Efficacy and toxicity of treatment with the anti-CTLA-4 antibody ipilimumab in patients with metastatic melanoma after prior anti-PD-1 therapy.

BACKGROUND: Recent phase III clinical trials have established the superiority of the anti-PD-1 antibodies pembrolizumab and nivolumab over the anti-CTLA-4 antibody ipilimumab in the first-line treatment of patients with advanced melanoma. Ipilimumab will be considered for second-line treatment after the failure of anti-PD-1 therapy. METHODS: We retrospectively identified a cohort of 40 patients with metastatic melanoma who received single-agent anti-PD-1 therapy with pembrolizumab or nivolumab and were treated on progression with ipilimumab at a dose of 3 mg kg(-1) for a maximum of four doses. RESULTS: Ten percent of patients achieved an objective response to ipilimumab, and an additional 8% experienced prolonged (>6 months) stable disease. Thirty-five percent of patients developed grade 3-5 immune-related toxicity associated with ipilimumab therapy. The most common high-grade immune-related toxicity was diarrhoea. Three patients (7%) developed grade 3-5 pneumonitis leading to death in one patient. CONCLUSIONS: Ipilimumab therapy can induce responses in patients who fail the anti-PD-1 therapy with response rates comparable to previous reports. There appears to be an increased frequency of high-grade immune-related adverse events including pneumonitis that warrants close surveillance.

Characteristics of pyrexia in BRAFV600E/K metastatic melanoma patients treated with combined dabrafenib and trametinib in a phase I/II clinical trial.

BACKGROUND: Pyrexia is a frequent adverse event with combined dabrafenib and trametinib therapy (CombiDT), but little is known of its clinical associations, etiology, or appropriate management. PATIENTS AND METHODS: All patients on the BRF133220 phase I/II trial of CombiDT treated at the standard dose (150/2) were included for assessment of pyrexia (n = 201). BRAF and MEK inhibitor-naïve patients (n = 117) were included for efficacy analyses. Pyrexia was defined as temperature ≥38°C (≥100.4(°)F) or related symptoms. RESULTS: Fifty-nine percent of patients developed pyrexia during treatment, 24% of which had pyrexia symptoms without a recorded elevation in body temperature. Pyrexia was grade 2+ in 60% of pyrexia patients. Median time to onset of first pyrexia was 19 days, with a median duration of 9 days. Pyrexia patients had a median of two pyrexia events, but 21% had three or more events. Various pyrexia management approaches were conducted in this study. A trend was observed between dabrafenib and hydroxy-dabrafenib exposure and pyrexia. No baseline clinical characteristics predicted pyrexia, and pyrexia was not statistically significantly associated with treatment outcome. CONCLUSIONS: Pyrexia is a frequent and recurrent toxicity with CombiDT treatment. No baseline features predict pyrexia, and it is not associated with clinical outcome. Dabrafenib and metabolite exposure may contribute to the etiology of pyrexia. The optimal secondary prophylaxis for pyrexia is best studied in a prospective trial.

Response to MAPK pathway inhibitors in BRAF V600M-mutated metastatic melanoma.

WHAT IS KNOWN AND OBJECTIVE: The management of metastatic melanoma has changed significantly in the past decade with the development of immunotherapies and targeted molecular therapies. Trials of targeted therapies have focused mainly on patients with the most common BRAF V600 mutations, namely V600E/K substitutions, with very little information available on the benefit of targeted therapies on less commonly occurring mutations such as V600R/D and M. CASE SUMMARY: We present a 54-year-old man with metastatic melanoma harbouring a rare BRAF V600M mutation, who experienced clinical and radiological response to combined therapy with the BRAF inhibitor dabrafenib and MEK inhibitor trametinib. WHAT IS NEW AND CONCLUSION: As our understanding of these therapies evolves and an increasing number of patients have mutational testing performed, there is a clear imperative--as highlighted by this case--to test for rarer mutations and facilitate their inclusion both in everyday practice and in future clinical trials.

Multiplex immunohistochemistry accurately defines the immune context of metastatic melanoma.

A prospective study explored the heterogeneous nature of metastatic melanoma using Multiplex immunohistochemistry (IHC) and flow cytometry (FACS). Multiplex IHC data quantitated immune subset number present intra-tumoral (IT) vs the tumor stroma, plus distance of immune subsets from the tumor margin (TM). In addition, mIHC showed a close association between the presence of IT CD8+ T cells and PDL1 expression in melanoma, which was more prevalent on macrophages than on melanoma cells. In contrast, FACS provided more detailed information regarding the T cell subset differentiation, their activation status and expression of immune checkpoint molecules. Interestingly, mIHC detected significantly higher Treg numbers than FACS and showed preferential CD4+ T cell distribution in the tumor stroma. Based on the mIHC and FACS data, we provide a model which defines metastatic melanoma immune context into four categories using the presence or absence of PDL1+ melanoma cells and/or macrophages, and their location within the tumor or on the periphery, combined with the presence or absence of IT CD8+ T cells. This model interprets melanoma immune context as a spectrum of tumor escape from immune control, and provides a snapshot upon which interpretation of checkpoint blockade inhibitor (CBI) therapy responses can be built.

Enhancement of platelet recovery after myelosuppressive chemotherapy by recombinant human megakaryocyte growth and development factor in patients with advanced cancer.

PURPOSE: To explore the influence of dose and schedule on the ability of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) to abrogate thrombocytopenia after multiple cycles of chemotherapy and to mobilize peripheral-blood progenitor cells (PBPC). PATIENTS AND METHODS: In this open-label study, 68 patients with advanced cancer were randomized to receive PEG-rHuMGDF subcutaneously at different doses and durations before administration of carboplatin 600 mg/m(2), cyclophosphamide 1,200 mg/m(2), and filgrastim 5 microgram/kg/d. PEG-rHuMGDF was not given after the first cycle of chemotherapy but was given after the second and subsequent cycles. Chemotherapy was given every 28 days for up to six cycles. RESULTS: In patients who received the same dose of chemotherapy for at least two cycles, the platelet nadir was significantly higher (47.5 x 10(9)/L v 35.5 x 10(9)/L; P =.003) and duration of grade 3 or 4 thrombocytopenia significantly shorter (0 v 3 days; P =.004) when PEG-rHuMGDF was administered after chemotherapy. There was no evidence of an effect of PEG-rHuMGDF when it was given before chemotherapy. Platelet recovery after the first cycle of chemotherapy was no different for different PEG-rHuMGDF regimens, and there was no difference between patients treated with PEG-rHuMGDF and historical controls treated with identical chemotherapy. There was a modest dose-related increase in progenitor cell levels after administration of PEG-rHuMGDF alone. Peak levels of PBPC occurred later in cycle 2 than in cycle 1 but were not different in magnitude. CONCLUSION: PEG-rHuMGDF abrogated severe thrombocytopenia after dose-intensive chemotherapy. However, it had only a modest effect on progenitor cell levels and did not enhance progenitor cell mobilization after chemotherapy and filgrastim.

Comparative study of the pharmacokinetics and toxicity of high-dose epirubicin with or without dexrazoxane in patients with advanced malignancy.

PURPOSE: We evaluated the toxicity and pharmacokinetics of the combination of dexrazoxane with epirubicin at dexrazoxane/epirubicin dose ratios of 5 to 9:1 in a controlled, crossover phase I study in patients with advanced malignancy. PATIENTS AND METHODS: Thirty-eight patients with a variety of malignancies were enrolled. Assessable patients received two cycles of chemotherapy consisting of epirubicin alone and in combination with dexrazoxane. Comparisons were made between the toxicity and pharmacokinetics of epirubicin in the two treatment arms, using each patient as his or her own control. Dexrazoxane and epirubicin were delivered at dose levels of 600/120 mg/m2, 900/120 mg/m2, 900/135 mg/m2, 900/150 mg/m2, and 1,200/135 mg/m2, respectively. Twenty-six patients completed two cycles of chemotherapy and were therefore assessable. RESULTS: The maximum-tolerated doses (MTDs) of dexrazoxane/epirubicin were 1,200/135 mg/m2, with the dose-limiting toxicities being neutropenia, infection, and stomatitis. There was no difference in the nadir neutrophil or platelet counts between single-agent and combination treatment at any of the dose levels. Severe vomiting and stomatitis occurred less frequently following administration of epirubicin and dexrazoxane when compared with epirubicin alone (P = .01 and .02, respectively). Prior administration of higher doses (900 mg/m2 and 1,200 mg/m2) of dexrazoxane increased the systemic clearance of epirubicin, resulting in a decrease in the area under the curve (AUC). Elimination half-life, maximum plasma concentration (Cmax), and apparent volume of distribution of epirubicin were not significantly affected by dexrazoxane. Left ventricular ejection fraction (LVEF) decreased by greater than 10% in two patients, but neither developed clinical or radiologic evidence of cardiac failure. CONCLUSION: This study demonstrates that dexrazoxane can be safely combined with escalating doses of epirubicin at dose ratios of 5 to 9:1 without having an adverse impact on toxicity. Studies are need to determine the optimal dose ratio for cardioprotection and to explore further the pharmacokinetic interactions of the two drugs at increasing doses of epirubicin supported by hematopoietic growth factors.

Phase I and pharmacokinetic study of KRN8602 alone and with filgrastim in patients with advanced cancer.

PURPOSE: To determine the recommended dose, toxicity profile, and pharmacokinetics of KRN8602 (MX2-hydrochloride), a novel morpholino anthracycline with potent cytotoxicity against anthracycline-sensitive and resistant experimental tumors in vitro and in vivo. PATIENTS AND METHODS: KRN8602 was administered alone in increasing doses to patients with advanced cancer or high-grade gliomas until dose-limiting toxicity (DLT) was observed in three or more of five patients treated in a dose level. Because neutropenia was dose limiting, further escalation was investigated with filgrastim support. RESULTS: Fifty-six assessable patients completed at least one cycle of chemotherapy. The recommended dose of KRN8602 alone was 40 mg/m2. Dose escalation was limited by neutropenia. The recommended dose of KRN8602 with filgrastim was 70 mg/m2, and limiting toxicities were neutropenia, diarrhea, and vomiting. The most commonly experienced nonhematologic toxicity was nausea and vomiting. Alopecia and mucositis were infrequent and mild. Pharmacokinetic parameters showed substantial variation, although the area under the plasma concentration-time curve (AUC) and maximum concentration both increased with dose. There was no relationship between pharmacokinetic parameters and toxicity. CONCLUSION: KRN8602 at doses of 40 mg/m2 when administered alone and 70 mg/m2 when administered with filgrastim appeared to be manageable. The major DLTs were neutropenia and, at higher doses, diarrhea and vomiting. The efficacy of this drug is currently being tested in phase II studies.

Randomized phase III study of temozolomide versus dacarbazine in the treatment of patients with advanced metastatic malignant melanoma.

PURPOSE: To compare, in 305 patients with advanced metastatic melanoma, temozolomide and dacarbazine (DTIC) in terms of overall survival, progression-free survival (PFS), objective response, and safety, and to assess health-related quality of life (QOL) and pharmacokinetics of both drugs and their metabolite, 5-(3-methyltriazen-1-yl)imidazole-4-carboximide (MTIC). PATIENTS AND METHODS: Patients were randomized to receive either oral temozolomide at a starting dosage of 200 mg/m(2)/d for 5 days every 28 days or intravenous (IV) DTIC at a starting dosage of 250 mg/m(2)/d for 5 days every 21 days. RESULTS: In the intent-to-treat population, median survival time was 7.7 months for patients treated with temozolomide and 6.4 months for those treated with DTIC (hazards ratio, 1.18; 95% confidence interval [CI], 0.92 to 1.52). Median PFS time was significantly longer in the temozolomide-treated group (1.9 months) than in the DTIC-treated group (1.5 months) (P =.012; hazards ratio, 1.37; 95% CI, 1.07 to 1.75). No major difference in drug safety was observed. Temozolomide was well tolerated and produced a noncumulative, transient myelosuppression late in the 28-day cycle. The most common nonhematologic toxicities were mild to moderate nausea and vomiting, which were easily managed. Temozolomide therapy improved health-related QOL; more patients showed improvement or maintenance of physical functioning at week 12. Systemic exposure (area under the curve) to the parent drug and the active metabolite, MTIC, was higher after treatment with oral temozolomide than after IV administration of DTIC. CONCLUSION: Temozolomide demonstrates efficacy equal to that of DTIC and is an oral alternative for patients with advanced metastatic melanoma.

Multicycle high-dose chemotherapy and filgrastim-mobilized peripheral-blood progenitor cells in women with high-risk stage II or III breast cancer: five-year follow-up.

PURPOSE: To determine the safety and efficacy of multiple cycles of dose-intensive, nonablative chemotherapy in women with poor-prognosis breast cancer. PATIENTS AND METHODS: Women with stage II breast cancer and 10 or more involved nodes or four or more involved nodes and estrogen receptor-negative tumors and women with stage III disease received three cycles of epirubicin 200 mg/m2 and cyclophosphamide 4 g/m2, with progenitor cell and filgrastim support every 28 days (n = 79) or 21 days (n = 20). Patients were reviewed at least twice yearly thereafter. Twenty-six patients had bone marrow and apheresis collections assessed for the presence of micrometastatic tumor cells. RESULTS: Ninety-nine women (median age, 43 years; range, 24 to 60 years) were treated. Ninety-two completed all three cycles of chemotherapy. The major toxicity was severe, reversible myelosuppression that was more prolonged with successive cycles, and this did not differ between patients given treatment every 28 days and those treated every 21 days. Febrile neutropenia occurred in 176 (61%) of 287 cycles. Severe mucositis (grade 3 or 4) occurred in 23% of cycles but tended to be short-lived and was reversible. The cardiac ejection fraction fell by a median of 4% during treatment, and three patients developed evidence of cardiac failure after chemotherapy. Two patients (2%) died of acute toxicity. Three of 26 patients had evidence of circulating micrometastatic tumor cells. The actuarial distant disease-free and overall survival rates at 60-month follow-up were 64% (95% confidence interval [CI], 53% to 75%) and 67% (95% CI, 56% to 78%), respectively. CONCLUSION: Multiple cycles of dose-intensive, nonablative chemotherapy is a feasible and safe approach. Disease control and survival are similar to those in other studies of myeloablative chemotherapy in poor-prognosis breast cancer. The regimen is being evaluated in a randomized trial of the International Breast Cancer Study Group.

Rapid hematopoietic recovery after multicycle high-dose chemotherapy: enhancement of filgrastim-induced progenitor-cell mobilization by recombinant human stem-cell factor.

PURPOSE: To assess the mobilization potential and safety of recombinant human stem-cell factor (SCF) when coadministered with filgrastim to untreated women with poor-prognosis breast cancer. PATIENTS AND METHODS: Eligible women had breast cancer with 10 or more positive axillary nodes, or estrogen receptor-negative tumor with 4 positive nodes, or stage III disease. Patients were randomized to receive SCF plus filgrastim or filgrastim alone. Filgrastim 12 microg/kg daily was administered for 6 days by continuous subcutaneous infusion. SCF was administered by daily subcutaneous injection at 5, 10, or 15 microg/kg concurrent with filgrastim for 7 days, or 10 microg/kg daily starting 3 days before filgrastim for a total of 10 days (SCF pretreatment). Apheresis was performed on days 5, 6, and 7 of filgrastim administration. Patients then had three cycles of epirubicin 200 mg/m2 and cyclophosphamide 4 g/m2 every 28 days, each supported by one third of the apheresis product. RESULTS: Sixty-two women were treated. Greater yields occurred in patients who received SCF 10 microg/kg daily plus filgastim than those who received filgrastim alone (P=.013 for CD34+ cells; P=.07 for granulocyte-macrophage colony-forming cells [GM-CFCs]). The difference was more marked with SCF-pretreatment than concurrent SCF. Fewer aphereses were required to reach the predetermined target of peripheral-blood progenitor/stem cells (PBPCs) in women who received SCF. SCF was generally well tolerated. Hematologic recovery was rapid after each of the three cycles of chemotherapy. There was no difference in recovery between the different treatment groups. CONCLUSION: Mobilization of PBPCs by filgrastim is significantly enhanced by coadministration of SCF, and commencing SCF before filgrastim can optimize this effect. SCF has the potential to reduce the number of aphereses required to collect a target number of PBPCs.

Specific targeting, biodistribution, and lack of immunogenicity of chimeric anti-GD3 monoclonal antibody KM871 in patients with metastatic melanoma: results of a phase I trial.

PURPOSE: KM871 is a chimeric monoclonal antibody against the ganglioside antigen GD3, which is highly expressed on melanoma cells. We conducted an open-label, dose escalation phase I trial of KM871 in patients with metastatic melanoma. PATIENTS AND METHODS: Seventeen patients were entered onto one of five dose levels (1, 5, 10, 20, and 40 mg/m2). Patients received three infusions of KM871 at 2-week intervals, with the first infusion of KM871 trace-labeled with indium-111 (111In) to enable assessment of biodistribution in vivo. Biopsies of metastatic melanoma sites were performed on days 7 to 10. RESULTS: Fifteen of 17 patients completed a cycle of three infusions of KM871. No dose-limiting toxicity was observed during the trial; the maximum-tolerated dose was therefore not reached. Three patients (at the 1-, 5-, and 40-mg/m2 dose levels) developed pain and/or erythema at tumor sites consistent with an inflammatory response. No normal tissue uptake of 111In-KM871 was observed, and tumor uptake of 111In-KM871 was observed in all lesions greater than 1.5 cm (tumor biopsy 111KM871 uptake results: range, 0.001% to 0.026% injected dose/g). The ratio of maximum tumor to normal tissue was 15:1. Pharmacokinetic analysis revealed a 111In-KM871 terminal half-life of 7.68 +/- 2.94 days. One patient had a clinical partial response that lasted 11 months. There was no serologic evidence of human antichimeric antibody in any patient, including one patient who received 16 infusions over a 12-month period. CONCLUSION: This study is the first to demonstrate the biodistribution and specific targeting of an anti-GD3 antibody to metastatic melanoma in patients. The long half-life and lack of immunogenicity of KM871 makes this antibody an attractive potential therapy for patients with metastatic melanoma.

Pharmacology of the colony-stimulating factors.

Leukocyte production is influenced by a family of glycoproteins called colony-stimulating factors. Two of these have been purified, cloned and produced in quantities sufficient for clinical use. Granulocyte colony-stimulating factor (G-CSF) preferentially stimulates neutrophil production and has been shown to reduce the duration of neutropenia following chemotherapy. G-CSF therapy also has beneficial effects in a variety of other neutropenic states. Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates neutrophil, monocyte and eosinophil production and function. GM-CSF is associated with more diverse haematological and clinical effects. George Morstyn and colleagues summarize the promising results from the early clinical trials with these new therapeutic agents.

Reactivity to human islets and fetal pig proislets by peripheral blood mononuclear cells from subjects with preclinical and clinical insulin-dependent diabetes.

A simple, direct assay for T-lymphocyte reactivity to islet antigen(s) in human insulin-dependent diabetes mellitus (IDDM) should facilitate preclinical diagnosis and the evaluation of intervention therapy to avert autoimmune-mediated beta-cell destruction. In subjects with preclinical or clinical IDDM, we measured the reactivity of peripheral blood mononuclear cells (PBMCs) incubated over 6 days with either adult human islets or fetal pig proislets, or other fetal pig tissues, and with human insulin. With islets, the stimulation index (SI) of [3H]thymidine uptake by PBMCs exceeded the mean + 2SD of control subjects in 6 of 6 preclinical subjects (SI 8.7 +/- 3.7), 7 of 11 clinical subjects (SI 5.2 +/- 3.4), and 1 of 12 control subjects (SI 2.7 +/- 1.7); with insulin, the responses were less in frequency and magnitude, being 4 of 6 (2.7 +/- 1.6), 3 of 11 (2.2 +/- 1.1), and 0 of 12 (1.20 +/- 0.55), respectively. The mean responses to islets of PBMCs from preclinical and clinical subjects differed significantly from control subjects (P less than 0.02 by 2-tailed Kruskal-Wallis test). Secretion of granulocyte macrophage colony-stimulating factor by PBMCs over 6 days was assayed in the preclinical group and generally paralleled the uptake of [3H]thymidine. PBMC reactivity to islets appeared to be at least as sensitive a marker of preclinical IDDM as autoantibodies to a 64,000-Mr protein, presumably the enzyme glutamic acid decarboxylase, in fetal pig proislets. In conclusion, islet-reactive T lymphocytes in subjects with preclinical and clinical IDDM can be identified in bulk culture of PBMCs.(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of proliferative responses to granulocyte-macrophage colony-stimulating factor (GM-CSF) in acute myeloid leukaemia using a fluorescent ligand for the nucleoside transporter.

Nucleoside transporter expression has been linked to proliferation in a variety of haemopoietic cell types. Granulocyte-macrophage colony-stimulating factor (GM-CSF) was given for 72 h before commencing chemotherapy in 15 patients with relapsed or refractory acute myeloid leukaemia (AML) and in 11 patients serial bone marrows were taken for measurement of [3H]thymidine labelling index, Ki-67 positivity and maximal binding of 5-(SAENTA-x8)-fluorescein, a flow cytometry ligand which enumerates nucleoside transporter sites. GM-CSF caused proliferation of marrow myeloblasts in eight of 11 patients, while in three patients there was no change in proliferative indices. The expression of nucleoside transporters increased up to 4-fold in the myeloblasts from the patients showing a proliferative response to GM-CSF but there was no increase in transporters on the myeloblasts from the three non-responding patients. A close correlation was found between the fold increase in nucleoside transporter expression and the fold increase in labelling index of marrow myeloblasts (r = 0.86, n = 9, p < 0.01). In one patient with acute megakaryoblastic leukemia, GM-CSF caused parallel increases in labelling index, Ki-67 positivity and numbers of nucleoside transporters on peripheral blood blast cells. Thus induction of proliferation by cytokine increases the expression of nucleoside transporters on leukaemic myeloblasts studied in serial samples from the same source (bone marrow or blood). The suitability of 5-(SAENTA-x8)-fluorescein for two colour flow cytometric analysis allows the rapid enumeration of nucleoside transporters in the myeloblast compartment of heterogeneous marrow samples.

Adjuvant treatment of high-risk breast cancer using multicycle high-dose chemotherapy and filgrastim-mobilized peripheral blood progenitor cells.

Women with primary breast cancer associated with extensive axillary node involvement or large primary tumors have a very poor prognosis despite treatment with standard-dose adjuvant chemotherapy. In an attempt to improve the outlook of these patients, we investigated the safety and feasibility of delivering three cycles of high-dose epirubicin and cyclophosphamide supported with filgrastim-mobilized peripheral blood progenitor cells (PBPC). Fifteen previously untreated women, median age 50 (range, 30-58) years, with poor prognosis early stage breast cancer received filgrastim (12 microgram/kg daily for 6 days) prior to chemotherapy to mobilize progenitor cells. Patients were then given three cycles of epirubicin (200 mg/m2) and cyclophosphamide (4 g/m2) at planned 28-day intervals, each followed by infusion of one third of the PBPC collected and daily administration of filgrastim (5 microgram/kg s.c.). Three leukaphereses collected a median of 114.9 (range, 22.7-273.5) x 10(4) granulocyte-macrophage-colony-forming cells/kg body weight. Hemopoietic recovery was rapid after each cycle, and there was no correlation between the rate of recovery and the number of granulocyte-macrophage-colony-forming cells infused. There was a small but significant progressive delay in recovery from hematological and nonhematological toxicities across the three cycles. Left ventricular ejection fraction fell to below 50% in eight (53%) patients, but none developed congestive cardiac failure. Two patients did not complete three cycles because of insufficient PBPC for a third cycle (n = 1) and 2-mercaptoethane sodium sulfonate- related drug reaction during the second cycle (n = 1). There were no deaths during the study or during the follow-up period (median, 70 weeks; range, 50-85 weeks), and no late toxicities occurred. Therefore, we concluded that the delivery of multiple cycles of nonmyeloablative, dose-intensive chemotherapy supported by PBPC and filgrastim is safe, and may be widely applicable to a variety of common chemosensitive cancers with a poor prognosis. The efficacy of three cycles of high-dose epirubicin and cyclophosphamide is to be compared with standard-dose chemotherapy in a randomized trial in patients with high-risk, operable stage II and III breast cancer.

A serum-free culture model for studying the differentiation of human dendritic cells from adult CD34+ progenitor cells.

The antigen-presenting capacity of dendritic cells (DCs) makes them attractive potential cellular adjuvants for vaccination strategies. Currently, most in vitro culture systems for the production of these DCs include serum. However, this is undesirable because serum contains growth factors that vary between individuals and could affect DC development. Unless the patient's own serum is used, foreign antigens and the risk of infection will detract from the usefulness of these cells in clinical strategies. In this study we investigated the production of DCs from CD34+ progenitor cells of cancer patients or normal donors under serum-free conditions. We have established a model system for the investigation of DC development and maturation. Dendritic cells that developed from myeloid precursors accumulated after 2 weeks in an intermediate CD1a , CD80-, CD83-, CD86- stage. Intermediate DCs adhered to plastic surfaces, expressed Birbeck granules, and were negative for CD2 and CD14. In the presence of granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-alpha, interleukin-4 promoted the development of these stages. Spontaneous maturation of intermediate DCs into fully activated DCs expressing CD83 and costimulatory molecules occurred asynchronously over the ensuing 2 to 3 weeks. This maturation involved increased expression of CD80, CD83, CD86, CMRF-44, HLA-A, -B, -C, and -DR as well as downregulation of CD1a and CD11b. Activated DCs are characterized by the lack of adherence to plastic surfaces and the absence of Birbeck granules. By day 28, these cells were nonphagocytic, potent antigen-presenting cells with an irreversible phenotype. This serum-free system offers advantages in that the process of differentiation and maturation of committed DCs is extended over a period of more than 28 days, allowing investigators to study the effects of individual cytokines or other supplements during distinct phases of DC development in a defined environment.

Cloned stromal cell lines derived from human Whitlock/Witte-type long-term bone marrow cultures.

We report a human bone marrow culture technique that initially parallels the murine Whitlock/Witte culture system. As in the murine system, B cells predominate over other cell types, and all differentiation stages from pre-B to plasma cell are observed. Although these human long-term cultures pass through stages resembling phases I to III of murine Whitlock/Witte cultures, no outgrowth of nonadherent cells was seen after cultures had reached the "crisis" phase unless Epstein-Barr virus (EBV)-transformants appeared. The stromal cells persisted well beyond crisis, but they could not be maintained and passaged as cell lines, limiting their use in molecular analysis. Transfection of these stromal cells with plasmid DNA containing the simian virus 40 (SV40) early region yielded 124 cloned cell lines. Analysis of these lines showed that all expressed SV40 large T antigen, but they retained most phenotypic markers found on non-transformed stromal cells. When adherent and T-cell-depleted bone marrow cells were cultured on either nontransformed stromal layers or transformed cell lines they proliferated actively and soon yielded predominantly lymphoid nonadherent populations. Moreover, prolonged survival of acute lymphoblastic leukemia cells of pre-B phenotype was regularly achieved on both normal and transformed adherent cell layers. Although the liquid culture system favored lymphocytes, transformed stroma supported colony formation by both human and murine hemopoietic progenitors when marrow was added in agar medium. This was not explained by colony-stimulating factor (CSF) production, because striking heterogeneity in the levels of granulocyte CSF (G-CSF) and granulocyte-macrophage CSF (GM-CSF) secretion by the lines was noted. Some lines that did not produce detectable CSF demonstrated good support of fresh bone marrow growth and acute lymphoblastic leukemia (ALL) cell survival. The heterogeneity of these cell lines and their capacity to support hemopoiesis suggest that they will be useful in studying the molecular basis of in vitro lymphohemopoiesis in man.

Clinical experience with recombinant human granulocyte colony-stimulating factor and granulocyte macrophage colony-stimulating factor.

Bacterially synthesized human granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF) have been studied to determine if they could prevent or reduce the neutropenia caused by chemotherapy. Our studies suggest that 10 micrograms/kg/day of G-CSF administered as a continuous subcutaneous infusion abrogates the neutropenia associated with a standard dose of melphalan. G-CSF produced a rapid increase of neutrophil levels, was well-tolerated, and was associated with only one frequent adverse effect: bone pain. GM-CSF, administered in doses ranging from 3 to 15 micrograms/kg/day subcutaneously, appeared to be useful in abrogating the neutropenia associated with chemotherapy, producing elevations in neutrophils, eosinophils, and monocytes. Although GM-CSF was relatively well-tolerated, several adverse effects, including bone pain, rashes, and fluid retention, were observed. The initial dose of GM-CSF in some patients produced a reaction that was characterized by hypoxia.

Haemopoietic growth factor control of normal and neoplastic cellular proliferation.

Endotoxin induces the appearance of granulocyte colony stimulating factor (G-CSF) and macrophage (M)-CSF in the serum of mice. No GM-CSF was detectable in endotoxin serum. Phytohaemagglutinin stimulated human T-lymphocytes to secrete three major forms of GM-CSF. All of the haemopoietic growth factors (HGFs) are active in vivo, but the cellular responses are different for each HGF. Prolonged administration of G-CSF increases the number of circulating neutrophils and does not lead to adverse side-effects. Chronic exposure to GM-CSF induces an accumulation of activated macrophages which can cause considerable tissue destruction. Although the 'src-like' oncogenes induce HGF production by chicken myeloblasts, factor-dependent murine cells can be converted to a tumorigenic phenotype without autocrine growth factor production.

The effects of dose and route of administration on the pharmacokinetics of granulocyte-macrophage colony-stimulating factor.

The pharmacokinetics of granulocyte-macrophage colony stimulating factor (GM-CSF) (0.3-30 micrograms/kg) were studied after subcutaneous bolus (n = 16) or intravenous bolus (n = 5) injection or 2 h intravenous infusion (n = 12). Each method of administration gave a different GM-CSF concentration-time profile. Highest peak serum concentrations (Cmax) followed the intravenous bolus, and the time GM-CSF persisted at a concentration greater than 1 ng/ml (t greater than 1 ng/ml) was longer after a subcutaneous than after an intravenous injection. Area under the concentration-time curve (AUC), Cmax and t greater than 1 ng/ml all increased with dose for each method of administration. After intravenous administration, there was a two-phase decline in concentration. The half-life (t1/2) of the terminal phase following an intravenous bolus ranged from 0.24 to 1.18 h and, following intravenous infusion, from 0.62 to 9.07 h and appeared to increase with dose. The apparent clearance was greatest following subcutaneous injection at doses below 3 micrograms/kg, suggesting a saturable mechanism or different bioavailability. Only 0.001%-0.2% of the injected dose appeared in the urine as immunoreactive GM-CSF.