, expression of several osteoblast genes was sharply reduced during G-CSF treatment; alkaline phosphatase, osteocalcin, stromal-derived factor 1, osteoprotegerin, and bone sialoprotein were all decreased (7.9, 19, 3.8, 14.2, and 4.1-fold decreased; n=5–12 each group, p<.05). These findings are consistent with either a true loss of osteoblasts or induction of osteoblast quiescence. To distinguish between these possibilities, the fate of osteoblasts during G-CSF treatment was assessed by in vivo BrdU labeling. At baseline, 34.9 ± .8% of osteoblasts were labeled after 16 days of BrdU administration. In untreated mice, this percentage decreased to 14.8% ± 0.05% after 9 days. However, in mice harvested 4 days after a 5-day course of G-CSF, the percentage of BrdU positive osteoblasts decreased to 6.5% ± 0.9% (n=2 each group, p<.05). These data suggest that G-CSF treatment increases the turnover of osteoblasts rather than inducing their quiescence. Since apoptosis is one potential mechanism of osteoblast turnover, we measured caspase 3 activation in G-CSF-treated mice. No increased apoptosis was observed [percent activated caspase 3 positive cells + SEM: 8.0% ± 1.5% (untreated),10.5% ± 0.5% (day 3), 5.8% ± 1.2% (day 4); n=2–4 each group, p=ns]. The sharp decrease in osteoblast number suggests that HSC activity might be altered by G-CSF treatment. To address this possibility, competitive repopulation assays were performed with HSC harvested from G-CSF or control mice. In mice competitively reconstituted with control HSC, the percentage of donor cells was 33.7% ± 4.7% at 4 months after transplantation. In contrast, in mice receiving G-CSF treated HSC, the percentage of donor cells was only 10.0% ± 2.6% (n=3–4 each group, p<.05). As this loss in repopulation potential may result from decreased HSC number or function, we estimated HSC number by measuring Kit+ lineage− Sca+ CD34− cells. We found no significant difference in this fraction between treated and untreated mice [number KLS CD34− per femur + SEM: 966 ± 229 (untreated) versus 945 ± 183 (treated); n=6–7 each group, p=ns]. These results suggest that the diminished repopulation potential of G-CSF treated bone marrow results from impaired HSC function rather than reduced HSC number. Finally, we hypothesized that the loss of long-term repopulation potential in HSC from G-CSF treated mice may result from the loss of factors in the bone marrow niche that maintain HSC quiescence. We therefore measured BrdU uptake in the KLS fraction of bone marrow cells. Preliminary results show a trend toward increased BrdU uptake in KLS cells from G-CSF treated mice (48.5% ± 9.2%) compared with untreated mice (26.5% ± 1.4%; n=2 each group, p=.14). In summary, these results suggest a model in which G-CSF induces increased osteoblast turnover, disruption of the stem cell microenvironment, and loss of HSC quiescence.

Interferon-alpha protects Philadelphia-negative progenitors from exhaustion in chronic myeloid leukemia patients with cytogenetic response.

Normal immature hematopoietic progenitors are relatively well preserved in most patients newly diagnosed with chronic myeloid leukemia, but tend to decline rapidly with time. Such exhaustion could reflect a suppressive effect of the Philadelphia positive clone expansion and/or be induced by Interferon-alpha treatment. A total of 51 CML patients were classified into three groups. Newly diagnosed untreated patients were group A (n=30). Of the 21 treated individuals with Interferon-alpha, for at least 12 months, 15 showed no cytogenetic response (group B) while six showed persisting major/complete response (group C). Patients belonging to groups A and B were mobilized with chemotherapy plus G-CSF while patients of group C received a short course of G-CSF only. Patients responding to IFN-alpha (group C) showed comparable numbers of bone marrow Ph- long-term culture initiating cells to those of newly diagnosed individuals (group A): 8.5 (<1-65)/10(6) MNC vs 10.5 (<1-30), while non-responders had markedly lower numbers: <1 (<1-5). The amount of Ph- LTC-IC collected was significantly lower in patients of group B 1.8 (0-325)x10(2)/kg than in patients of either group A 31.3 (0-952)x10(2)/kg (P<0.002) or group C 109 (8-259)x10(2)/kg (P<0.01). Interestingly, five patients of group B who had 100% Ph+ metaphases, but Ph- progenitors in their bone marrow, mobilized normal amounts of Ph(-) progenitors. These findings suggest that the decline of normal hematopoietic progenitors, currently observed in the majority of CML patients, is not induced by IFN-alpha treatment, but it is likely due to the expanding leukemic clone. They also indicate that normal hematopoietic reservoir is consistently preserved in patients given IFN-alpha early after diagnosis and achieving a stable cytogenetic response.

Autologous bone marrow transplantation in non-Hodgkin's lymphoma patients: effect of a brief course of G-CSF on harvest and recovery.

This study compares harvest and hematological recovery data of 100 lymphoma patients who underwent BM harvest either after a short course of G-CSF (16 microg/kg for 3 days) (n = 57) or in steady-state conditions (n = 43). G-CSF allowed the attainment of a significantly higher median number of total nucleated cells x 10(8)/kg (4.4, range 1.4-17, vs 2.1, range 0.6-4.2; P < 0.0001), mononuclear cells x 10(8)/kg (0.55, range 0.20-1.4, vs 0.41, range 0.15-0.76, P < 0.0001) and CFU-GM/ml (310, range 10-5500, vs 80, range 10-3800, P = 0.008), with lower volumes of blood collected (17.5 ml/kg, range 8-31 vs 21.0, range 15-30, P = 0.0001). Hematological recovery was faster in patients who received pre-treated BM (median time to PMN >0.5 x 10(9)/l and to platelets >20 x 10(9)/l was 12, range 10-14, and 13, range 10-18, days, respectively) than in those autotransplanted with steady-state BM (median time to PMN >0.5 x 10(9)/l and to platelets >20 x 10(9)/l 13, range 10-18 and 14, range 10-20 days, respectively, P = 0.004 and P = 0.01). Transfusional requirement was significantly different and patients of the G-CSF group needed shorter hospitalization (17 days, range 12-24, vs 20 days, range 14-32; P = 0.02). These data suggest that treating patients with G-CSF before BM harvest improves the quality of the harvest and accelerates engraftment and hematological recovery.

Blood stem cell allografting.

The emergence of peripheral blood stem cells as the preferred source of autologous rescue in high-dose therapy has indicated their potential benefits for allogeneic transplantation. Adequate mobilization with minimal side-effects can be achieved in normal donors using a short course of G-CSF. Allogeneic stem cells provide long-term engraftment that appears to be more rapid than that seen with marrow, but this observation awaits confirmation in a prospective randomized study. The cellular composition of stem cell and bone marrow allografts is very different, with greater numbers of CD34+ progenitors, T lymphocytes, and natural killer cells in stem cell harvests. Although infused T-cell numbers are increased, no difference in acute graft-versus-host disease has been documented between stem cell and marrow allografts. Any differences in chronic graft-versus-host disease or relapse risk await longer follow-up. It is likely that the use of allogeneic stem cells will expand both in matched sibling and mismatched transplantation.