Continuous Granulocyte Colony-stimulating Factor Research Articles

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

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

Role of CSF3R mutations in the pathomechanism of congenital neutropenia and secondary acute myeloid leukemia.

Acquired mutations in the intracellular part of CSF3R (colony stimulating factor 3 receptor, granulocyte) have been detected with a frequency of more than 30% in severe congenital neutropenia (CN) patients. CN is a preleukemic syndrome with a risk of approximately 20% to develop leukemia. More than 80% of CN patients who develop acute myeloid leukemia or myelodysplastic syndrome reveal CSF3R mutations, suggesting that they are involved in leukemogenesis. Using deep-sequencing technology, we were able to analyze large cohorts of CN patients for the entire CSF3R sequence as well as to identify cell clones carrying mutations in the intracellular part of CSF3R with very high sensitivity. Acquisition of CSF3R mutations is a CN-specific phenomenon and is associated with inherited mutations causing CN or cyclic neutropenia, such as ELANE mutations. In the group of CN patients negative for known germ-line mutations, biallelic CSF3R mutations were identified. In addition, CSF3R mutant clones are highly dynamic and may disappear and reappear during continuous granulocyte colony-stimulating factor (G-CSF) therapy. The time between the first detection of CSF3R mutations and overt leukemia is highly variable.

Growth factor release by vesicular phospholipid gels: in-vitro results and application for rotator cuff repair in a rat model

BackgroundBiological augmentation of rotator cuff repair is of growing interest to improve biomechanical properties and prevent re-tearing. But intraoperative single shot growth factor application appears not sufficient to provide healing support in the physiologic growth factor expression peaks. The purpose of this study was to establish a sustained release of granulocyte-colony stimulating factor (G-CSF) from injectable vesicular phospholipid gels (VPGs) in vitro and to examine biocompatibility and influence on histology and biomechanical behavior of G-CSF loaded VPGs in a chronic supraspinatus tear rat model.MethodsG-CSF loaded VPGs were produced by dual asymmetric centrifugation. In vitro the integrity, stability and release rate were analyzed. In vivo supraspinatus tendons of 60 rats were detached and after 3 weeks a transosseous refixation with G-CSF loaded VPGs augmentation (n = 15; control, placebo, 1 and 10 μg G-CSF/d) was performed. 6 weeks postoperatively the healing site was analyzed histologically (n = 9; H&E by modified MOVIN score/Collagen I/III) and biomechanically (n = 6).ResultsIn vitro testing revealed stable proteins after centrifugation and a continuous G-CSF release of up to 4 weeks. Placebo VPGs showed histologically no negative side effects on the healing process. Histologically in vivo testing demonstrated significant advantages for G-CSF 1 μg/d but not for G-CSF 10 μg/d in Collagen III content (p = 0.035) and a higher Collagen I/III ratio compared to the other groups. Biomechanically G-CSF 1 μg/d revealed a significant higher load to failure ratio (p = 0.020) compared to control but no significant differences in stiffness.ConclusionsBy use of VPGs a continuous growth factor release could be obtained in vitro. The in vivo results demonstrate an improvement of immunohistology and biomechanical properties with a low dose G-CSF application via VPG. The VPG itself was well tolerated and had no negative influence on the healing behavior. Due to the favorable properties (highly adhesive, injectable, biocompatible) VPGs are a very interesting option for biologic augmentation. The study may serve as basis for further research in growth factor application models.

Guidelines for pediatric management of severe chronic neutropenia

For many years, there was no effective treatment for severe chronic neutropenia. Patients were simply prescribed antibiotics, when they developed fever and infections. Recombinant human granulocyte colony-stimulating factor (G-CSF) dramatically changed this situation, when it became available for clinical studies in 1986. Soon thereafter evidence from small trials suggested that G-CSF was useful for treatment of cyclic, congenital, and idiopathic neutropenia. A randomized controlled trial then established that daily subcutaneous G-CSF, given in doses to achieve a blood neutrophil count of approximately 2.0 3 10/L, could reduce the occurrence of fever, mouth ulcers, infections, and antibiotic use in these patients [1]. Although this trial proved the efficacy of G-CSF therapy as a preventive therapy for severe chronic neutropenia, it did not provide precise guidelines for the best ways to care for these patients. In this issue of the Journal, Fioredda et al. [2] report consensus guidelines for therapy and follow-up of children with congenital and acquired neutropenia. The report is based on a careful review of the literature and grading the evidence in 59 reports by an expert panel of Italian hematologists. The evidence in these reports was scored by the experts on a scale of 1–9. Statements receiving a mean score that was greater than 7 were regarded as appropriate/necessary clinical practices. Statements about treatment are referenced, and the level of evidence and the level and strength of the consensus are also provided. The panel is to be commended for the careful way they conducted and presented these guidelines. Since 1994, another group, the Advisory Board for the Severe Chronic Neutropenia International Registry (SCNIR or Registry), has also engaged in developing treatment recommendations and guidelines for these patients [3–7]. The Registry’s efforts are based on data from patients enrolled in the original Phase 2 and Phase 3 trials and prospectively collected data from a much larger group of patients enrolled subsequently in the Registry. The board meets annually and communicates regularly about individual patients. Annually, the board reviews new and cumulative clinical observations, by diagnostic category, by specific/genetic diagnosis, and by geographic origin. It also discusses adverse events, reviews, and revises treatment recommendations, and periodically published reports from this longitudinal, observational study. The Italian report includes the reports and observations of the SCNIR and largely confirms its recommendations. This is important, because the SCNIR was originally sponsored by Amgen, a US manufacturer of G-CSF, and the physicians directing the SCNIR have a potential conflict of interest in making recommendations regarding this agent. By contrast, the expert panel for this report does not have this conflict, but it reached the same conclusions regarding G-CSF therapy based on its review of evidence. This report provides recommendations about several controversial issues that differ somewhat from the recommendations and statements for the SCNIR. For example, for patients with glycogen storage disease, the report does not mention the problem of severe splenomegaly, when these patients receive G-CSF therapy; the SCNIR advises physicians of this concern and advises using the lowest possible G-CSF dose to minimize this problem. The report advises that Shwachman–Diamond syndrome may be at greater risk of leukemic transformation and therefore advises ‘‘on-demand’’ G-CSF. The recommendation is not referenced, and the relationship of G-CSF treatment and the risk of leukemic transformation are not known. The SCNIR recommends continuous G-CSF for patients with severe neutropenia and recurrent fever and infections, regardless of the type of neutropenia. The report is also limited to children, but approximately half of the patients now in the SCNIR are now of adult age. The SCNIR makes recommendations for the continuum of ages, including both children and adults. Table 2 provides expert recommendations about the frequency of biochemical parameters and abdominal ultrasound; the SCNIR has not evaluated or made recommendations about these tests. Overall, however, these are minor points, and these guidelines are very useful for improving the care of patients with these rare hematological diseases.

Effective Treatment of Elderly Acute Myeloid Leukaemia (AML) with Continuous Combined Granulocyte Colony Stimulating Factor (G-CSF) and Single Oral Chemotherapeutic Agents.

Advanced age is a poor prognostic factor in acute myeloid leukaemia (AML). There is evidence that continuous low-dose chemotherapy with oral agents can achieve partial remission in AML and may be suitable as palliative therapy in elderly patients. Such low-dose treatment is usually complicated by drug-related neutropenia and thrombocytopenia. Granulocyte colony stimulating factor (G-CSF) may lessen the associated neutropenia and some anecdotal reports suggest G-CSF alone may be able to induce remission in AML. The study aim was to assess the benefits of combining an oral chemotherapeutic agent with continuous G-CSF. We describe a retrospective audit of 12 elderly patients with AML, diagnosed between 2000 and 2002, who were deemed to be either unfit for induction chemotherapy or who had failed to respond to intravenous chemotherapy. Patients were treated with continuous oral mercaptopurine, thioguanine, or hydroxyurea with concomitant G-CSF on three to seven days/week. The median age at diagnosis was 80 years (range 70–89 years). Eight (67%) had a preceding myelodysplastic syndrome. Eleven of the twelve patients died within the study period described, with a median survival of 9 months (95%CI 4.32–13.75). Eleven patients (92%) had a response to treatment, with blast disappearance obtained within 14 days of starting treatment. All patients achieved an increase in neutrophil count >0.5x109/L. Neutrophil recovery was attained within a mean of 13 days of treatment. From the onset of treatment, patients had neutrophils greater than 0.5 x109/L for 71% of the time, despite being on continuous cytotoxic treatment. Five patients (42%) had a platelet response with a rise in count above 100x109/L. A total of nine (75%) patients experienced a period of platelet transfusion independence and four (33%) patients became red cell transfusion independent for a mean of 4.6 and 4.8 months respectively. The analysis of this small cohort suggests that G-CSF administered in combination with one of the above oral chemotherapeutic agents, may be a novel way of providing treatment to elderly patients with AML which is both tolerable and of potential survival benefit.

The CXCL12–CXCR4 chemotactic pathway as a target of adjuvant breast cancer therapies

Dose-dense adjuvant breast cancer chemotherapy is a new treatment strategy that aims to improve tumour control by using more frequent cytotoxic dosing together with continuous granulocyte colony-stimulating factor (G-CSF) to minimize neutropaenia. In addition to stimulating neutrophil proliferation, G-CSF mobilizes neutrophils from the bone marrow through proteolytic disruption of the chemokine receptor CXCR4 and its chemotactic ligand CXCL12. As breast cancers also express CXCR4 and oestrogen induces CXCL12, the success of dose-dense treatment could partly reflect inhibition of CXCR4-dependent micrometastatic homing and/or paracrine survival, and suggests a benefit of adjuvant oestrogen suppression for patients with oestrogen-receptor-negative, CXCR4-positive disease.

Sustained receptor activation and hyperproliferation in response to granulocyte colony-stimulating factor (G-CSF) in mice with a severe congenital neutropenia/acute myeloid leukemia-derived mutation in the G-CSF receptor gene.

In approximately 20% of cases of severe congenital neutropenia (SCN), mutations are found in the gene encoding the granulocyte colony-stimulating factor receptor (G-CSF-R). These mutations introduce premature stop codons, which result in truncation of 82-98 COOH-terminal amino acids of the receptor. SCN patients who develop secondary myelodysplastic syndrome and acute myeloid leukemia almost invariably acquired a GCSFR mutation, suggesting that this genetic alteration represents a key step in leukemogenesis. Here we show that an equivalent mutation targeted in mice (gcsfr-Delta715) results in the selective expansion of the G-CSF- responsive progenitor (G-CFC) compartment in the bone marrow. In addition, in vivo treatment of gcsfr-Delta715 mice with G-CSF results in increased production of neutrophils leading to a sustained neutrophilia. This hyperproliferative response to G-CSF is accompanied by prolonged activation of signal transducer and activator of transcription (STAT) complexes and extended cell surface expression of mutant receptors due to defective internalization. In view of the continuous G-CSF treatment of SCN patients, these data provide insight into why progenitor cells expressing truncated receptors clonally expand in vivo, and why these cells may be targets for additional genetic events leading to leukemia.

Liver transplantation for glycogen storage disease types I, III, and IV.

Glycogen storage disease (GSD) types I, III, and IV can be associated with severe liver disease. The possible development of hepatocellular carcinoma and/or hepatic failure make these GSDs potential candidates for liver transplantation. Early diagnosis and initiation of effective dietary therapy have dramatically improved the outcome of GSD type I by reducing the incidence of liver adenoma and renal insufficiency. Nine type I and 3 type III patients have received liver transplants because of poor metabolic control, multiple liver adenomas, or progressive liver failure. Metabolic abnormalities were corrected in all GSD type I and type III patients, while catch-up growth was reported only in two patients. Whether liver transplantation results in reversal and/or prevention of renal disease remains unclear. Neutropenia persisted in both GSDIb patients post liver transplantation necessitating continuous granulocyte colony stimulating factor treatment. Thirteen GSD type IV patients were liver transplanted because of progressive liver cirrhosis and failure. All but one patient have not had neuromuscular or cardiac complications during follow-up periods for as long as 13 years. Four have died within a week and 5 years after transplantation. Caution should be taken in selecting GSD type IV candidates for liver transplantation because of the variable phenotype, which may include life-limiting extrahepatic manifestations. It remains to be evaluated, whether a genotype-phenotype correlation exists for GSD type IV, which may aid in the decision making. Liver transplantation should be considered for patients with glycogen storage disease who have developed liver malignancy or hepatic failure, and for type IV patients with the classical and progressive hepatic form.

Perturbed Granulopoiesis in Mice With a Targeted Mutation in the Granulocyte Colony-Stimulating Factor Receptor Gene Associated With Severe Chronic Neutropenia

Mutations in the granulocyte colony-stimulating factor (G-CSF) receptor gene are found in a number of patients with severe chronic neutropenia predisposed to acute myeloid leukemia. These mutations result in the absence of the C-terminal domain of the G-CSF-R, a region which has been implicated in differentiation signaling. We generated mice with an equivalent mutation (gcsfr-triangle Delta715) by homologous and Cre-mediated recombination in embryonic stem cells. Both wt/triangle Delta715 and triangle Delta715/triangle Delta715 mice have significantly reduced numbers of blood neutrophils compared with their wt/wt littermates. However, under continuous G-CSF administration mutant mice develop peripheral neutrophil counts that significantly exceed those of wild-type littermates. These findings indicate that depending on G-CSF levels in mice, the triangle Delta715 mutation can contribute both to neutropenia and to neutrophilia.

Inhibitory effects on in vitro cell growth of human urothelial tumor cell lines under the combined administration of hematopoietic growth factors and clinically relevant antineoplastic agents.

In five of eight human transitional carcinoma cell (TCC) lines a proliferative response has been reported during exposure to interleukin-3 (IL-3), granulocyte-macrophage colony stimulating factor (GM-CSF) and granulocyte colony stimulating factor (G-CSF). To elucidate possible growth-modulating effects of these factors combined with clinically relevant antineoplastic agents, cells of the human TCC lines EJ28 and T24 were exposed to methotrexate (MTX), vinblastine (VBL), doxorubicin (DXR) and cisplating (CDDP) with and without single or continuous exposure to IL-3, GM-CSF and G-CSF at concentrations of 1-100 ng/ml. Compared with cells exposed only to chemotherapy, significant inhibitory effects occurred as a result of continuous exposure to IL-3 or GM-CSF at the highest activities with CDDP and MTX in the T24 and EJ28 lines; continuous G-CSF administration (100 ng/ml) in combination with MTX led to significant growth inhibition in the EJ28 line. In contrast, no significant growth modulation was found on combined administration of DXR or VBL with any one of the three colony stimulating factors tested.