Low-dose Granulocyte-macrophage Colony-stimulating Factor Research Articles

GM-CSF-expanded CD1dhiCD5+ B cells suppress experimental autoimmune myasthenia gravis (THER3P.886)

Abstract IL-10-competent subset within CD1dhiCD5+ B cells, also known as B10 cells, has been shown to regulate autoimmune diseases. Whether B10 cells can prevent or suppress the development of experimental autoimmune myasthenia gravis (EAMG) has not been studied. In this study, we investigated whether low dose granulocyte macrophage-colony stimulating factor (GM-CSF) can expand B10 cells in vivo, and whether adoptive transfer of CD1dhiCD5+ B cells would prevent or suppress EAMG. We found that treatment of EAMG mice with low-dose GM-CSF increased the proportion of CD1dhiCD5+ B cells and B10 cells. In vitro co-culture studies revealed that CD1dhiCD5+ B cells altered T cell cytokine profile but did not directly inhibit T cell proliferation. On the other hand, CD1dhiCD5+ B cells inhibited B cell proliferation and its autoantibody production in an IL-10-dependent manner. Adoptive transfer of CD1dhiCD5+ B cells to mice could prevent disease and suppress ongoing EAMG. This was associated with downregulation of mature dendritic cell markers and expansion of regulatory T cells resulting in the suppression of acetylcholine receptor (AChR)-specific T cell and B cell responses. Thus, our data have provided significant insights into the mechanisms underlying the tolerogenic effects of B10 cells in EAMG. These observations suggest that in vivo or in vitro expansion of CD1dhiCD5+ B cells or B10 cells may represent an effective strategy in the treatment of human myasthenia gravis.

Aberrant phosphorylation of STAT5 by granulocyte-macrophage colony-stimulating factor in infant cytomegalovirus infection mimicking juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia (JMML) progenitor cells exhibit in vitro hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF). Phospho-specific flow cytometry using anti-phosphorylated STAT5 antibody is a new method recently reported to detect GM-CSF hypersensitivity of cells. However, colony assays using methylcellulose medium to measure GM-CSF-hypersensitivity remain as the current gold standard. Interestingly, cytomegalovirus (CMV) infection in infancy often presents with a variety of clinical symptoms that mimic JMML, with CMV giving a positive result by colony assay. We wanted to determine whether aberrant STAT5 activation occurs in CMV infection by using phospho-specific flow cytometry, and to ascertain whether this method is effective at discriminating CMV infection from JMML. Peripheral blood mononuclear cells from patients with JMML and CMV infection displayed an elevated proportion of p-STAT5 cells after low-dose GM-CSF stimulation when compared with cells from normal individuals. However, we found no significant differences in the percentage of p-STAT5 positive cells from patients with CMV infection and JMML at any doses of the GM-CSF doses used. We conclude that patients with CMV infection cannot be discriminated from patients with JMML by this new diagnostic method.

Abstract 5618: Arming the melanoma sentinel lymph node against tumor spread: The effects of local administration of combined low-dose CpG-B PF-3512676 and GM-CSF

Abstract The sentinel lymph node (SLN) procedure has proven a useful prognostic tool for the assessment of melanoma relapse and mortality risk, but is also of great value for the assessment of immunological interventions. Since early melanoma development is accompanied by a suppressed immune state of the SLN, there is a strong rationale for therapeutic immune modulation of the SLN in order to strengthen local as well as systemic cell-mediated anti-tumor immunity. Previous Phase II studies showed promising immunostimulatory effects on the melanoma SLN of Granulocyte/Macrophage-Colony Stimulating Factor (GM-CSF) and the CpG-B oligodeoxynucleotide PF-3512676. The present 3-arm Phase II study was designed to determine the effects of combined low-dose GM-CSF+CpG-B administration. Stage I-III melanoma patients (n=28) were randomized to receive i.d. injections around the primary tumor excision site of saline, CpG-B alone (1 mg), or CpG-B combined with GM-CSF (100 μg), 7 and 2 days prior to surgical excision of the SLN. Flowcytometric DC analyses revealed significantly increased maturation of all identifiable cDC and pDC SLN subsets upon administration of CpG-B with or without GM-CSF, but remarkably, the combination resulted in significantly higher levels of co-stimulatory molecules on both cDC and pDC. In addition, a significant CpG-related increase in frequencies of CD1a−CD11chiCD14+/− cDC subsets was observed in the SLN, possibly resulting from DC and/or monocyte mobilisation from blood. Indeed, increased activation of pro-inflammatory 6-sulfo LacNac+slanDC, monocytes, and pDC was observed in peripheral blood upon administration of CpG-B alone, and additionally of CD1c+ cDC when combined with GM-CSF. In the case of slanDC this activation was accompanied by significantly reduced frequencies. Peripheral blood rates of CD14+DRlo myeloid suppressors remained unchanged after saline or combined CpG and GM-CSF administration, but significantly decreased after administration of CpG-B alone. T cell analyses revealed a CpG-related increase in CTLA-4 and FoxP3 levels on Tregs in SLN, without changes in their actual rates. Nevertheless, higher intracellular Th1 cytokine levels were detected in ex vivo expanded T cells of CpG-administered patients than of patients receiving saline or CpG-B+GM-CSF. In keeping with this, also higher frequencies were found in SLN of MAA-specific CD8+ T cells in low dose CpG-B administered patients (n=4) than in saline controls (n=5), as determined by tetramer binding and cytokine release. We conclude that the imunomodulatory effects observed in melanoma SLN upon local administration of combined low-dose GM-CSF and CpG-B are mostly attributable to CpG-B and that they are consistent with the induction of protective cell-mediated anti-tumor immunity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5618.

Therapeutic Potential of in vitro Expansion of Foxp3+ Tregs Utilizing GM-CSF-treated Bone Marrow Dentritic Cell Precusors in Experimental Myathenia Gravis (143.6)

Abstract We have previously shown that regulatory T cells (Tregs) generated in vivo by the selective activation of particular subsets of dendritic cells (DCs) in response to low-dose granulocyte-macrophage colony-stimulating factor (GM-CSF) have the capacity to prevent and reverse experiemental autoimmune myasthenia gravis (EAMG). To further understand the underlying mechanisms of GM-CSF-induced Treg mobilization, we studied the effects of GM-CSF on T cell/DC co-cultures using matured splenic DCs as well bone marrow (BM) - derived DC precursors in vitro. Compared to splenic DCs, bone marrow-derived DC precursors, when cultured in the presence of GM-CSF, gave rise to a population of CD11c+CD11bhighCD8α- DCs (BMDCs) which were able to expand Foxp3+ regulatory T cells (Tregs) in the absence of TCR stimulation. Additionally, the BMDCs secreted high levels of TGF-β which was required for adaptive differentiation of naive T cells to Foxp3+ Tregs upon TCR stimulation. Adoptive transfer of expanded Tregs effectively suppressed ongoing EAMG. These results strongly indicate that BMDCs differentiated in the presence of GM-CSF can expand natural Tregs and induce adaptive Tregs through different mechanisms. Expansion of polyclonal and antigen-specific Tregs in vitro using this strategy may represent an effective treatment for restoring immune tolerance and ameliorating EAMG.

Granulocyte-Macrophage Colony-Stimulating Factor, Interferon Alpha and Interleukin-2 as Adjuvant Treatment for High-Risk Renal Cell Carcinoma

This prospective, non-randomized study assessed toxicity and potential efficacy of low-dose granulocytemacrophage colony-stimulating factor (GM-CSF), interferon alpha (IFN) and interleukin 2 (IL-2) postoperatively in patients with high-risk renal cell carcinoma (RCC). Eligibility requirements were resected locally advanced (T3b-4 or N1–2) or metastatic (M1) RCC, no prior systemic therapy, and excellent organ function. Patients received treatment during 6 months: GM-CSF, 1 mcg/kg, 3 tiw, s.c., first week of each month; IFN, 10 MIU, 3 tiw, s.c., second week, and IL- 2, 1 MIU, 3 tiw, i.v., third week. Fourth week of each month was free from treatment. The primary end-point was diseasefree survival (DFS). Secondary end-points were progression rate, overall survival (OS), safety, and toxicity. Using the method of Dixon and Simon, the accrual goal was 35 patients. 35 patients were enrolled onto the study (28m/7f; median age = 46, range 21-71; ECOG 0-1; 14 (40%) pts with stage III and 21 (60%) pts with stage IV). Toxicity was minimal characterized by mainly flu-like syndrome and erythema in injection site. Median DFS was 14.1 months (95% CI, 3–20.2 months). At a median follow-up time of 5.1 years, progression rate was 65.7%, and OS was 40%. The median OS was 53.0 months (95% CI, 40.6–65.4 months). 5-years DFS and OS were similar between locally advanced and N2/M1 groups. Although the toxicities seen in this trial were low grade and regimen was well tolerated, low-dose GM-CSF, IFN and IL-2 did not improve DFS in comparison with historical control in patients with resected high-risk RCC.

Phase II Multicenter Trial of Maintenance Biotherapy After Induction Concurrent Biochemotherapy for Patients With Metastatic Melanoma

Biochemotherapy improves responses in metastatic melanoma, but not overall survival, in randomized trials. We developed a maintenance biotherapy regimen after induction biochemotherapy in an attempt to improve durability of responses and overall survival. One hundred thirty-three chemotherapy-naïve patients with metastatic melanoma without CNS metastases were treated at 10 melanoma centers. The biochemotherapy induction regimen included cisplatin, vinblastine, dacarbazine, decrescendo interleukin-2 (IL-2), and interferon alfa-2b with granulocyte-macrophage colony-stimulating factor (GM-CSF) cytokine support. Patients not experiencing disease progression were eligible for maintenance biotherapy with low-dose IL-2 and GM-CSF followed by intermittent pulses of decrescendo IL-2 over 12 months. Patients were observed for response, progression-free survival, toxicity, and overall survival. The response rate to induction biochemotherapy was 44% (95% CI, 35% to 52%; complete response, 8%; partial response, 36%; stable disease, 29%). The median number of biochemotherapy cycles was four, and the median number of maintenance biotherapy cycles was five. The median progression-free survival was 9 months, and the median survival was 13.5 months. The 12-month and 24-month survival rates were 57% and 23%, respectively. Twenty percent of patients remain alive (12 without disease), with median follow-up of 30 months (95% CI, 25+ to 45+ months). Thirty-nine percent of patients developed CNS metastases. The median times to CNS progression and death were 8 months and 5 months, respectively. Maintenance biotherapy after induction biochemotherapy seems to prolong progression-free survival and improve overall survival compared with recent multicenter trials of biochemotherapy or chemotherapy. The regimen should be studied in a randomized clinical trial in patients with advanced metastatic melanoma. CNS progression remains a formidable challenge.

Bevacizumab (BEV) and continuous low-dose granulocyte-macrophage colony-stimulating factor (GM-CSF) in patients with advanced solid tumors: final results of a prospective clinical trial

e14544 Background: BEV is potent inhibitor of VEGF-mediated tumor angiogenesis. VEGF is also crucial in antineoplastic immunortolerance by blocking the generation of dendritic cells (DC). VEGF inhibition may thus result in an improved anti-cancer immune response which may be further augmented by GM-CSF, a powerful stimulator of DC generation. In a previous study, GM-CSF was able to produce clinical responses in patients (pts) suffering from refractory carcinomas (Kurbacher et al., Oncology 2005). In this trial, we investigated BEV+GM-CSF in pts with advanced solid neoplasms. Methods: 26 pts have been enrolled: epithelial ovarian cancer, n=12; breast cancer, n= 6; primary peritoneal carcinoma, n=3; hormone-refractory prostate cancer, n=2; miscellaneous, n=3. All pts have been considered refractory to or non-treatable by chemotherapy due to their baseline bone marrow and/or organ functions. Fourteen pts had previously received BEV as part of other antineoplastic regimens. BEV was applied at a fixed dose of 6 mg/kg q2w. The starting dose of GM-CSF (sargramostim) was 125 μg/d. In cases not responding and/or not achieving moderate leukocytosis (< 20 G/L), GM-CSF was increased every 4 weeks at 25 μg increments up to a maximum of 250 μg/d. Toxicity was documented according to the NCI-CTC score, responses were recorded according to the RECIST or Rustin criteria. Results: Grade (G) 3–4 hypertension was seen in 3 pts, with 1 pt experiencing cerebral stroke 6 weeks after cessation of BEV+GM-CSF. Other G3–4 toxicities were fatigue in two pts, and fever, pain, myalgia, and central nervous symptoms in either one pt. Other side-effects like injection-site reactions, proteinuria, anemia, thrombocytopenia, and constipation did not exceed G1–2. The objective response rate was 31% (1 CR, 7 PR, 10 SD, and 8 PD); the rate of pts benefiting was 69%. Eight pts failing prior BEV benefited from BEV+GM-CSF. Until today, 13 pts have died. The median time to progression is 92 days and the median overall survival is 302 days. Conclusions: BEV+GM-CSF is an active and generally well tolerated non-cytotoxic regimen in pts with advanced solid tumors. Thus, large-scaled studies of this promising treatment are warranted. No significant financial relationships to disclose.

A pilot study of bevacizumab (Bev) and continous low-dose granulocyte-macrophage colony-stimulating factor (GM-CSF) in heavily pretreated patients with refractory solid tumors: Preliminary results

3031 Bev is an important inhibitor of VEGF, a mediator of tumor-induced angiogenesis. Inhibiting generation of dendritic cells (DC) from blood and tissue precursors, VEGF is also a crucial immunosuppressive agent in anti-tumoral immunotolerance. Beyond anti-angiogenesis, inhibition of VEGF may results in an improved antineoplastic immune response, which may be further improved by GM-CSF known to effectively stimulate DCs. GM-CSF has formerly be shown to induce objective responses in refractory gynecologic and breast carcinomas [Kurbacher et al. Oncology 2005]. This ongoing prospective clinical trial was initiated to investigate the combination of Bev and GM-CSF in patients (pts) with heavily pretreated solid tumors. Toxicity was documented according to the NCI-CTC scale, responses were recorded according to the RECIST or the Rustin criteria. Statistical pre-study considerations forced termination of the trial if no responses were observed among the first 12 pts recruited. Until now, 10 pts are included: epithelial ovarian carcinoma, n=4; metastatic breast cancer, n=3; peritoneal papillary-serous carcinoma, n=2; salivary gland adenocarcinoma, n=1. Regarding their history, all pts have been considered chemo-resistant or non-treatable due to their underlying bone marrow and/organ functions. Bev was applied at a fixed dose of 6 mg/kg q2w. GM-CSF was administered sc at 125 μg/d. GM-CSF was increased every 4 weeks at 25 μg/d increments until a maximum dose of 250 μg/d until occurence of objective tumor response or leukocytosis <20,000 cells/μL. Among the 10 pts treated so far, 2 partial responses (PR), 5 stable diseases (SD), and 3 progressive diseases (PD) have been recorded accounting for an objective response rate of 20%. Median time to progression is 16 weeks, with 7 pts still alive. Toxicity was generally mild and never therapy-limiting: NCI-CTC grade (G) 2 fatigue in 3 pts, G2 flu-like symptoms and G2 in either 1 pt, G1 injection site reactions in 3 pts. Preliminary results of this trial have shown Bev plus GM-CSF to produce a promising clinical activity in heavily pretreated pts with solid tumors. Thus, pts accrual is still continuing. No significant financial relationships to disclose.

Identification of a New Subset of Myeloid Suppressor Cells in Peripheral Blood of Melanoma Patients With Modulation by a Granulocyte-Macrophage Colony-Stimulation Factor–Based Antitumor Vaccine

Phenotypic and functional features of myeloid suppressor cells (MSC), which are known to serve as critical regulators of antitumor T-cell responses in tumor-bearing mice, are still poorly defined in human cancers. Here, we analyzed myeloid subsets with suppressive activity present in peripheral blood of metastatic melanoma patients and evaluated their modulation by a granulocyte-macrophage colony-stimulating factor (GM-CSF)--based antitumor vaccine. Stage IV metastatic melanoma patients (n = 16) vaccinated with autologous tumor-derived heat shock protein peptide complex gp96 (HSPPC-96) and low-dose GM-CSF provided pre- and post-treatment whole blood specimens. Peripheral-blood mononuclear cells (PBMCs) were analyzed by flow cytometry, separated into cellular subsets, and used for in vitro proliferation assays. PBMCs from stage-matched metastatic melanoma patients (n = 12) treated with non-GM-CSF-based vaccines (ie, HSPPC-96 alone or interferon alfa/melanoma-derived peptides) or sex- and age-matched healthy donors (n = 16) were also analyzed for comparison. The lack of or low HLA-DR expression was found to identify a CD14+ cell subset highly suppressive of lymphocyte functions. CD14+HLA-DR-/lo cells were significantly expanded in all metastatic melanoma patients, whereas they were undetectable in healthy donors. Suppressive activity was mediated by transforming growth factor beta (TGF-beta), whereas no involvement of the arginase and inducible nitric oxide synthase pathways could be detected. CD14+HLA-DR-/lo cells, as well as spontaneous ex vivo release and plasma levels of TGF-beta, were augmented after administration of the HSPPC-96/GM-CSF vaccine. No enhancement of the CD14+-mediated suppressive activity was found in patients receiving non-GM-CSF-based vaccines. CD14+HLA-DR-/lo cells exerting TGF-beta-mediated immune suppression represent a new subset of MSC potentially expandable by the administration of GM-CSF-based vaccines in metastatic melanoma patients.

Regulation of cyclooxygenase activity in cultured endometrial stromal cells by granulocyte-macrophage colony-stimulating factor

To assess the ability of granulocyte-macrophage colony-stimulating factor (GM-CSF) to regulate cyclooxygenase (COX) enzyme activity and prostaglandins (PGs) synthesis, specifically PGE2 production in stromal cells, neither of which have been addressed in the literature. Prospective study. Department of obstetrics and gynecology at a university hospital. Human luteal phase endometrium was obtained from surgical specimens (n = 6) for clinical indications. Confluent stromal cells were stimulated with GM-CSF. Expression of COX mRNA, COX enzyme activity, and PGE2 level in cultured stromal cells. Confluent stromal cell cultures treated with P and E2 for 9 days were stimulated with GM-CSF. After treatment of 12 hours, low-dose GM-CSF (0.001-0.1 ng/mL) increased COX-2 mRNA levels in stromal cell, whereas high dose GM-CSF (1-100 ng/mL) decreased COX-1 and COX-2 mRNA levels. After treatment of 48 hours, low concentrations of GM-CSF (0.001-0.1 ng/mL) increased total COX and COX-2 enzyme activity, whereas high concentrations of GM-CSF (1-100 ng/mL) inhibited COX and COX-2 activity; The PGE2 levels decreased by 31% to 393.3 pg/mL (P < .05) with concentrations of GM-CSF increasing from 1 ng/mL to 100 ng/mL. There appeared to be a biphasic pattern of COX-2 enzyme response to GM-CSF with low concentrations increasing activity and high concentrations inhibiting activity. It is possible that GM-CSF may provide critical regulation of PG production in the preimplantation period.

Accumulation of amphotericin B in human macrophages enhances activity against Aspergillus fumigatus conidia: quantification of conidial kill at the single-cell level.

A cytofluorometric assay that allowed assessment of damage to phagocytosed Aspergillus fumigatus conidia at the single-cell level was developed. After ingestion by monocyte-derived macrophages (MDMs), conidia were reisolated by treatment of the cells with streptolysin O, a pore-forming toxin with lytic properties on mammalian cells but not on fungi. The counts obtained by staining of damaged conidia with propidium iodide and quantification by cytofluorometry correlated with colony counts. By the use of this method, we demonstrate that MDMs differentiated in vitro by low-dose granulocyte-macrophage colony-stimulating factor and gamma interferon have only a limited capacity to damage Aspergillus conidia in vitro. The killing rate 12 h after phagocytosis was found to be only 10 to 15%. However, intracellular loading of the phagocytes with amphotericin B (AmB) dose dependently enhanced the anticonidial activity. Preincubation of macrophages with only 1 microg of AmB per ml resulted in an uptake of 18 fg of AmB/cell, leading to killing rates of 50 to 60%. The experimental protocol provides a new tool for the rapid quantification of anticonidial activity against A. fumigatus in vitro. Intracellular accumulation of AmB may represent an important factor underlying the efficacy of this antifungal drug in the prophylaxis and treatment of Aspergillus infections.

Neutropenia Associated With T-Cell Large Granular Lymphocyte Leukemia: Long-Term Response to Cyclosporine Therapy Despite Persistence of Abnormal Cells

AbstractT-cell large granular lymphocyte (T-LGL) leukemia is clinically indolent, but is associated with severe neutropenia in approximately 50% of cases. The pathogenesis of the neutropenia is unclear. We report reversal of severe neutropenia associated with T-LGL leukemia in five patients treated with cyclosporine (CSA). All five had persistent neutrophil counts below 0.5 × 109/L, two had agranulocytosis, and four had recurrent infections. Increased populations of LGL were present in blood and marrow, with a T-LGL immunophenotype (CD3+CD8+CD16±CD56±CD57+) shown by multiparameter flow cytometry, and clonal T-cell receptor (TCR) gene rearrangements in two of two pretreatment blood samples studied. CSA was initiated at doses of 1 to 1.5 mg/kg orally every 12 hours, with subsequent dose adjustments based on trough serum levels. Four patients attained normal neutrophil counts with CSA alone; one required addition of low-dose granulocyte-macrophage colony-stimulating factor. Time to attainment of 1.5 × 109/L neutrophils ranged from 21 to 75 days. Attempts to taper and withdraw CSA resulted in recurrent neutropenia. Three patients have maintained normal neutrophil counts on continued CSA therapy for 2, 8, and 8.5 years. Two patients died 1.7 and 4.6 years after initiation of CSA despite normal neutrophil counts—one of metastatic melanoma and one of complications after aortofemoral bypass surgery. Despite resolution of neutropenia, increased populations of T-LGL cells have persisted in all patients during CSA therapy, as shown by morphology and flow cytometry and by the presence of clonal TCR gene rearrangements in four patients' posttreatment blood samples. We conclude that CSA is an effective therapy for neutropenia associated with T-LGL leukemia, and that resolution of neutropenia despite persistence of abnormal cells implies that CSA may inhibit T-LGL secretion of yet unidentified mediators of neutropenia.

Neutropenia Associated With T-Cell Large Granular Lymphocyte Leukemia: Long-Term Response to Cyclosporine Therapy Despite Persistence of Abnormal Cells

T-cell large granular lymphocyte (T-LGL) leukemia is clinically indolent, but is associated with severe neutropenia in approximately 50% of cases. The pathogenesis of the neutropenia is unclear. We report reversal of severe neutropenia associated with T-LGL leukemia in five patients treated with cyclosporine (CSA). All five had persistent neutrophil counts below 0.5 × 109/L, two had agranulocytosis, and four had recurrent infections. Increased populations of LGL were present in blood and marrow, with a T-LGL immunophenotype (CD3+CD8+CD16±CD56±CD57+) shown by multiparameter flow cytometry, and clonal T-cell receptor (TCR) gene rearrangements in two of two pretreatment blood samples studied. CSA was initiated at doses of 1 to 1.5 mg/kg orally every 12 hours, with subsequent dose adjustments based on trough serum levels. Four patients attained normal neutrophil counts with CSA alone; one required addition of low-dose granulocyte-macrophage colony-stimulating factor. Time to attainment of 1.5 × 109/L neutrophils ranged from 21 to 75 days. Attempts to taper and withdraw CSA resulted in recurrent neutropenia. Three patients have maintained normal neutrophil counts on continued CSA therapy for 2, 8, and 8.5 years. Two patients died 1.7 and 4.6 years after initiation of CSA despite normal neutrophil counts—one of metastatic melanoma and one of complications after aortofemoral bypass surgery. Despite resolution of neutropenia, increased populations of T-LGL cells have persisted in all patients during CSA therapy, as shown by morphology and flow cytometry and by the presence of clonal TCR gene rearrangements in four patients' posttreatment blood samples. We conclude that CSA is an effective therapy for neutropenia associated with T-LGL leukemia, and that resolution of neutropenia despite persistence of abnormal cells implies that CSA may inhibit T-LGL secretion of yet unidentified mediators of neutropenia.

Differential dose‐related haematological effects of GM‐CSF in pancytopenia: evidence supporting the advantage of low‐ over high‐dose administration in selected patients

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multifunctional haematopoietin which can promote production of several blood cell lineages, though the predominant target cells are neutrophils, monocytes, and their precursors. Occasional undesirable clinical effects include eosinophilia, an increase in blasts, or thrombocytopenia. Here, we describe four patients who were treated with GM-CSF, at subcutaneous doses significantly lower than are conventional, and experienced an unusual response pattern. Three patients had severe pancytopenia associated with chronic lymphocytic leukaemia (CLL) or myelodysplastic syndrome (MDS) and exhibited an unexpected switch in the responsive lineage on high- versus very low-dose therapy. The two CLL patients developed marked eosinophilia (up to 10.0 x 10(9) cells/l) without an increase in neutrophils on 125-300 micrograms/m2/d of GM-CSF. In contrast, when the dose was lowered to 10 micrograms/m2/d, the neutrophils rose to physiological levels, without significant eosinophilia. The MDS patient showed a rapid rise in peripheral blasts (baseline level = 0; post-therapy level = 5.0 x 10(9)/l), without a change in other cell types, when receiving 60 micrograms/m2/d of GM-CSF. After GM-CSF was held, blasts returned to baseline levels; reinstituting therapy at the very low dose of 6 micrograms/m2/d was followed by an increase in platelet counts from 50 to 185 x 10(9)/l with only a minor increase in blasts. The fourth patient, who suffered from severe aplastic anaemia complicated by recurrent gastrointestinal haemorrhage, was only treated with the low-dose regimen. He showed a predominant platelet effect with counts rising from 9 to 169 x 10(9)/l. Very low-dose GM-CSF therapy was devoid of constitutional side effects. The biological implications of these GM-CSF responses are discussed. Our results indicate that, in some patients, GM-CSF may stimulate different target cells depending on the dose. Therefore, in contrast to the results of administration of many classical drugs, there may not always be a direct relationship between the amount of GM-CSF given and the optimal effect.

A Phase I/II Trial of Zidovudine, Interferon- , and Granulocyte-Macrophage Colony-Stimulating Factor in the Treatment of Human Immunodeficiency Virus Type 1 Infection

Twenty-four patients infected with human immunodeficiency virus type 1 (HIV-1) who had CD4+ counts of 0.2-0.5 x 10(9) cells/l received granulocyte-macrophage colony-stimulating factor (GM-CSF) in combination with zidovudine plus escalating doses of daily subcutaneous interferon-alpha. Mean neutropenia-inducing doses of interferon-alpha were 9.4 x 10(6) and 10.6 x 10(6) IU/day for groups receiving 100 or 200 mg zidovudine every 4 h, respectively. Mean GM-CSF doses used to reverse neutropenia were 0.64 and 0.63 microgram/kg/day for these two groups, respectively, although the mean minimum effective GM-CSF dose for both was only 0.30 microgram/kg/day. Serum p24 antigen declined greater than 70% in all 5 antigenemic patients. Toxicities included a dose-dependent increase in lymphokine-like side effects (100%), anorexia and weight loss (42%), fatigue (42%), and anemia (50%). While toxicities of the combination can be significant, low-dose GM-CSF readily ameliorated neutropenia associated with zidovudine and interferon-alpha therapy without adversely affecting the antiviral properties of the combination.

Effects of low doses of recombinant human granulocyte‐macrophage colony stimulating factor (GM‐CSF) in patients with myelodysplastic syndromes

There has been no previously published experience with granulocyte-macrophage colony stimulating factor (GM-CSF) doses less than 12 micrograms/m2 daily in patients with myelodysplastic syndromes, and most observations have been made at doses greater than or equal to 120 micrograms/m2 daily. We administered 5 micrograms/m2 daily by subcutaneous injection to 29 such patients increasing the dose in patients who did not show a haematologic response. Doses of 5 or 10 micrograms/m2 ('low-dose GM-CSF') produced an increase in neutrophils in 14/29 patients. Response was significantly (P = 0.03) more frequent in patients who had a higher pre-treatment neutrophil count (e.g. 11/16 in patients with greater than or equal to 0.5 x 10(9)/l). A rise in blasts followed administration of low-dose GM-CSF in five patients, all with either refractory anaemia with excess blasts (RAEB) or refractory anaemia with excess blasts in transformation (RAEBT). Platelets decreased in five patients, four of whom had no change in blasts, reverting to baseline when GM-CSF was discontinued. We and others have previously observed similar rises in blasts or decreases in platelets at doses of 120 micrograms/m2 daily. Low-dose GM-CSF produced no constitutional side effects. Our results suggest that low doses of GM-CSF might be initially employed in neutropenic patients with myelodysplastic syndromes who present with pretreatment neutrophil counts greater than 0.5 x 10(9)/l. Increasing the dose, and hence the risk of extramedullary toxicity, only in patients who do not respond to the low dose. Patients who present with lower pre-treatment neutrophil counts might begin treatment at doses above 10 micrograms/m2, but below the 120 micrograms/m2 commonly employed, which may be necessary in relatively few patients.

Capillary leak syndrome during low dose granulocyte-macrophage colony-stimulating factor (rh GM-CSF) treatment of a patient in a continuous febrile state

A 20 years old man with peripheral primitive neuroectodermal tumor involving the bone marrow received 12 Gy fractionated total body irradiation, 140 mg/m2 melphalan, 1800 mg/m2 etoposide, and 1500 mg/m2 carboplatin for consolidation of first remission. Thereafter, 250 micrograms/m2/day recombinant human granulocyte-macrophage colony-stimulating factor (rh GM-CSF) (Behring Werke) were administered as continuous infusion 4 days after infusion of autologous bone marrow and peripheral stem cells to accelerate granulocyte reconstitution for control of a continued febrile state. The clinical picture of capillary leak syndrome developed with weight gain, pleural effusions and peripheral edema. The patient's condition stabilized after discontinuation of rh GM-CSF. Eight days later he died of invasive aspergillosis. The clinical course of our patient suggests a potentially fatal toxic effect of rh GM-CSF, even in low dose, in the setting of septicemia or fungemia.

High-dose melphalan and granulocyte-macrophage colony-stimulating factor for refractory multiple myeloma

High-dose melphalan has induced remissions in about 40% of patients with refractory myeloma, but the mortality has been high, at about 20%, due to complications of prolonged granulocytopenia. In an attempt to stimulate earlier granulocyte recovery, recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) was administered subcutaneously to 23 patients with refractory myeloma who had been treated with melphalan at a high dose of 100 mg/m2. Thirty-nine percent of patients achieved marked tumor cytoreduction by at least 75%, 2 died within 2 months from infectious complications during severe neutropenia; and median durations of relapse-free and overall survival were 7 and 10+ months, respectively. The nine patients presenting with both advanced age over 50 years and a long history of prior therapy of over 1 year required significantly longer median times of 31 days for granulocytes and of 63 days for platelets to reach safe levels of at least 500/microL and 50,000/microL, respectively, than the 14 remaining patients who had none or only one of these adverse features (21 and 26 days, respectively). In a historic control of 43 patients treated previously with high-dose melphalan but without GM-CSF, hematologic recovery to the aforementioned levels of granulocytes and platelets proceeded over almost 5 weeks, regardless of age and prior treatment exposure. Thus GM-CSF seems to hasten marrow recovery, especially in patients with adequate normal marrow stem-cell reserve as defined by younger age or less prior therapy. While not shortening the duration of neutropenia, GM-CSF dose increments (from 0.25 to 0.5 to 0.75 mg/m2) increased the incidence of severe toxicity from 0% to almost 40%, especially among older patients. These results support the usefulness of low-dose GM-CSF (0.25 mg/m2) in stimulating marrow recovery in selected patients with adequate marrow reserve treated with high-dose melphalan for refractory multiple myeloma.

High-Dose Melphalan and Granulocyte-Macrophage Colony-Stimulating Factor for Refractory Multiple Myeloma

Abstract High-dose melphalan has induced remissions in about 40% of patients with refractory myeloma, but the mortality has been high, at about 20%, due to complications of prolonged granulocytopenia. In an attempt to stimulate earlier granulocyte recovery, recombinant human granulocyte- macrophage colony-stimulating factor (GM-CSF) was administered subcutaneously to 23 patients with refractory myeloma who had been treated with melphalan at a high dose of 100 mg/m2. Thirty-nine percent of patients achieved marked tumor cytoreduction by at least 75%, 2 died within 2 months from infectious complications during severe neutropenia; and median durations of relapse-free and overall survival were 7 and 10+ months, respectively. The nine patients presenting with both advanced age over 50 years and a long history of prior therapy of over 1 year required significantly longer median times of 31 days for granulocytes and of 63 days for platelets to reach safe levels of at least 500/microL and 50,000/microL, respectively, than the 14 remaining patients who had none or only one of these adverse features (21 and 26 days, respectively). In a historic control of 43 patients treated previously with high-dose melphalan but without GM-CSF, hematologic recovery to the aforementioned levels of granulocytes and platelets proceeded over almost 5 weeks, regardless of age and prior treatment exposure. Thus GM-CSF seems to hasten marrow recovery, especially in patients with adequate normal marrow stem-cell reserve as defined by younger age or less prior therapy. While not shortening the duration of neutropenia, GM-CSF dose increments (from 0.25 to 0.5 to 0.75 mg/m2) increased the incidence of severe toxicity from 0% to almost 40%, especially among older patients. These results support the usefulness of low-dose GM-CSF (0.25 mg/m2) in stimulating marrow recovery in selected patients with adequate marrow reserve treated with high-dose melphalan for refractory multiple myeloma.