Cancer Chemotherapy & Biotherapy: Principles & Practices, 4th Edition

Hematopoietic Growth Factors

William P. Petros

INTRODUCTION

Hematopoietic growth factors (HGFs) are an important therapeutic class of agents for patients being treated with cytotoxic chemotherapy. Appropriate prescribing of these compounds has resulted in reductions in transfusion requirements and prevention of myelosuppression-related complications. The primary function of these acidic glycoprotein molecules is augmentation of the production and activation of hematopoietic cells. They may also play a role in immune response.

The HGFs have many uses in oncology (Table 35.1). The focus of this chapter is on evaluating the cytokines with known clinical importance for hematopoiesis in the patient with cancer. Most of the justification for the current role of HGFs in therapeutics is directly linked to both the degree of interpatient variability in chemotherapy-induced myelosuppression and the role of dose intensity in the treatment of a particular malignancy. At the present time, these agents should not be used to increase chemotherapy doses indiscriminately and without scientific justification.

CHEMISTRY AND PHARMACEUTICS

Chemically, the HGFs are glycoproteins typically consisting of a primary chain of over 100 amino acids that are intermittently bridged by disulfide bonds. The tertiary structure of the protein plays an important role in its biologic activity. Some HGFs circulate as dimers. The molecular weight of these molecules (approximately 19 to 90 kd) is dependent on both the primary chemical structure and the extent of glycosylation. Several endogenous forms of a particular HGF may be found in the circulation varying by the degree of glycosylation—a posttranscriptional event. Recombinant HGFs may be glycosylated depending on the type of expression system used in their manufacture. Three expression systems have been used in HGF synthesis: bacterial, yeast, and mammalian cell lines. Bacterial systems, such as E. coli, do not produce glycosylated products; whereas yeast cells do provide some degree of glycosylation. Mammalian cells produce glycosylation that is more similar to the human pattern. It is not yet clear if differences in glycosylation translate into clinically important effects. In vitro studies suggest that glycosylation may alter the receptor binding, the elimination patterns, and/or the immunogenicity of cytokines.1, 2 Thus individual generic names have been assigned depending on the expression vector used in the manufacturing process (e.g., see GM-CSF in Table 35.2). The unknown clinical significance of variations in glycosylation and the use of different activity assay methods by the various manufacturers make dosage comparisons difficult even between generic types of an HGF.

EPO is a good example of how glycosylation can alter the pharmacologic properties of an HGF. Site-directed mutagenesis of the gene encoding EPO was utilized; the mutation added two N-glycosylation sites, producing the product darbepoetin.3 This resulted in a 23% increase in molecular weight compared with recombinant EPO but a threefold prolongation of the serum circulation time due to protection from metabolic degradation.4

Another approach to increasing the circulation time of recombinant proteins involves increasing the molecular size or hydrodynamic volume by conjugation with polyethylene glycol (PEG). This effectively constrains renal filtration of the drug and thus circumvents much of the metabolic degradation typically occurring during tubular reabsorption. Using this approach, a currently marketed form of G-CSF, pegfilgrastim, was created by covalently binding a 20-kd PEG molecule to the N-terminal methionine residue.

TABLE 35.1 CURRENT AND POTENTIAL USES OF HEMATOPOIETIC GROWTH FACTORS IN ONCOLOGY

Aid to hematopoietic reconstitution following myelosuppressive therapy
Bone marrow failure
Drug-induced (not chemotherapy-induced) neutropenia
Regional therapy for localized infections
Immunostimulation
In vivo cellular expansion of hematopoietic stem cells
Ex vivo cellular expansion
Generation of immune effector cells (e.g., dendritic cells)
Immune thrombocytopenic purpura
Transporters for toxins to treat hematologic malignancies

HEMATOPOIETIC GROWTH FACTOR RECEPTORS

Polymorphisms in genes that regulate HGF receptors have been reported, and some data suggest this type of variability has physiologic significance.5Glycoprotein receptors for HGFs are transcribed and expressed in a variety of hematologic and nonhematologic cells, including many cancer cell lines. The clinical implications of HGF receptor expression on malignant cells are unknown; however, one study showed that the ability of multiple malignant cell lines to transcribe genes encoding for a cytokine receptor is, by itself, insufficient to render these cells responsive to cytokine stimulation.6 Numerous clinical trials of G-CSF and GM-CSF for prevention or reversal of chemotherapy-associated neutropenia assessed the effect of these cytokines on patient survival, but not as much attention has been paid to the effects of EPO until recently. It is known that EPO activates antiapoptotic pathways and has a role in angiogenesis. Functional receptors for the cytokine are present in breast, prostate, and various pediatric tumors.7, 8 Placebo-controlled clinical trials of EPO in patients with breast cancer or head and neck found worse cancer control in those receiving EPO; unfortunately, the studies were not powered to address this issue in a prospective manner.9, 10 Additional studies focusing on the effects of EPOs on cancer progression are underway.

HGF receptors contain either one subunit (e.g., EPO, G-CSF, TPO) or multiple subunits (e.g., GM-CSF, IL-3, IL-6), and frequently subunits are shared between HGF receptors. Most common is a unique α subunit and a shared β subunit, with the latter essential for both high-affinity binding and signal transduction. Cytokine binding to cell surface receptors results in receptor clustering (oligomerization), activation, and generation of intracellular signals. In general, the HGF receptors lack intrinsic tyrosine kinase domains (except for M-CSF, SCF, and FLT-3); however, cytoplasmic tyrosine kinases have been implicated in signal transduction pathways for some of these cytokines.11 HGF receptor density is both cell and maturation specific. For a particular HGF, occupancy of only a small percentage of available receptors is required to adequately stimulate a hematopoietic cell.12 Exposure to exogenously administered cytokines may result in altered regulation of that cytokine's receptor or the receptors of other cytokines, leading to synergistic or antagonistic effects.13

The ultimate physiologic effect of circulating HGFs is determined by both the cytokine concentration in blood and the presence of receptor antagonists and/or inhibitors (Fig. 35.1). A number of studies have investigated the relevance of soluble forms of the HGF receptors.14 The potential biologic effects of these molecules are numerous and include ligand stabilization before binding, receptor competition for ligand, receptor down-regulation, and cell sensitization that enhances the response to a ligand (Fig. 35.2).15 Diverse biologic responses to soluble receptors may be a function of their concentration. The level of these molecules is tightly regulated and thought to be primarily produced by proteolytic cleavage of the transmembrane receptor's extracellular ligand-binding domain or by alternative splicing of a truncated mRNA for the receptor.16 Receptor antagonists that block cell-surface receptors and compounds that down-modulate cell surface receptors may also inhibit cytokine activity. Elevated serum concentrations of various soluble receptors have been associated with inflammation or malignant diseases.

MECHANISM OF ACTION AND BIOLOGIC EFFECTS

Multiple biologic and biochemical effects are influenced by HGFs; thus, an overall paradigm as to their mechanism(s) of action is not well established. In addition to their ability to augment activation and proliferation, these cytokines aid in committing cells to a restricted differentiation pathway as well as increase the survival of some cells. The interpretation of biologic effects resulting from administering an HGF is sometimes complex because these effects may be attributable to the HGF's ability to alter the endogenous concentrations of other cytokines.

Animal models that are genetically deficient in a particular HGF have helped define the putative roles of HGFs. For example, homozygous G-CSF–deficient mice are viable, fertile, and superficially healthy but display chronic neutropenia, whereas those rendered GM-CSF–deficient do not have altered hematopoiesis.17,18

TABLE 35.2 PHARMACEUTICAL CHARACTERISTICS OF HEMATOPOIETIC GROWTH FACTORS

Cytokine

Other Names

Generic Names

Expression Vector

Brand Names

No. of Amino Acids

Human Chromosome Location

Normal Endogenous Sources

G-CSF

Granulocyte colony-stimulating factor

Filgrastim
Lenograstim

E. coli
CHO

Neupogen (Amgen)
Granocyte (Chugai)

174

17

Monocytes/macrophages, fibroblasts, endothelial cells, keratinocytes

GM-CSF

Granulocyte-macrophage colony-stimulating factor

Sargramostim
Molgramostim
Regramostim

Yeast
E. coli
CHO

Leukine (Immunex)
Leucomax (Schering)

127

5

T-lymphocytes, monocytes/macrophages, fibroblasts, endothelial cells, osteoblasts, epithelial cells

EPO

Erythropoietin

Epoetin-α
Epoetin-α
Epoetin-β
Darbepoetin-α

CHO
CHO
CHO
CHO

Epogen (Amgen)
Procrit/Eprex (Ortho)
NeoRecormon (Roche)
Aranesp (Amgen)

165

7

Renal cells, hepatocytes

IL-11

Interleukin-11

Oprelvekin

E. coli

Neumega (Genetics Institute)

178

19

Stromal fibroblasts, trophoblasts

SCF

Stem cell factor, steel factor, mast cell growth factor, c-kit ligand

Ancestim

E. coli

Stemgen (Amgen)

165

12

Endothelial cells, fibroblasts, circulating mononuclears, bone marrow stromal cells

TPO

Thrombopoietin, megakaryocyte growth and development factor

332

3

Liver, kidney

CHO, Chinese hamster ovary cells; CSF, colony-stimulating factor.

Figure 35.1 Potential routes of cytokine action and clearance. (▼, cytokine receptor antagonist; P, protease; V, receptors for cytokine [soluble or attached to cell membrane]; Y, antibodies to cytokine.)

 

 

Figure 35.3 depicts the hematopoietic effects elicited by the various HGFs. Although such representation is useful as a basic learning tool, the true nature of the system, which produces hundreds of billions of cells per day in steady-state conditions, is undoubtedly much more complex than shown here. Interactions between recombinant and endogenous cytokines may play an important role in their biologic effects.19

Best established are the effects of HGFs such as EPO and G-CSF on the terminal differentiation of erythrocytes and neutrophils, respectively. The in vitro sensitivity of bone marrow cells to stimulation by G-CSF may be related to the patient's age, with a lower sensitivity noted in older subjects.20 Other important effects include a reduction in the time taken for newly produced neutrophils to be released from the bone marrow into the circulation and increases in antibody-dependent cytotoxic capacity. The former effect occurs primarily with G-CSF, as compared with GM-CSF.21 Administration of G-CSF following chemotherapy stimulates primitive hematopoietic progenitors to appear in the peripheral blood to such a degree that they may exceed the concentrations of these cells in normal bone marrow.22 Interestingly, G-CSF receptor expression appears not to be required for mobilization of early hematopoietic progenitor cells by that cytokine.23 Molecules such as G-CSF also enhance responses such as membrane depolarization, release of arachidonic acid, and generation of superoxide anions by neutrophils.

The therapeutic effects of EPO include maintenance of the induction, proliferation, and differentiation of erythroid progenitors from the bone marrow. Clearly, the primary effects of EPO are on the erythroid lineage; however, it may also play a role in the stimulation of early multipotent progenitors.24 Its erythropoietic activity may be at least partly attributable to induction of heme synthetic enzymes such as porphobilinogen deaminase. Other data suggest EPO may act by suppressing apoptosis of colony-forming units.

Figure 35.2 Some potential pharmacologic interactions of soluble colony-stimulating factor receptors. A: Proteolytic cleavage of soluble receptors results in a down-modulation of the membrane-bound receptor. B: Ligand binding to soluble receptor results in prolongation of ligand effect by stabilizing it in extracellular space. C: Competition between soluble and membrane-bound receptors for ligand results in decreased signal. D: Association of soluble receptors with ligand and nonbinding receptor subunits produces ligand sensitivity in cells without expression of membrane-bound receptor. (Reproduced with permission from Heaney ML, Golde DW. Soluble cytokine receptors. Blood 1996;87:847–857.)

Figure 35.3 Representation of myeloid hematopoietic differentiation. Cytokines capable of stimulating specific cells are listed below such cells. See Table 35.2 for other names of cytokines shown here. (BFU-E, burst-forming unit, erythroid; CFU-GEMM, colony-forming unit–granulocyte-erythrocyte-megakaryocyte macrophage; CFU-GM, colony-forming unit–granulocyte-macrophage; EPO, erythropoietin; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL, interleukin; M-CSF, macrophage colony-stimulating factor; SCF, stem cell factor; TPO, thrombopoietin.)

 

The primary effect of GM-CSF on hematopoietic cells lies in its ability to augment the survival and proliferation of cells in the granulocytic and macrophage lineages as well as maintain megakaryocyte progenitors at high concentrations. In vitro studies have shown that GM-CSF increases the proportion of hematopoietic cells entering the S phase of the cell cycle and results in a dose-dependent shortening in the length of the cell cycle. Increases in granulocyte life span, metabolic functional activity, and antibody-dependent cellular cytotoxicity have been noted with in vitro incubations. These may be particularly important for the monocyte/macrophage cell lines because production of other cytokines seems to be an important function of these cells. HGFs may display both acute and subacute effects on the peripheral blood concentrations of myeloid cells. For example, leukocyte concentrations initially decline following administration of GM-CSF, most likely as a result of margination of cells induced to express Mo1 leukocyte cell surface adhesion antigen.25 Other alterations of cellular function by GM-CSF include inhibition of neutrophil migration to sterile inflammatory fields.26 GM-CSF is a potent stimulator (in vitro and in vivo) of dendritic cells, which are important initiators of primary immune responses.27 The clinical implications of these effects are under study.

The glycoprotein thrombopoietin (TPO), also known as the “c-mpl ligand” or “megakaryocyte growth and development factor” (MGDF), is thought to play a very important, early, and relatively specific role in the regulation of platelet production. Mice deficient in mpl have mature, functional platelets, albeit at a concentration 15% of normal.28 TPO has been shown to stimulate blast colony formation in cells obtained from patients with acute myelogenous leukemia (AML), but solid tumors do not routinely express the mpl receptor.29 Production of TPO is thought to occur predominantly in the liver at a constant rate, whereas circulating concentrations are regulated by platelet receptor–mediated clearance of the cytokine. One negative aspect of TPO is its potential to inhibit platelet release from mature megakaryocytes.30

IL-11 is known for its properties as a stimulator of megakaryopoiesis, perhaps interacting at a later stage than TPO. However, it also has been shown to synergistically act with early and later acting cytokines in various stages of hematopoiesis. In addition, it is expressed and has activity in many other tissues, including those of the CNS, GI tract, and testes.

SCF is a very early acting cytokine that is a ligand for the oncogene c-kit. Stimulation of in vitro proliferation has been demonstrated in mast cells as well as early and intermediate bone marrow progenitors. SCF can also protect hematopoietic cells from radiation-induced damage.

CLINICAL PHARMACOLOGY

Endogenous production of HGFs occurs in a wide variety of both hematopoietic and nonhematopoietic cells (Table 35.2). Some of these cytokines are found in detectable quantities in blood; however, many factors may influence their concentrations, including concurrent drug therapy, disease, and cell homeostasis.31,32 For example, concentrations of G-CSF and TPO increase during periods of neutropenia and thrombocytopenia, respectively. This initially led to speculation that such cytopenias cause enhanced production of these cytokines. However, the effect was a result of the reduction of the receptor-based HGF clearance (see later). Exogenously administered cytokines or other drugs may also influence endogenous cytokine concentrations in patients with cancer. We have noted relatively higher concentrations of M-CSF, IL-6, and TNF-α in patients receiving GM-CSF than in those on G-CSF following autologous bone marrow transplantation.33 These observations are in accordance with in vitro studies indicating that recombinant GM-CSF can induce neutrophil and macrophage production of multiple cytokines.34 Blood obtained following administration of G-CSF to human volunteers has been shown to yield an increase in the concentrations of antiinflammatory cytokines upon ex vivo stimulation.35 Endogenous cytokine concentrations have also been helpful in evaluating the efficacy19, 33 and toxicity33 of HGF therapy.

Assay Methodology

Most published pharmacokinetic studies of HGFs are conducted by ELISA or RIA quantitative techniques. Use of an automated plate reader with these immunoassays allows the measurement of approximately 75 samples in several hours or less. ELISA assays provide easy and sensitive quantitative measurement of cytokine concentrations; however, they determine the immunoactivity, not necessarily the biological activity, of proteins. False-positive results are possible when a molecule that has been rendered biologically inactive remains intact at the assay's antibody binding site. However, currently used biological assays are often too cumbersome, costly, and variable for extensive studies of cytokines at multiple sample time points. Furthermore, endogenous inhibitors sometimes found in clinical samples may influence them. Neither assay type can distinguish endogenous HGFs from an exogenously administered recombinant forms, although approaches capable of distinguishing them are emerging.36

Pharmacokinetics

A variety of processes may influence the pharmacokinetic disposition of recombinant proteins such as the HGFs, as summarized in Figure 35.1. Some characteristics may be predictable by the molecular weight and/or glycosylation, while others may vary depending on receptor concentrations at particular time points.

Absorption

Subcutaneous administration of HGFs generally has been shown to produce lower bioavailability. This effect is thought to be secondary to degradation of the recombinant protein by subcutaneous proteases. Conversely, subcutaneous administration may result in greater clinical efficacy than short intravenous infusions. One explanation for these effects may be secondary to the low yet prolonged blood concentrations achieved with subcutaneous delivery. A study evaluating this effect through a clinical comparison of intravenous and subcutaneous administration of GM-CSF was conducted in patients with myelodysplasia.37 Twenty patients were randomly assigned to receive GM-CSF (yeast) either by 2-hour IV infusion or subcutaneously every 12 hours. Treatment lasted for 2 weeks, followed by a 2-week washout; thereafter patients were crossed over to the alternative administration route. Optimal hematopoietic stimulation occurred with the subcutaneous route. Severe toxicity occurred at a similar rate in each group.

Distribution

HGFs typically display relatively small volumes of distribution, approximating that of the plasma volume. As expected, studies that are designed to evaluate multiple compartment pharmacokinetics have described rapid distribution phases, followed by more prolonged elimination. Peak concentrations generally follow a linear dose-dependency.

Metabolism/Excretion

Serum HGF concentrations may be influenced by exogenous drug administration, increased endogenous production, or reduced elimination. Decay of HGFs from the circulation may occur via a variety of possible mechanisms, including: attachment of the ligand to the cell surface receptor, with subsequent endocytosis; metabolism by proteolytic enzymes (especially in the liver); and urinary excretion by glomerular filtration, followed by reabsorption and catabolism (Fig. 35.1). The pattern and elimination pathways of exogenously administered cytokines may be affected by the dose; the administration route and schedule; the degree of glycosylation/pegylation of the recombinant protein; the specific receptors available; the production of antibodies; and, in some cases, renal function. Receptors for G-CSF are of sufficient quantity in patients with a normal or recovering WBC count to provide an important mechanism of clearing the protein. These effects are more pronounced with the pegylated form of filgrastim, since the additional molecular size hinders the protein's renal filtration and thus its subsequent availability for metabolism (Fig. 35.4).38 In addition, it appears that administration of G-CSF during neutropenia up-regulates the number of receptors per cell and thus enhances its own clearance.39 Therefore, the serum profile of filgrastim shows greater fluctuation (peak to trough) during times when the patient is starting to recover from neutropenia than in the midst of neutropenia. Disposition of the pegylated form of filgrastim shows comparatively little decay during neutropenia until the WBC count begins its rebound.

Figure 35.4 Influence of the kidney on pharmacokinetics of pegylated versus nonpegylated proteins. Plasma disposition of filgrastim (Panel A) or pegfilgrastim (Panel B) following IV administration of 100 µg/kg to either normal (closed symbols) or nephrectomized (open symbols) rats. (Data from Yang B-B, Lum PK, Hayashi MM, et al. Polyethylene glycol modification of filgrastim results in decreased renal clearance of the protein in rats. J Pharm Sci 2004;93:1367–1373.)

The presence of other proteins, such as receptor antagonists or modulators of receptor expression, may also significantly affect receptor-mediated clearance. Dramatic reductions in the systemic clearance of a cytokine have been observed to follow concurrent administration of antibodies to that cytokine.40 This result may seem paradoxical; however, the substantially higher molecular weight of the antibody-HGF complex could limit its ability to be filtered by the glomerulus.

A summary of the pharmacokinetic parameters from published studies of selected CSFs is provided in Table 35.3. Factors potentially affecting the variability in the studies include assay methods, patient age, CSF dosage, receptor concentrations (e.g., WBC count), and the expression vector of the recombinant protein.

The effects of organ dysfunction on the pharmacokinetics of these proteins have not been extensively evaluated. It has previously been reported that systemic clearance and urinary excretion of regramostim is altered in bone marrow transplant patients with increased serum creatinine levels following an ablative regimen that included cisplatin.41 The hypothesis was that the tubular toxicity produced by cisplatin alters the renal metabolism of regramostim and that this is modified further by reduced creatinine clearance. It is of importance to note that even though the data in Table 35.3 are summarized for multiple recombinant forms of each product (with different degrees of glycosylation) for purposes of comparing different CSFs, one cannot necessarily extrapolate such data to other GM-CSF molecules with different degrees of glycosylation.

Limited clinical data are available that evaluate the effects of glycosylation on these molecules. For example, it has been firmly established that EPO needs terminal sialic acid residues on the oligosaccharides to protect the molecule from immediate proteolytic attack.42 However, other HGFs, such as GM-CSF, do not require glycosylation to be of practical clinical use. Hovgaard et al. published a comparison between molgramostim (E. coli-derived and nonglycosylated) and regramostim (Chinese hamster ovary-derived and glycosylated) in a small series of patients.43 This non-crossover study found a significantly shorter distributional half-life, a higher peak serum concentration, and a lower AUC in patients receiving intravenous molgramostim. Comparison of the products following subcutaneous administration yielded a quicker time to peak concentration and a shorter duration of detectable levels with molgramostim (Fig. 35.5). While a pharmacodynamic study will need to be completed, the data thus far suggest that differences in glycosylation may limit interchangeability of dosing data for products such as these.

CLINICAL DATA FOR INDIVIDUAL DRUGS

A number of factors must be taken into consideration when interpreting the clinical results of HGF trials. Neutrophil recovery is obviously influenced by the chemotherapy regimen and dosages selected. Individual patient factors are also important, as evidenced by descriptions of reduced HGF efficacy in patients with extensive prior myelosuppressive therapy. Likewise HGFs such as G CSF or GM-CSF, which are thought to act primarily on more mature cell types, may not be very effective in patients receiving even low doses of stem cell toxins such as thiotepa.44 Early HGF studies typically did not include concomitant administration of prophylactic antibiotics, a practice that is currently more common.

TABLE 35.3 SUMMARY OF PHARMACOKINETIC STUDIES OF HEMATOPOIETIC GROWTH FACTORS

CSF

Route

N

Half-life (hr)

Tmax (hr)

Cl (mL/min/kg)

G-CSF

SQ

37

2.5–5.8

4–8

19–56

Peg G-CSF

SQ

10

27–47

72–120

0.04–0.68

G-CSF

IV

58

(α 8a, β 1.8) 1.3–5.1

NA

4–21

GM-CSF

SQ

55

1.6–5.8

2.7–20

249–312

GM-CSF

IV

63

(α 5–20a, β 1.1–2.5) 1.1–2.4

NA

9.9–178

EPO

SQ

125

9–38

12–28

N/A

EPO

IV

135

4–11.2

NA

2.8–6.7

DARBO

SQ

14

33–49

54–86

0.062

DARBO

IV

28

18–25

NA

0.027–0.033

IL-11

SQ

18

6.9

3.2

NAb

Cl, systemic clearance (values are “apparent” for SQ route); CSF, colony-stimulating factor; DARBO, darbepoetin alpha; EPO, erythropoietin; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL, interleukin; N, number of patients; NA, not applicable; Peg, pegylated; Tmax, time of maximal concentration after SQ injection.
Data presented are ranges of mean values in the reviewed studies (refs. 41, 43, 173, 175)
aValues are in minutes.
bClearance of IL-11 in infants and children is 1.2-fold to 1.6-fold higher than in adults or adolescents.

Administration of stem cell transfusions obviously influences reconstitution. The number and type of cells (e.g., CD34+ selected peripheral blood progenitors, umbilical cord blood, etc.) can also cause sufficient heterogeneity in reconstitution so as to make any comparisons difficult.

Caution needs to be exercised in the use of these products, since concomitant administration of cycle-specific chemotherapy and G-CSF or GM-CSF has led to enhanced myelosuppression with some regimens.45, 46

Erythropoietin

Multiple factors may account for anemia in patients with cancer, although the predominant causes are thought related to the cancer itself or to cytotoxic chemotherapy. The etiology can be explained by both increased red cell destruction and reduced red cell production. Erythropoiesis is inversely correlated to disease stage in malignancies, such as multiple myeloma, where deficient red cell production is thought to be the primary mechanism.47 Chemotherapy may obviously alter the bone marrow's ability to produce erythrocytes while also eliciting toxic effects to the kidney. Renal toxicity is important because almost all endogenous EPO is produced in the peritubular interstitial cells of the kidney and is regulated by an oxygen sensor (Fig. 35.6). Although the etiology of these effects is thought to be related to platinum analog toxicity, some data suggest that alternative mechanisms are important.48 More specifically, investigators have noted an apparent deficiency in endogenous serum EPO associated with malignancy-related anemia that is not entirely explained by clinically apparent renal dysfunction. Platinum-associated anemia may be due to reduced EPO production secondary to toxic effects on EPO-producing renal cells or possibly effects of platinum on cytochrome P450 hemoproteins.49 Both inappropriately low and surprisingly high EPO concentrations have been reported following chemotherapy.50, 51 Such variability may be partly due to acute paradoxical elevations of endogenous EPO concentrations immediately following chemotherapy.52 The measurement of endogenous concentrations of EPO may be used to select anemic cancer patients who would achieve the greatest therapeutic benefit from administration of the recombinant protein. However, this practice is no longer common.53, 54 Efficacy of EPO is dependent on adequate iron stores. Aggressive therapy with intravenous iron has been shown to improve the hemoglobin response in cancer patients treated with recombinant EPO.55

Abels summarized the results from a randomized, placebo-controlled study that used recombinant EPO for anemia (HCT < 32%) in patients with cancer.56, 57Recombinant EPO (100 to 150 U/kg per day) was administered three times weekly for 8 to 12 weeks or until the patient's hematocrit reached 38 to 40%. Patients were divided into three cohorts depending on whether they were receiving chemotherapy and whether the regimen included cisplatin. The criteria for RBC transfusion were not standardized but appeared similar between the groups. As shown in Table 35.4, the patients treated with EPO in each group demonstrated statistically significant increases in HCT and a higher frequency of correction of anemia; however, the transfusion requirements were not significantly different. When the two cohorts that received chemotherapy were combined, significant reductions in the number of patients transfused (28% vs. 46%) and the number of units transfused per patient (1.04 vs. 1.81) were evident following the first month of therapy in the EPO group. Others have confirmed these findings.58

Figure 35.5 Serum disposition of molgramostim (derived from Escherichia coli) (dashed line) and regramostim (derived from Chinese hamster ovary cells) (solid line) after subcutaneous injection in patients treated with 5.5 or 8.0 µg/kg per day, respectively. (GM-CSF, granulocyte-macrophage colony-stimulating factor; rhGM-CSF, recombinant human granulocyte-macrophage colony-stimulating factor.) (Reproduced with permission from Hovgaard D, Mortensen BT, Schifter S, et al. Comparative pharmacokinetics of single-dose administration of mammalian and bacterially derived recombinant human granulocyte-macrophage colony-stimulating factor. Eur J Haematol 1993;50:32–36.)

Randomized trials of prophylactic EPO therapy have also demonstrated efficacy in the prevention of anemia during treatment with a cyclophosphamide, epirubicin, and fluorouracil combination chemotherapy regimen for breast cancer or a platinum-based combination for small cell lung cancer.59, 60 Similar results have also been found with the use of the longer-acting darbepoetin analog in prevention of cancer/chemotherapy-associated anemia.61, 62

A randomized (but not placebo-controlled) evaluation of EPO therapy following allogeneic BMT has been reported in 28 patients with leukemia.63 EPO (100 to 150 U/kg per day) was administered for 30 days posttransplant. EPO therapy significantly accelerated the appearance of reticulocytes and reduced the RBC transfusion requirements (12 units in the control group vs. 4 units in the treatment group); however, median hemoglobin levels were unaffected. Interestingly, patients in the EPO group also demonstrated significantly quicker platelet recovery, which translated into a reduction in the number of platelet transfusions; however, more patients in the control group experienced venoocclusive disease, thus complicating the interpretation. Some preclinical studies indicate that chronic administration of high-dose EPO may produce competition between erythrocytic and megakaryocytic cell lines, resulting in thrombocytopenia.64 Subsequent randomized trials in the allogeneic and autologous BMT settings have failed to show a substantial prophylactic benefit from EPO administration.65, 66

Figure 35.6 Feedback control of erythropoietin and erythrocyte production. (BFU-E, burst-forming unit, erythroid; CFU- E, colony-forming unit, erythroid.) (Reproduced with permission from Erslev AJ. Erythropoietin. N Engl J Med 1991;324: 1339–1344.)

TABLE 35.4 RESULTS OF RANDOMIZED, PLACEBO-CONTROLLED TRIAL OF EPO IN PATIENTS WITH MALIGNANCIES

 

EPO

Placebo

I

II

III

I

II

III

Number of patients

63

79

64

55

74

61

Change in HCT (mg/dL)

+2.8a

+6.9a

+6.0a

–0.1

+1.1

+1.3

Correction of anemia (% with HCT (38)

20.6a

40.5a

35.9a

3.6

4.1

1.6

Patients transfused (%)

33.3

40.5

53.1

38.2

48.6

68.9

Mean transfusions per patient

1.52

2.03

3.56

2.19

2.75

4.01

HCT, hematocrit.
Patients in group I were not receiving chemotherapy. Patients in group II received nonplatinum-based chemotherapy. Patients in group III underwent chemotherapy regimens that included cisplatin.
aValues significantly different from placebo group.
Reproduced with permission from Abels RI. Use of recombinant human erythropoietin in the treatment of anemia in patients who have cancer. Semin Oncol 1992;19(Suppl 8):2935.

Guidelines suggest a trigger hemoglobin value of 10 g/dL for the initiation of EPO; however, comorbid factors such as cardiac disease should be taken into account and may indicate a lower target.67 A 6- to 8-week trial of EPO-based therapy is typically sufficient for the assessment of response (1 to 2 g/dL rise in hemoglobin).

A variety of EPO dosing schedules have been evaluated. Studies conducted in healthy volunteers suggest that the thrice weekly subcutaneous regimen provides better erythropoietic response than the same total dose given once weekly;68 however, the initial report of a large, open-label, once weekly EPO regimen in cancer patients did demonstrate efficacy.69 The most common regimen in current use for cancer patients is 40,000 units per week administered subcutaneously.

Darbepoetin was initially approved as a once weekly injection. However, subsequent data support extending the interval to every 2 weeks (200 µg subcutaneously), and this is the most common darbepoetin regimen in use today. Further extension of the dosing interval is being explored, along with more complicated regimens (front-loading). The goal of the latter trials is to accelerate the hemoglobin response by use of weekly darbepoetin administration and then extend the interval to every 3 weeks or longer once a target hemoglobin is achieved.

Granulocyte Colony-Stimulating Factor

Clinical data from phase I/II studies of G-CSF or GM-CSF have been reviewed.70 Many randomized, placebo-controlled phase III clinical trials have evaluated the efficacy of using G-CSF as a prophylactic for febrile neutropenia following myelosuppressive chemotherapy (Table 35.5).71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82 In general, these trials have demonstrated a significant acceleration of neutrophil recovery using an HGF, with some trials leading to a reduction in hospitalization for neutropenic fever (Fig. 35.7). One must realize that chemotherapy regimens expected to produce substantial myelosuppression were selected for many of these studies in order to optimize potential differences between the groups. In some trials, prophylaxis with G-CSF also improved other events related to myelosuppression, such as infections and mucositis.

Most published HGF clinical trials were not designed to evaluate differences achieved in chemotherapy dose intensity. Nonrandomized and randomized studies have demonstrated an increased ability to give chemotherapy cycles on the planned time schedule with use of G-CSF or GM-CSF prophylaxis.83, 84, 85, 86, 87Some phase I studies of new cytotoxic agents with myelosuppression as the dose-limiting toxicity have been successful in achieving additional dose escalations with the aid of prophylactic HGFs.88 The European randomized study of filgrastim with CAE (cyclophosphamide, Adriamycin, etoposide) in small cell lung cancer75 differed from the US trial72 in that it did not allow patients on the placebo arm to receive filgrastim if they became neutropenic and febrile on a previous cycle. This enabled evaluation of the effect of filgrastim on the dose intensity of CAE. Twenty-nine percent of patients treated with filgrastim had chemotherapy doses reduced secondary to myelosuppression, compared with 61% of patients receiving placebo. While this difference was statistically significant, the median percentage of the prescribed dose given (mg/m2 per week) was approximately 88% for the patients on placebo, compared with 96% for patients treated with filgrastim. The results of this and other studies89 demonstrate that HGFs alone will probably not allow substantial increases in dose intensity unless some type of cellular support is incorporated into the regimen. However, it does appear that HGFs may facilitate compression of chemotherapy schedules (i.e., improve dose density).90

TABLE 35.5 RESULTS OF RANDOMIZED, PLACEBO-CONTROLLED CLINICAL TRIALS OF G-CSF OR GM-CSF PROPHYLAXIS AFTER CHEMOTHERAPY IN PATIENTS NOT RECEIVING HEMATOPOIETIC CELLULAR SUPPORT

Reference

Cancer Diagnosis

Chemotherapy

CSF Given

N

N+

H+

Ab+

ID

71

Leukemias

ME

G-CSF (E. coli)

108

NR

NR

72

SCLC

CAE

G-CSF (E. coli)

211

NR

73

Urogenital

Various

G-CSF (E. coli)

77

NR

NR

NR

75

SCLC

CAE

G-CSF (E. coli)

130

NC

74

NHL

VAPEC-B

G-CSF (E. coli)

80

NC

NC

NC

76

Pediatric ALL

Standard + VP16/ARAC

G-CSF (E. coli)

164

NC

77

Solid tumors, lymphoma

Various

G-CSF (E. coli)a

138

NC

NC

NC

78

Breast

FEC

G-CSF (CHO)

120

79

AML

DNM, ARA-C

G-CSF (CHO)

173

NR

NR

NC

80

AML (>54 yr)

DNM, ARA-C

G-CSF (E. coli)

234

NC

NC

81

AML

DNM, ARA-C, VP-16

G-CSF (E. coli)

521

NC

129

NHL

COP-BLAM

GM-CSF (E. coli)

182

128

Ovarian

CC

GM-CSF (CHO)

15

NR

NR

NR

130

NSCLC

MVP

GM-CSF (NR)

52

NR

NR

NR

131

AML (>59 yr)

DNM, ARA-C

GM-CSF (E. coli)

388

NC

NR

NC

133

AML (>55 yr)

DNM, ARA-C

GM-CSF (yeast)

124

NC

NR

132

Breast

FAC

GM-CSF (yeast)

142

NC

NR

NR

↓, decreased; Ab+, use of antibiotics; ALL, acute lymphoblastic leukemia; AML, acute myelogenous leukemia; ARA-C, cytosine arabinoside; BLAM, bleomycin sulfate, doxorubicin hydrochloride, and procarbazine; CAE, cyclophosphamide, doxorubicin hydrochloride, and etoposide; CC, carboplatin and cyclophosphamide; COP, cyclophosphamide, vincristine sulfate, prednisone; CSF, colony-stimulating factor; DNM, daunomycin; FAC, 5-fluorouracil, doxorubicin hydrochloride, and cyclophosphamide; FEC, fluorouracil, etoposide, and cisplatin; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; H+, duration of hospitalization; ID, frequency of infectious complications; ME, behenoylcytosine arabinoside and etoposide; MVP, mitomycin, vinblastine sulfate, and cisplatin; N, number of patients; N+, duration of neutropenia; NC, no significant change; NHL, non-Hodgkin's lymphoma; NR, not reported or no data provided regarding statistical significance; NSCLC, non–small cell lung cancer; SCLC, small cell lung cancer; VAPEC-B, vincristine, doxorubicin, prednisolone, etoposide, cyclophosphamide, bleomycin; VP-16, etoposide.
aCSF initiated when absolute neutrophil count was <500/mm3

A randomized, open-label study of filgrastim during part of the induction chemotherapy (cyclophosphamide, cytarabine, methotrexate, 6-MP) for 76 patients with acute lymphoblastic leukemia (ALL) found substantial improvement in the duration of neutropenia, fewer interruptions in the chemotherapy schedule, and no change in disease-free survival at a median of 20 months follow-up.91 Interestingly, a randomized study of lenograstim as part of induction chemotherapy (cytarabine plus idarubicin or amsacrine) in 640 patients with AML found improvements in disease-free survival among patients who were on lenograstim and had standard-risk disease.92

Figure 35.7 Neutrophil concentrations in 129 patients randomly assigned to receive either granulocyte colony-stimulating factor (G-CSF) or placebo after cyclophosphamide, doxorubicin hydrochloride, and etoposide (CAE) chemotherapy. (Reproduced with permission from Trillet-Lenoir V, Green J, Manegold C, et al. Recombinant granulocyte colony stimulating factor reduces the infectious complications of cytotoxic chemotherapy. Eur J Cancer 1993;29A:319–324.)

The most common administration technique for filgrastim (and one that is approved by the FDA) is to initiate the drug approximately 24 hours following chemotherapy. Some relatively small trials have attempted to delay the initiation for several days, without obvious negative effects.93 A single administration of pegfilgrastim (100 mcg/kg or 6 mg total dose) following chemotherapy appears to be equivalent to the full 10- to 14-day course of daily (5 mcg/kg) filgrastim injections, based on randomized, double-blind trials.94, 95 Delay in the institution of filgrastim prophylaxis for afebrile patients until the ANC is 500/mm3 or lower has been shown to shorten the period of neutropenia but not change the rate or duration of hospitalization.77

Use of G-CSF (or placebo) to treat febrile neutropenia was assessed in a double-blind, randomized trial of 218 patients who were also receiving standard antibiotic therapy. Use of G-CSF was associated with an acceleration of neutrophil recovery and a shortening in the duration of neutropenic fever; however, the overall hospitalization duration was unaffected.96

G-CSF has been used in several phase I, II, and III trials for acceleration of hematopoietic recovery following stem cell transplantation (Table 35.6).97, 98, 99,100 In general, these trials indicate that G-CSF administered following high-dose chemotherapy and autologous bone marrow transplantation will improve the rate of peripheral blood neutrophil recovery once it begins, but G-CSF has no substantial effect on the approximately 8 day period of absolute leukopenia or on platelet recovery. The proportion of patients with febrile neutropenia has not been substantially altered, although the number of days of febrile neutropenia has been reduced with G-CSF. Studies have also administered G-CSF for several days before BMT in order to produce high concentrations of progenitor cells in the peripheral blood (PBPCs) for subsequent reinfusion.101, 102, 103 The premise is that the PBPCs will express receptors for terminally acting HGFs (such as G-CSF) and thus be immediately able to proliferate, providing some neutrophils during the 8-day period of absolute leucopenia. G-CSF is the most common cytokine utilized to generate in vivo production of PBPCs, either alone or following administration of myelosuppressive standard-dose chemotherapy (Table 35.6). A somewhat unexpected benefit of this therapy is a reduction in platelet transfusion requirements. Such effects may be explained in part by a dose-dependent increase in the early peripheral blood myeloid progenitors (CD34+) noted in the PBPC product.

Administration of G-CSF following autologous PBPC reinfusion aids engraftment.104, 105, 106, 107, 108 Most of the original investigations initiated HGF therapy immediately following administration of the bone marrow; however, some studies have suggested that a 5- to 6-day delay in G-CSF use may achieve similar efficacy.109, 110 Caution should be exercised in translating these data to situations where progenitor cells are being administered, due to their quicker hematopoietic reconstitution and potentially different interactions with HGFs; however, both similar or conflicting results have been found in other trials.111,112, 113 Use of G-CSF following allogeneic transplantation is controversial. A large retrospective evaluation and a meta-analysis agree that HGFs accelerate myeloid recovery. However, some data suggest a detrimental effect on platelet recovery and graft versus host disease.114, 115 Effects on the latter could vary depending on the stem cell source.116, 117

Other controversial issues being studied include the optimal timing of leukapheresis and the concomitant use of other cytokines to aid trilineage engraftment. HGF-primed PBPCs have also been used to compress the schedule of multicyclic cytotoxic chemotherapy regimens such as those involving treatment with ifosfamide, carboplatin, and etoposide (ICE).118

Determining the optimal schedule, dose, and combination of G-CSF with other cytokines is a focus of interest among institutions. A randomized chemotherapy-based priming study evaluated sequential administration of GM-CSF followed by G-CSF compared with either agent alone. The G-CSF–containing arms yielded better mobilization and more beneficial outcomes (less transfusions, myelosuppression, etc.) compared with the GM-CSF–treated patients.119 A combination of G-CSF with cyclophosphamide is routinely used to increase the generation of CD34+ cells; however, a randomized study found greater efficacy and less toxicity with G-CSF alone in patients with multiple myeloma.120 A randomized, crossover study of progenitor cell mobilization with chemotherapy (high-dose cyclophosphamide) plus G-CSF versus GM-CSF plus G-CSF demonstrated more efficient generation of progenitors with the chemotherapy combination.121Several studies have shown that combining G-CSF with the early acting cytokine SCF results in more efficient progenitor cell mobilization and reduces apheresis requirements.122

A wide variety of G-CSF doses and administration techniques have been used in clinical trials. One randomized study of over 100 patients was not able to demonstrate differences in filgrastim doses of 5 versus 10 µg/kg per day for PBPC mobilization.123 The most common filgrastim dose for prophylaxis of myelosuppression following standard or high-dose chemotherapy is 5 µg/kg per day. Lower doses of lenograstim (2 µg/kg per day) appear to have efficacy similar to that of standard doses (5 µg/kg per day).124 A crossover study of filgrastim (E. coli-derived) and lenograstim (CHO-derived) suggests higher hematopoietic activity of the former with equimolar dosing.125 Discontinuation of G-CSF during its use in the recovery phase following myelosuppressive chemotherapy will typically result in a rebound depression of the WBC count by approximately 50% within 24 hours. Pegfilgrastim can be given as a fixed dose (6 mg) once per chemotherapy cycle. The current labeling indicates that the drug should be given 24 hours after chemotherapy and at least 14 days before the next cycle of chemotherapy. Studies are ongoing to evaluate administration of pegfilgrastim on the same day as select chemotherapy.

TABLE 35.6 RESULTS OF RANDOMIZED CLINICAL TRIALS OF G-CSF OR GM-CSF IN ASSOCIATION WITH HIGH-DOSE CHEMOTHERAPY AND STEM CELL TRANSPLANTATION

Reference

Cancer Type

Stem Cell Source

CSF Given Post Cells

N

WBC Recovery

Days ANC >500 vs. Control

PLT Recovery

Fever

136

Leukemia

Allo-BM

GM-CSF (CHO)

20a

3

NC

137

Lymphoma

Auto-BM

GM-CSF (Yeast)

128a

+

7

NC

NC

138

Lymphoma/ALL

Auto-BM

GM-CSF (E. coli)

81a

+

13

NC

NC

139

Lymphoma

Auto-BM

GM-CSF (E. coli)

61a

+

7

NC

NC

140

Lymphoma

Auto-BM

GM-CSF (E. coli)

69a

+

4

NC

NR

141

Leukemia/etc.

Allo-BM

GM-CSF (CHO)

57a

+

4

NC

NC

142

NHL

Auto-BM

GM-CSF (E. coli)

91a

+

7

NC

NC

143

Hodgkin's

Auto-BM

GM-CSF (E. coli)

24a

+

NR

+

NR

144

Various

Allo-BM

GM-CSF (Yeast)

109a

+

4

NC

NC

145

Various

Allo-BM

GM-CSF (E. coli)

53a

+

4

NC

NC

97

NR

Allo-BM

G-CSF (CHO)

53a

+

10

NR

NR

100

Various

Allo-PBPC

G-CSF (E. coli)

54a

+

4

NC

NR

117

Various

Allo-PBPC

G-CSF (E. coli)

42a

+

3

NC

NR

99

Lymphoma

Auto-BM

G-CSF (E. coli)

43

+

8

NC

98

Lymphoma

Auto-BM

G-CSF (E. coli)

54

+

7

NC

NC

150

Various

Auto-PBPC

GM-CSF E. coli)

50a

NC

NR

NC

105

Various

Auto-PBPC

G-CSF (NR)

41

+

5.5

NC

NC

108

Lymphoma

Auto-PBPC

G-CSF (E. coli)

23

+

3.5

NC

NC

106

Various (peds)

Auto-PBPC

G-CSF (E. coli)

63

+

1

NC

NC

107

Lymphoma/myeloma

Auto-PBPC

G-CSF (E. coli)

38a

+

4

NC

NC

174

Various

Auto-PBPC

G-CSF (NR) + GM-CSF (NR)

37a

+

6

NC

NC

+, augmented;–, worsened; ↑, increased; ↓, decreased; ALL, acute lymphoblastic leukemia; allo-BM, allogeneic bone marrow transplant; ANC, absolute neutrophil count; auto-BM, autologous bone marrow transplant; auto-PBPC, autologous peripheral blood progenitor cells; CHO, Chinese hamster ovary; CSF, colony-stimulating factor; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; NC, no significant change; NHL, non-Hodgkin's lymphoma; NR, not reported or no data provided regarding statistical significance; PLT, platelet; WBC, white blood cell count.
aPlacebo-controlled.

Some evidence suggests that use of G-CSF in children with ALL during intensive therapy with etoposide may be associated with a higher frequency of secondary myeloid malignancies.126 This potential effect should be prospectively evaluated as trials attempt to increase dose density with use of growth factors in other diseases. Other adverse effects thought secondary to G-CSF administration have been fairly mild in the clinical trials reported. Bone pain is the most frequent complaint, often occurring near the time of maximal hematopoiesis. Concurrent administration of G-CSF with cycle-specific chemotherapy may paradoxically worsen myelosuppressive effects.127

Granulocyte-Macrophage Colony-Stimulating Factor

GM-CSF has been used in numerous phase I, II, and III trials following standard doses of myelosuppressive chemotherapy, where it does seem to reduce the occurrence of febrile neutropenia (Table 35.5).128, 129, 130, 131, 132 A large phase III placebo-controlled study of sargramostim as prophylaxis for neutropenic fever was conducted in elderly patients with AML. In addition to the benefit of enhanced neutrophil recovery and reduction of infections, the sargramostim-treated group also displayed significantly longer survival.133

A randomized, placebo-controlled study that evaluated the initiation of molgramostim in patients only at the onset of chemotherapy-induced febrile neutropenia could not demonstrate substantial clinical or monetary benefit.134

In another randomized, placebo-controlled study of molgramostim, 240 patients with AML were given the cytokine (or placebo) during and after induction chemotherapy (idarubicin plus cytarabine).135 Treatment with GM-CSF shortened the time to neutrophil recovery and improved disease-free survival in those 55 to 64 years old.

Randomized, placebo-controlled clinical trials involving GM-CSF have also been conducted in the setting of high-dose chemotherapy with stem cell support (Table 35.6).136, 137, 138, 139, 140, 141, 142, 143, 144, 145 The results demonstrate that GM-CSF has an ability to accelerate neutrophil recovery after a few days of absolute leukopenia when bone marrow alone is used as the sole stem cell source (Fig. 35.8). Patients who were previously exposed to drugs that deplete stem cells (e.g., carmustine or busulfan) experienced less benefit from GM-CSF.146 Most studies could not discern an effect of the HGF on the rate of documented bacterial infections. The impact of GM-CSF on platelet recovery is inconsistent, and use of GM-CSF has generally not solved the problem of transfusion dependence. GM-CSF therapy has shown no significant effect on the incidence or severity of graft versus host disease in patients receiving allogeneic transplants.

As with G-CSF, a number of nonrandomized and randomized trials have found GM-CSF (alone or following chemotherapy) useful to prime PBPCs for subsequent leukapheresis.101, 147, 148, 149, 150 Kritz et al. randomly assigned patients to receive GM-CSF–primed PBPCs or no cellular support following cytotoxic chemotherapy, with autologous marrow rescue given if needed on day 15. The study was stopped early due to a substantial difference in myeloid and platelet recovery between the groups, in favor of the PBPC-treated patients.151 While this study was small and had short follow-up, it demonstrated the potential ability of GM-CSF to improve hematopoiesis through PBPCs alone.

Figure 35.8 Neutrophil concentrations in 129 patients randomly assigned to receive either granulocyte-macrophage colony-stimulating factor (GM-CSF) or placebo after high-dose chemotherapy with autologous bone marrow transplantation. (Reproduced with permission from Nemunaitis J, Rabinowe SN, Singer JW, et al. Recombinant granulocyte-macrophage colony-stimulating factor after autologous bone marrow transplantation for lymphoid cancer. N Engl J Med 1991;324:1773–1778.)

Filgrastim and molgramostim appear to produce similar yields of CD34+ PBPCs when used in conjunction with chemotherapy for priming. However, filgrastim may shorten this process.152 Caballero et al. randomly assigned 42 patients with breast cancer to receive open-label filgrastim or molgramostim following STAMP I or V high-dose chemotherapy and autologous, filgrastim-stimulated PBPCs.153 All patients also received acetaminophen before the HGF doses and prophylactic antibiotics. The only differences noted between the arms were slightly faster platelet recovery and shorter hospitalization (by 2 days) in the filgrastim arm.

Dose-related adverse effects with GM-CSF include capillary leak syndrome, central vein thrombosis, and hypotension. Effects seen over a variety of doses include fever, pleuritis, myalgia, bone pain, pulmonary infiltrates, rash, and thrombophlebitis. Some patients have experienced a syndrome of transient hypoxia and hypotension following the first dose but not subsequent doses of GM-CSF.154 Most randomized studies that used the standard dose (250 mcg/m2per day) and the currently marketed sargramostim version (vs. molgramostim) have shown only mild adverse effects. GM-CSF is a known inducer of other endogenous cytokines, which are thought to account for at least some of the adverse effects. As with G-CSF, the simultaneous administration of GM-CSF and cycle-specific chemotherapy or radiation therapy has worsened myelosuppression.155, 156

Clinical Synopsis of G-CSF AND GM-CSF

Introduction of G-CSF, and to a lesser extent GM-CSF, into routine clinical practice has profoundly influenced the treatment of chronic neutropenias and the generation of PBPCs for collection and subsequent reinfusion following high-dose chemotherapy. However, the most frequent use of these agents has been in prophylaxis of chemotherapy-induced neutropenic fever, an indication for which the outcome data are less clear. There is still not much evidence that administration of an HGF improves disease-free or overall survival, even for tumor types typically thought to be chemosensitive. Thus, routine HGF use (i.e., as primary prophylaxis) is not justified for most patients. Attempts to define the populations that benefit from HGFs are underway. For example, elderly patients are more likely to have chemotherapy dose reductions, probably as a result of myelosuppression.157 HGFs are being evaluated in this clinical setting. The majority of current HGF prescriptions are for secondary prophylaxis (i.e., written for patients who experienced myelosuppression on a previous cycle of chemotherapy.) There are even less data showing any effect of HGFs on disease-free or overall survival for such an indication. Use of HGFs in the treatment of neutropenic fever is unlikely to improve patient outcome.

Use of HGFs for priming PBPCs does decrease toxicity, both in the priming period and posttransplant, in addition to reducing length of hospital stay and cost of therapy. There is probably some benefit to the administration of HGFs following stem cell infusion; however, the degree of the effect is most likely dependent on the quality and quantity of the cells infused. Older studies that report differences in hospitalization should be interpreted with caution, given the changes in the outpatient treatment of neutropenic fever, even following high-dose chemotherapy.

Interleukin-11

Preclinical and in vitro studies indicate that IL-11 directly stimulates megakaryocytes. Oprelvekin (recombinant IL-11) was the first cytokine to reach the market for the prevention of chemotherapy-induced thrombocytopenia, and this occurred only 3 years after the initial research application. Phase I evaluations in patients with cancer demonstrated an impressive ability to increase steady-state platelet counts by over twofold.158

The ability of recombinant IL-11 to prevent thrombocytopenia was evaluated in a randomized, placebo-controlled trial of 77 patients with breast cancer who had not previously experienced severe chemotherapy-induced thrombocytopenia. Patients received two cycles of doxorubicin/cyclophosphamide followed by G-CSF and study drug on each cycle. Using the limit of 20,000 platelets/µL, 43% of patients on placebo and 30% on IL-11 required a platelet transfusion. The mean number of transfusions was 2.2 for the placebo group versus 0.8 for the IL-11 group.159

A randomized, placebo-controlled evaluation of IL-11 for secondary prophylaxis of thrombocytopenia was conducted in 93 patients with cancer who previously received platelet transfusions for chemotherapy-induced toxicity. As expected, greater than 90% of the patients in the placebo arm required a platelet transfusion, compared with 72% of the IL 11–treated patients. The mean number of transfusions was 3.3 for the placebo group versus 2.2 for the IL-11 group.160

Administration of IL-11 did not appear to substantially alter platelet recovery or transfusion requirements in 80 patients with breast cancer enrolled in a randomized, placebo-controlled study following high-dose chemotherapy and infusion with G-CSF–primed PBPCs.161

Approximately 60% of patients treated with IL-11 experience some degree of generalized edema, thought to be secondary to increased retention of sodium. In addition, atrial arrhythmias, tachycardia, conjunctival injection, and worsening of effusions can occur. Constitutional symptoms such as myalgia, arthralgia, and fatigue were the dose-limiting toxicities during phase I trials.

Thrombopoietin

Murine studies suggested a complex thrombopoietin (TPO) dose versus platelet response curve, perhaps due to inhibitory effects on platelet progenitors at high doses.162 Murine and nonhuman primate studies demonstrated a synergism between TPO and G-CSF in the acceleration of neutrophil and platelet recovery following myelosuppressive chemotherapy, with no evidence of lineage competition.

Clinical studies of recombinant TPO were initially conducted with either the full-length glycosylated TPO molecule (rTPO, Genetech/Pfizer) or a truncated, pegylated derivative (rMGDF, Amgen). A clinical study of rMGDF in 17 patients with cancer (not currently receiving chemotherapy) demonstrated a dose-dependent increase in platelet counts. Those receiving the highest doses achieved from a 51 to 584% increase in platelet counts. The effect was clinically evident following 6 days of therapy, and the counts continued to rise several days after drug discontinuation.163 Platelets generated in these patients appeared to have normal function based on in vitro assays. Similar data were found with the full-length rTPO molecule.164

Two other studies administered recombinant rMGDF to a total of 94 patients with lung cancer both prior to and following a carboplatin/cyclophosphamide regimen. A significant shortening in the time to platelet nadir and a quicker platelet recovery were noted.165, 166 Similar effects were seen in a gynecologic cancer population being treated with carboplatin and the full-length rTPO molecule.167 A placebo-controlled trial of rMGDF in patients being treated for AML was not able to demonstrate an impact on thrombocytopenia.168 Utilization of rMGDF following autologous bone marrow transplantation demonstrated that its thrombopoietic effects were delayed and not impressive.169

rTPO and rMGDF were generally well tolerated in these early studies, with essentially no evidence of dose-limiting adverse effects, nor were any fever or flu-like symptoms discernable; however, a few patients did experience thrombotic events. The role of the study drug in such processes is unclear.

Clinical development of rMGDF was halted when some patients in cancer trials and normal, healthy volunteers given the cytokine began to demonstrate neutralizing antibodies to TPO. A similar phenomenon has occurred when some other recombinant growth factors, molgramostim and PIXY321 (an IL-3/GM-CSF fusion protein), have been used without immunosuppressive chemotherapy, resulting in abrogation of their hematopoietic activities.170, 171 Novel peptide and nonpeptide, potentially less immunogenic molecules are also being investigated.172

REFERENCES

1. Moonen P, Mermod JJ, Ernst JM, et al. Increased biological activity of deglycosylated recombinant human granulocyte/ macrophage colony-stimulating factor produced by yeast or animal cells. Proc Natl Acad Sci 1987;84: 4428–4431.

2. Fukuda M, Sasaki H, Fukuda MN. Erythropoietin metabolism and the influence of carbohydrate structure. Contrib Nephrol 1989;76:8–89.

3. Elliott S, Lorenzini T, Asher S, et al. Enhancement of therapeutic protein in vivo activities through glycoengineering. Nat Biotechnol 2003;21:414–421.

4. Allon M, Kleinman K, Walczyk M, et al. Pharmacokinetics and pharmacodynamics of darbepoetin alfa and epoetin in patients undergoing dialysis. Clin Pharmacol Ther 2002;72:546–555.

5. Zeng SM, Murray JC, Widness JA, et al. Association of single nucleotide polymorphisms in the thrombopoietin-receptor gene, but not the thrombopoietin gene, with differences in platelet count. Am J Hematol 2004;77:12–21.

6. Guillaume T, Sekhavat M, Rubinstein DB, et al. Transcription of genes encoding granulocyte-macrophage colony-stimulating factor, interleukin 3, and interleukin 6 receptors and lack of proliferative response to exogenous cytokines in nonhematopoietic human malignant cell lines. Cancer Res 1993; 53:3139–3144.

7. Arcasoy MO, Amin K, Karayal AF, et al. Functional significance of erythropoietin receptor expression in breast cancer. Lab Invest 2002;82:911–918.

8. Batra S, Perelman N, Luck LR, et al. Pediatric tumor cells express erythropoietin and a functional erythropoietin receptor that promotes angiogenesis and tumor cell survival. Lab Invest 2003;83:1477–1487.

9. Leyland-Jones B; BEST Investigators and Study Group. Breast cancer trial with erythropoietin terminated unexpectedly. Lancet Oncol 2003;4:459–460.

10. Henke M, Laszig R, Rube C, et al. Erythropoietin to treat head and neck cancer patients with anaemia undergoing radiotherapy: randomised, double-blind, placebo-controlled trial. Lancet 2003 18;362:1255–1260.

11. Miyajima A, Mui ALF, Ogorochi T, et al. Receptors for granulocyte-macrophage colony-stimulating factor, interleukin-3, and interleukin-5. Blood 1993;82:1960–1974.

12. Park LS, Urdal DL. Colony-stimulating factor receptors. Trans Proc 1989;21:54–56.

13. Jacobsen SEW, Ruscetti FW, Dubois CM, et al. Induction of colony-stimulating factor receptor expression on hematopoietic progenitor cells: proposed mechanism for growth factor synergism. Blood 1992;80:678–687.

14. Sayani, F, Montero-Julian FA, Ranchin V, et al. Identification of the soluble granulocyte-macrophage colony stimulating factor receptor protein in vivo. Blood 2000;95:461–469.

15. Heaney ML, Golde DW. Soluble hormone receptors. Blood 1993;82:1945–1948.

16. Porteu F, Nathan C. Shedding of tumor necrosis factor receptors by activated human neutrophils. J Exp Med 1990; 172: 599–607.

17. Lieschke GJ, Grail D, Hodgson G, et al. Mice lacking granulocyte colony-stimulating factor have chronic neutropenia, granulocyte and macrophage progenitor cell deficiency, and impaired neutrophil mobilization. Blood 1994;84:1737–1746.

18. Dranoff G, Crawford AD, Sadelain M, et al. Involvement of granulocyte-macrophage colony-stimulating factor in pulmonary homeostasis. Science 1994;264:713–716.

19. Petros WP, Rabinowitz J, Gibbs JP, et al. Effect of endogenous TNF-alpha on recombinant G-CSF stimulated hematopoiesis in mice and humans. Pharmacotherapy 1998;18:816–823.

20. Chatta GS, Andrews RG, Rodger E, et al. Hematopoietic progenitors and aging: alterations in granulocytic precursors and responsiveness to recombinant human G-CSF, GM-CSF, and IL-3. J Gerontol 1993;48:M207–212.

21. Lord BI, Gurney H, Chang J, et al. Haemopoietic cell kinetics in humans treated with rGM-CSF. Int J Cancer 1992;50:26–31.

22. Pettengell R, Testa NG, Swindell R, et al. Transplantation potential of hematopoietic cells released into the circulation during routine chemotherapy for non-Hodgkin's lymphoma. Blood 1993;82:2239–2248.

23. Liu F, Poursine-Laurent J, Link DC. Expression of the G-CSF receptor on hematopoietic progenitor cells is not required for their mobilization by G-CSF. Blood 2000;95:3025–3031.

24. Jaar B, Baillou C, Viron B, et al. Long-term effects of recombinant human erythropoietin on bone marrow progenitor cells. Nephrol Dial Transplant 1993;8:614–620.

25. Arnaout MA, Wang EA, Clark SC, et al. Human recombinant granulocyte-macrophage colony-stimulating factor increases cell-cell adhesion and surface expression of adhesion-promoting surface glycoproteins on mature granulocytes. J Clin Invest 1986;7:597–601.

26. Peters WP, Stuart A, Affronti ML, et al. Neutrophil migration is defective during recombinant human granulocyte-macrophage colony-stimulating factor infusion after autologous bone marrow transplantation in humans. Blood 1988;72:1310–1315.

27. Demir G, Klein HO, Tuzuner N. Low dose daily rhGM-CSF application activates monocytes and dendritic cells in vivo. Leuk Res 2003;27:1105–1108.

28. Gurney AL, Carver-Moore K, de Sauvage FJ, et al. Thrombocytopenia in c-mpl–deficient mice. Science 1995;265: 1445–1447.

29. Graf G, Dehmel U, Drexler HG. Expression of TPO and TPO receptor MPL in human leukemia-lymphoma and solid tumor cell lines. Leuk Res 1996;20:831–838.

30. Choi ES, Hokom MM, Chen JL, et al. The role of megakaryocyte growth and development factor in terminal stages of thrombopoiesis. Br J Haematol 1996;95:227–233.

31. Rabinowitz J, Petros WP, Peters WP. Cytokine kinetics: clinical pharmacology studies complementing recombinant growth factor trials. Cancer Bull 1994;46:40–47.

32. Milsits K, Beyer J, Siegert W. Serum concentrations of G-CSF during high-dose chemotherapy with autologous stem cell rescue. Bone Marrow Transplant 1993;11:372–377.

33. Rabinowitz J, Petros WP, Stuart AR, et al. Characterization of endogenous cytokine concentrations after high-dose chemotherapy with autologous bone marrow support. Blood 1993; 81:2452–2459.

34. Lindemann A, Riedel D, Oster W, et al. Granulocyte-macrophage colony-stimulating factor induces cytokine secretion by human polymorphonuclear leukocytes. J Clin Invest 1989; 83:1308–1312.

35. Hartung T, Docke W-D, Gantner F, et al. Effect of granulocyte colony-stimulating factor treatment on ex vivo blood cytokine response in human volunteers. Blood 1995;85:2482–2489.

36. Pascual JA, Belalcazar V, de Bolos C, et al. Recombinant erythropoietin and analogues: a challenge for doping control. Ther Drug Monit 2004;26:175–179.

37. Rosenfeld CS, Sulecki M, Evans C, et al. Comparison of intravenous versus subcutaneous recombinant human granulocyte-macrophage colony-stimulating factor in patients with primary myelodysplasia. Exp Hematol 1991;19:273–277.

38. Johnston E, Crawford J, Blackwell S, et al. Randomized, dose-escalation study of SD/01 compared with daily filgrastim in patients receiving chemotherapy. J Clin Oncol 2000;18: 2522–2528.

39. Terashi K, Oka M, Ohdo S, et al. Close association between clearance of recombinant human granulocyte colony-stimulating factor (G-CSF) and G-CSF receptor on neutrophils in cancer patients. Antimicrob Agents Chemother 1999;43:21–24.

40. Tomlinson-Jones A, Ziltener HJ. Enhancement of the biologic effects of interleukin-3 in vivo by anti-interleukin-3 antibodies. Blood 1993;82:1133–1141.

41. Petros WP, Rabinowitz J, Stuart AR, et al. Disposition of recombinant human granulocyte-macrophage colony-stimulating factor in patients receiving high-dose chemotherapy and autologous bone marrow support. Blood 1992;80:1135–1140.

42. Fukuda M, Sasaki H, Fukuda MN. Erythropoietin metabolism and the influence of carbohydrate structure. Contrib Nephrol 1989;76:78–89.

43. Hovgaard D, Mortensen BT, Schifter S, et al. Comparative pharmacokinetics of single-dose administration of mammalian and bacterially-derived recombinant human granulocyte-macrophage colony-stimulating factor. Eur J Haematol 1993; 50:32–36.

44. O'Dwyer PJ, LaCreta FP, Schilder R, et al. Phase I trial of thiotepa in combination with recombinant human granulocyte-macrophage colony-stimulating factor. J Clin Oncol 1992;10: 1352–1358.

45. Petros WP, Crawford J. Safety of concomitant use of granulocyte colony-stimulating factor or granulocyte-macrophage colony-stimulating factor with cytotoxic chemotherapy agents. Curr Opinion Hematol 1997;4:213–216.

46. Tjan-Heijnen VCG, Biesma B, Festen J, et al. Enhanced myelotoxicity due to granulocyte colony-stimulating factor administration until 48 hours before the next chemotherapy course in patients with small-cell lung carcinoma. J Clin Oncol 1998; 16:2708–27014.

47. Beguin Y, Yerna M, Loo M, et al. Erythropoiesis in multiple myeloma: defective red cell production due to inappropriate erythropoietin production. Br J Haematol 1992;82:648–653.

48. Miller CB, Jones RJ, Piantadosi S, et al. Decreased erythropoietin response in patients with the anemia of cancer. N Engl J Med 1990;322:1689–1692.

49. Fandrey J, Seydel FP, Siegers CP, et al. Role of cytochrome P450 in the control of the production of erythropoietin. Life Sci 1990;47:127–134.

50. Smith DH, Goldwasser E, Volkes EE. Serum immunoerythropoietin levels in patients with cancer receiving cisplatin-based chemotherapy. Cancer 1991;68:1101–1105.

51. Birgegard G, Wide L, Simonsson B. Marked erythropoietin increase before fall in Hb after treatment with cytostatic drugs suggests mechanism other than anaemia for stimulation. Br J Haematol 1989;72:462–466.

52. Schapira M, Antin JH, Ransil BJ, et al. Serum erythropoietin levels in patients receiving intensive chemotherapy and radiotherapy. Blood 1990;76:2354–2359.

53. Osterborg A, Boogarets MA, Cimino R, et al. Recombinant human erythropoietin in transfusion-dependent anemic patients with multiple myeloma and non-Hodgkin's lymphoma: a randomized multicenter study. Blood 1996; 87:2675–2682.

54. Ludwig H, Fritz E, Leitgeb C, et al. Prediction of response to erythropoietin treatment in chronic anemia of cancer. Blood 1994;84:1056–1063.

55. Auerbach M, Ballard H, Trout JR, et al. Intravenous iron optimizes the response to recombinant human erythropoietin in cancer patients with chemotherapy-related anemia: a multicenter, open-label, randomized trial. J Clin Oncol 2004; 22: 1301–1307.

56. Abels RI. Use of recombinant human erythropoietin in the treatment of anemia in patients who have cancer. Semin Oncol 1992;19(Suppl 8):29–35.

57. Case DC, Bukowski RM, Carey RW, et al. Recombinant human erythropoietin therapy for anemic cancer patients on combination chemotherapy. J Natl Cancer Inst 1993;85:801–806.

58. Cascinu S, Fedeli A, Del Ferro E, et al. Recombinant human erythropoietin treatment in cisplatin-associated anemia: a randomized, double-blind trial with placebo. J Clin Oncol 1994; 12:1058–1062.

59. Del Mastro L, Venturini M, Lionetto R, et al. Randomized phase III trial evaluating the role of erythropoietin in the prevention of chemotherapy-induced anemia. J Clin Oncol 1997; 15:2715–2721.

60. De Campos E, Radford J, Steward W, et al. Clinical and in vitro effects of recombinant human erythropoietin in patients receiving intensive chemotherapy for small-cell lung cancer. J Clin Oncol 1995;13:1623–1631.

61. Hedenus M, Adriansson M, San Miguel J, et al. Efficacy and safety of darbepoetin alfa in anaemic patients with lymphoproliferative malignancies: a randomized, double-blind, placebo-controlled study. Darbepoetin Alfa 20000161 Study Group. Br J Haematol 2003;122:394–403.

62. Vansteenkiste J, Pirker R, Massuti B, et al. Double-blind, placebo-controlled, randomized phase III trial of darbepoetin alfa in lung cancer patients receiving chemotherapy. Aranesp 980297 Study Group. J Natl Cancer Inst 2002;94:1211–1220.

63. Steegmann JL, Lopez J, Otero MJ, et al. Erythropoietin treatment in allogeneic BMT accelerates erythroid reconstitution: results of a prospective controlled randomized trial. Bone Marrow Transplant 1992;10:541–546.

64. McDonald TP, Clift RE, Cottrell MB. Large, chronic doses of erythropoietin cause thrombocytopenia in mice. Blood 1992; 80:352–358.

65. Biggs JC, Atkinson KA, Booker V, et al. Prospective randomised double-blind trial of the in vivo use of recombinant human erythropoietin in bone marrow transplantation from HLA-identical sibling donors. Bone Marrow Transplant 1995;15:129–134.

66. Chao NJ, Schriber JR, Long GD, et al. A randomized study of erythropoietin and granulocyte colony-stimulating factor (G CSF) versus placebo and G-CSF for patients with Hodgkin's and non-Hodgkin's lymphoma undergoing autologous bone marrow transplantation. Blood 1994; 83:2823–2828.

67. Rizzo JD, Lichtin AE, Woolf SH, et al. Use of epoetin in patients with cancer: evidence-based clinical practice guidelines of the American Society of Clinical Oncology and the American Society of Hematology. J Clin Oncol. 2002; 20:4083–4107.

68. Cheung WK, Goon BL, Guilfoyle MC, et al. Pharmacokinetics and pharmacodynamics of recombinant human erythropoietin after single and multiple subcutaneous doses to healthy subjects. Clin Pharmacol Ther 1998;64:412–423.

69. Gabrilove JL, Cleeland CS, Livingston RB, et al. Clinical evaluation of once-weekly dosing of epoetin alfa in chemotherapy patients: improvements in hemoglobin and quality of life are similar to three-times-weekly dosing. J Clin Oncol 2001; 19:2875–2882.

70. Lieschke GJ, Burgess AW. Granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor [pt. 2]. N. Engl J Med 1992;327:99–106.

71. Ohno R, Tomonaga M, Kobayashi T, et al. Effect of G-CSF after intensive induction therapy in relapsed or refractory acute leukemia. N Engl J Med 1990;323:871–877.

72. Crawford J, Ozer H, Stoller R, et al. Reduction by granulocyte colony-stimulating factor of fever and neutropenia induced by chemotherapy in patients with small-cell lung cancer. N Engl J Med 1991;325:164–170.

73. Kotake T, Miki T, Akaza H, et al. Effect of recombinant granulocyte colony-stimulating factor on chemotherapy-induced neutropenia in patients with urogenital cancer. Cancer Chemother Pharmacol 1991;27:2553–2557.

74. Pettengell R, Gurney H, Radford JA, et al. Granulocyte colony-stimulating factor to prevent dose-limiting neutropenia in non-Hodgkin's lymphoma: a randomized controlled trial. Blood 1992;80:1430–1436.

75. Trillet-Lenoir V, Green J, Manegold C, et al. Recombinant granulocyte colony stimulating factor reduces the infectious complications of cytotoxic chemotherapy. Eur J Cancer 1993;29A: 319–324.

76. Pui C-H, Boyett JM, Hughes WT, et al. Human granulocyte colony-stimulating factor after induction chemotherapy in children with acute lymphoblastic leukemia. N Engl J Med 1997; 336:1781–1787.

77. Hartmann LC, Tschetter LK, Habermann TM, et al. Granulocyte colony-stimulating factor in severe chemotherapy induced afebrile neutropenia. N Engl J Med 1997;336:1776–1780.

78. Chevallier B, Chollet P, Merrouche Y, et al. Lenograstim prevents morbidity from intensive induction chemotherapy in the treatment of inflammatory breast cancer. J Clin Oncol 1995; 13:1564–1571.

79. Dombret H, Chastang C, Fenaux P, et al. A controlled study of recombinant human granulocyte colony-stimulating factor in elderly patients after treatment for acute myelogenous leukemia. N Engl J Med 1995;332:1678–1683.

80. Godwin JE, Kopecky KJ, Head DR, et al. A double-blind placebo-controlled trial of granulocyte colony-stimulating factor in elderly patients with previously untreated acute myeloid leukemia: a Southwest Oncology Group study (9031). Blood 1998;91: 3607–3615.

81. Heil G, Hoelzer D, Sanz MA, et al. A randomized, double-blind, placebo-controlled, phase III study of filgrastim in remission induction and consolidation therapy for adults with de novo acute myeloid leukemia. Blood 1997; 90:4710–4713.

82. Harousseau JL, Witz B, Lioure B, et al. Granulocyte colony-stimulating factor after intensive consolidation chemotherapy in acute myeloid leukemia: results of a randomized trial of the Groupe Ouest-Est Leucemies Aigues Myeloblastiques. J Clin Oncol 2000;18:780–787.

83. Gabrilove JL, Jakubowski A, Scher H, et al. Effect of granulocyte colony-stimulating factor on neutropenia and associated morbidity due to chemotherapy for transitional cell carcinoma of the urothelium. N Engl J Med 1988;318:1414–1422.

84. Bronchud MH, Howell A, Crowther D, et al. The use of granulocyte colony-stimulating factor to increase the intensity of treatment with doxorubicin in patients with advanced breast and ovarian cancer. Br J Cancer 1989;60:121–125.

85. Scinto AF, Ferraresi V, Campioni N, et al. Accelerated chemotherapy with high-dose epirubicin and cyclophosphamide plus r-met-HUG-CSF in locally advanced and metastatic breast cancer. Ann Oncol 1995; 6:665–671.

86. Piccart MJ, Bruning P, Wildiers J, et al. An EORTC pilot study of filgrastim (recombinant human granulocyte colony stimulating factor) as support to a high dose-intensive epiadriamycin-cyclophosphamide regimen in chemotherapy-naïve patients with locally advanced or metastatic breast cancer. Ann Oncol 1995;6:673–677.

87. Michel G, Landman-Parker J, Auclerc MF, et al. Use of recombinant human granulocyte colony-stimulating factor to increase chemotherapy dose-intensity: a randomized trial in very high-risk childhood acute lymphoblastic leukemia. J Clin Oncol 2000;18:1517–1524.

88. Rowinsky EK, Grochow LB, Sartorius SE, et al. Phase I and pharmacologic study of high doses of the topoisomerase I inhibitor topotecan with granulocyte colony-stimulating factor in patients with solid tumors. J Clin Oncol 1996;14:1224–1235.

89. Doorduijn JK, van der Holt B, van Imhoff GW, et al. CHOP compared with CHOP plus granulocyte colony-stimulating factor in elderly patients with aggressive non-Hodgkin's lymphoma. J Clin Oncol 2003;21:3041–3050.

90. Citron ML, Berry DA, Cirrincione C, et al. Randomized trial of dose-dense versus conventionally scheduled and sequential versus concurrent combination chemotherapy as postoperative adjuvant treatment of node-positive primary breast cancer: first report of Intergroup Trial C9741/Cancer and Leukemia Group B Trial 9741. J Clin Oncol 2003;21:1431–1439.

91. Ottmann OG, Hoelzer D, Gracien E, et al. Concomitant granulocyte colony-stimulating factor and induction chemoradiotherapy in adult acute lymphocytic leukemia: a randomized phase III trial. Blood 1995;86:444–450.

92. Lowenberg B, van Putten W, Theobald M, et al. Effect of priming with granulocyte colony-stimulating factor on the outcome of chemotherapy for acute myeloid leukemia. Dutch-Belgian Hemato-Oncology Cooperative Group; Swiss Group for Clinical Cancer Research. N Engl J Med 2003;349:743–752.

93. Rahiala J, Perkkio M, Riikonen P. Prospective and randomized comparison of early versus delayed prophylactic administration of granulocyte colony-stimulating factor (filgrastim) in children with cancer. Med Pediatr Oncol 1999;32:326–330.

94. Green MD, Koelbl H, Baselga J, et al. A randomized double-blind multicenter phase III study of fixed-dose single-administration pegfilgrastim versus daily filgrastim in patients receiving myelosuppressive chemotherapy. International Pegfilgrastim 749 Study Group. Ann Oncol 2003;14:29–35.

95. Holmes FA, O'Shaughnessy JA, Vukelja S, et al. Blinded, randomized, multicenter study to evaluate single administration pegfilgrastim once per cycle versus daily filgrastim as an adjunct to chemotherapy in patients with high-risk stage II or stage III/IV breast cancer. J Clin Oncol 2002;20:727–731.

96. Mahr DW, Lieschke GJ, Green M, et al. Filgrastim in patients with chemotherapy-induced febrile neutropenia. Ann Intern Med 1994;121:492.

97. Asano S, Masaoka T, Takaku F. Beneficial effect of human glycosylated granulocyte colony-stimulating factor in marrow-transplanted patients: results of multicenter phase II-III studies. Transplant Proc 1991;23:1701–1703.

98. Schmitz N, Dreger P, Zander AR, et al. Results of a randomized, controlled, multicentre study of recombinant human granulocyte colony-stimulating factor (filgrastim) in patients with Hodgkin's disease and non-Hodgkin's lymphoma undergoing autologous bone marrow transplantation. Bone Marrow Transplant 1995;15:261–266.

99. Stahel RA, Jost LM, Cerny T, et al. Randomized study of recombinant human granulocyte colony-stimulating factor after high-dose chemotherapy and autologous bone marrow transplantation for high-risk lymphoma malignancies. J Clin Oncol 1994;12:1931–1938.

100. Bishop MR, Tarantolo SR, Geller RB, et al. A randomized, double-blind trial of filgrastim (granulocyte colony-stimulating factor) versus placebo following allogeneic blood stem cell transplantation. Blood 2000;96:80–85.

101. Peters WP, Rosner G, Ross M, et al. Comparative effects of granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor on priming peripheral blood progenitor cells for use with autologous bone marrow after high-dose chemotherapy. Blood 1993;81:1709–1719.

102. Chao NJ, Schriber JR, Grimes K, et al. Granulocyte colony-stimulating factor “mobilized” peripheral blood progenitor cells accelerate granulocyte and platelet recovery after high-dose chemotherapy. Blood 1993;81:2031–2035.

103. Sheridan WP, Begley CG, Juttner CA, et al. Effect of peripheral-blood progenitor cells mobilised by filgrastim (G-CSF) on platelet recovery after high-dose chemotherapy. Lancet 1992;339:640–644.

104. Shimazaki C, Oku N, Uchiyama H, et al. Effect of granulocyte colony-stimulating factor on hematopoietic recovery after peripheral blood progenitor cell transplantation. Bone Marrow Transplant 1994;13:271.

105. Klumpp TR, Magan KF, Goldberg SL, et al. Granulocyte colony-stimulating factor accelerates neutrophil engraftment following peripheral-blood stem-cell transplantation: a prospective, randomized trial. J Clin Oncol 1995;13:1323–1327.

106. Kawano Y, Takaue Y, Mimaya J, et al. Marginal benefit/disadvantage of granulocyte colony-stimulating factor therapy after autologous blood stem cell transplantation in children: results of a prospective randomized trial. Blood 1998;92:4040–4046.

107. McQuaker IG, Hunter AE, Pacey S, et al. Low-dose filgrastim significantly enhances neutrophil recovery following autologous peripheral-blood stem-cell transplantation in patients with lymphoproliferative disorders: evidence for clinical and economic benefit. J Clin Oncol 1997;15:451–457.

108. Lee SM, Radford JA, Dobson L, et al. Recombinant human granulocyte colony-stimulating factor (filgrastim) following high-dose chemotherapy and peripheral blood progenitor cell rescue in high-grade non-Hodgkin's lymphoma: clinical benefits at no extra cost. Br J Cancer 1998;77:1294–1299.

109. Torres Gomez A, Jimenez MA, Alvarez MA, et al. Optimal timing of granulocyte colony-stimulating factor (G-CSF) administration after bone marrow transplantation: a prospective randomized study. Ann Hematol 1995;71:65–70.

110. Vey N, Molnar S, Faucher C, et al. Delayed administration of granulocyte colony-stimulating factor after autologous bone marrow transplantation: effect on granulocyte recovery. Bone Marrow Transplant 1994;14:779–782.

111. Faucher C, Le Corroller AG, Chabannon C, et al. Administration of G-CSF can be delayed after transplantation of autologous G-CSF–primed blood stem cells: a randomized study. Bone Marrow Transplant 1996;17:533–536.

112. Hornedo J, Sola C, Solano C, et al. The role of granulocyte colony-stimulating factor (G-CSF) in the post-transplant period. SOLTI Group. Bone Marrow Transplant 2002; 29: 737–743.

113. de Azevedo AM, Nucci M, Maiolino A, et al. A randomized, multicenter study of G-CSF starting on day +1 vs day +5 after autologous peripheral blood progenitor cell transplantation. Bone Marrow Transplant 2002;29:745–751.

114. Ho VT, Mirza NQ, Junco D, Okamura T, Przepiorka D. The effect of hematopoietic growth factors on the risk of graft-vs-host disease after allogeneic hematopoietic stem cell transplantation: a meta-analysis. Bone Marrow Transplant 2003;32: 771–775.

115. Ringden O, Labopin M, Gorin NC, et al. Treatment with granulocyte colony-stimulating factor after allogeneic bone marrow transplantation for acute leukemia increases the risk of graft-versus-host disease and death: a study from the Acute Leukemia Working Party of the European Group for Blood and Marrow Transplantation. J Clin Oncol 2004;22:416–423.

116. Morton J, Hutchins C, Durrant S. Granulocyte-colony-stimulating factor (G-CSF)–primed allogeneic bone marrow: significantly less graft-versus-host disease and comparable engraftment to G-CSF-mobilized peripheral blood stem cells. Blood 2001;98:3186–3191.

117. Przepiorka D, Smith TL, Folloder J, et al. Controlled trial of filgrastim for acceleration of neutrophil recovery after allogeneic blood stem cell transplantation from human leukocyte antigen-matched related donors. Blood 2001;97:3405–3410.

118. Pettengell R, Wall P, Thatcher N, et al. Multicyclic, dose-intensive chemotherapy supported by sequential reinfusion of hematopoietic progenitors in whole blood. J Clin Oncol 1995; 13:148–156.

119. Weaver CH, Schulman KA, Wilson-Relyea B, et al. Randomized trial of filgrastim, sargramostim, or sequential sargramostim and filgrastim after myelosuppressive chemotherapy for the harvesting of peripheral-blood stem cells. J Clin Oncol 2000; 18:43–53.

120. Desikan KR, Barlogie B, Jagannath S, et al. Comparable engraftment kinetics following peripheral-blood stem-cell infusion mobilized with granulocyte colony-stimulating factor with or without cyclophosphamide in multiple myeloma. J Clin Oncol 1998;16;1547–1553.

121. Koc ON, Gerson SL, Cooper BW, et al. Randomized cross-over trial of progenitor-cell mobilization: high-dose cyclophosphamide plus granulocyte colony-stimulating factor (G-CSF) versus granulocyte-macrophage colony-stimulating factor plus G-CSF. J Clin Oncol 2000;18:1824–1830.

122. Stiff P, Gingrich R, Luger S, et al. A randomized phase 2 study of PBPC mobilization by stem cell factor and filgrastim in heavily pretreated patients with Hodgkin's disease or non-Hodgkin's lymphoma. Bone Marrow Transplant 2000;26:471–481.

123. Andre M, Baudoux E, Bron D, et al. Phase III randomized study comparing 5 or 10 microg per kg per day of filgrastim for mobilization of peripheral blood progenitor cells with chemotherapy, followed by intensification and autologous transplantation in patients with nonmyeloid malignancies. Transfusion 2003; 43:50–57.

124. Toner GC, Shapiro JD, Laidlaw CR, et al. Low-dose versus standard-dose lenograstim prophylaxis after chemotherapy: a randomized, crossover comparison. J Clin Oncol 1998;16; 3874–3879.

125. Carlsson G, Ahlin A, Dahllof G, et al. Efficacy and safety of two different rG-CSF preparations in the treatment of patients with severe congenital neutropenia. Br J Haematol 2004;126: 127–132.

126. Relling MV, Boyett JM, Blanco JG, et al. Granulocyte colony-stimulating factor and the risk of secondary myeloid malignancy after etoposide treatment. Blood 2003;101:3862–3867.

127. Meropol NJ, Miller LL, Korn EL, et al. Severe myelosuppression resulting from concurrent administration of granulocyte colony-stimulating factor and cytotoxic chemotherapy. J Natl Cancer Inst 1992;84:1201–1203.

128. de Vries, EGE, Biesma B, Willemese PHB, et al. A double-blind placebo-controlled study with granulocyte-macrophage colony-stimulating factor during chemotherapy for ovarian carcinoma. Cancer Res 1991;51:116–122.

129. Gerhartz HH, Engelhard M, Meusers P, et al. Randomized, double-blind, placebo-controlled, phase III study of recombinant human granulocyte-macrophage colony-stimulating factor as adjunct to induction treatment of high-grade malignant non-Hodgkin's lymphomas. Blood 1993;82:2329–2339.

130. Eguchi K, Kabe J, Kudo S, et al. Efficacy of recombinant human granulocyte-macrophage colony-stimulating factor for chemotherapy-induced leukemia in patients with non-small-cell lung cancer. Cancer Chemother Pharmacol 1994; 34:37–43.

131. Stone RM, Berg DT, George SL, et al. Granulocyte-macrophage colony-stimulating factor after initial chemotherapy for elderly patients with primary acute myelogenous leukemia. N Engl J Med 1995;332:1671–1677.

132. Jones SE, Schottstaedt MW, Duncan LA, et al. Randomized double-blind prospective trial to evaluate the effects of sargramostim versus placebo in a moderate-dose fluorouracil, doxorubicin, and cyclophosphamide adjuvant chemotherapy program for stage II and III breast cancer. J Clin Oncol 1996; 14:2976–2983.

133. Rowe JM, Andersen JW, Mazza JJ, et al. A randomized placebo-controlled phase III study of granulocyte-macrophage colony-stimulating factor in adult patients (>55 to 70 years of age) with acute myelogenous leukemia: a study of the Eastern Cooperative Oncology Group (E1490). Blood 1995; 86:457–462.

134. Vellenga E, Uyl-de Groot CA, de Wit R, et al. Randomized placebo-controlled trial of granulocyte-macrophage colony-stimulating factor in patients with chemotherapy-related febrile neutropenia. J Clin Oncol 1996;14:619–627.

135. Witz F, Sadoun A, Perrin MC, et al. A placebo-controlled study of recombinant human granulocyte-macrophage colony-stimulating factor administered during and after induction treatment for de novo acute myelogenous leukemia in elderly patients. Groupe Ouest Est Leucemies Aigues Myeloblastiques (GOELAM). Blood 1998;91:2722–2730.

136. Powles R, Smith C, Milan S, et al: Human recombinant GM-CSF in allogeneic bone-marrow transplantation for leukemia: double-blind, placebo-controlled trial. Lancet 1990; 336:1417–1420.

137. Nemunaitis J, Rabinowe SN, Singer JW, et al. Recombinant granulocyte-macrophage colony-stimulating factor after autologous bone marrow transplantation for lymphoid cancer. N Engl J Med 1991; 324:1773–1778.

138. Link H, Boogaerts MA, Carella AM, et al. A controlled trial of recombinant human granulocyte-macrophage colony-stimulating factor after total body irradiation, high-dose chemotherapy, and autologous bone marrow transplantation for acute lymphoblastic leukemia or malignant lymphoma. Blood 1992; 80:2188–2195.

139. Khwaja A, Linch DC, Goldstone AH, et al. Recombinant human granulocyte-macrophage colony-stimulating factor after autologous bone marrow transplantation for malignant lymphoma: a British National Lymphoma Investigation double-blind, placebo-controlled trial. Br J Haematol 1992; 82:317–323.

140. Advani R, Chao NJ, Horning SJ, et al. Granulocyte-macrophage colony-stimulating factor as an adjunct to autologous hemopoietic stem cell transplantation for lymphoma. Ann Intern Med 1992;116:183–189.

141. De Witte T, Gratwohl A, Van Der Lely N, et al. Recombinant human granulocyte-macrophage colony-stimulating factor accelerates neutrophil and monocyte recovery after allogeneic T-cell–depleted bone marrow transplantation. Blood 1992; 79:1359–1365.

142. Gorin NC, Coiffier B, Hayat M, et al. Recombinant human granulocyte-macrophage colony-stimulating factor after high-dose chemotherapy and autologous bone marrow transplantation with unpurged and purged marrow in non-Hodgkin's lymphoma: a double-blind placebo-controlled trial. Blood 1992; 80:1149–1157.

143. Gulati SC, Bennett CL. Granulocyte-macrophage colony-stimulating factor as adjunctive therapy in relapsed Hodgkin disease. Ann Intern Med 1992;116:177–182.

144. Nemunaitis J, Rosenfeld CS, Ash R, et al. Phase III randomized, double-blind placebo-controlled trial of rhGM-CSF following allogeneic bone marrow transplantation. Bone Marrow Transplant 1995;15:949.

145. Hiraoka A, Masaoka T, Mizoguchi H, et al. Recombinant human non-glycosylated granulocyte-macrophage colony stimulating factor in allogeneic bone marrow transplantation: double-blind placebo-controlled phase III clinical trial. Jpn J Clin Oncol 1994; 24:205–211.

146. Rabinowe SN, Neuberg D, Bierman PJ, et al. Long-term follow-up of a phase III study of recombinant human granulocyte-macrophage colony-stimulating factor after autologous bone marrow transplantation for lymphoid malignancies. Blood 1993;81:1903–1908.

147. Boiron JM, Marit G, Faberes C, et al. Collection of peripheral blood stem cells in multiple myeloma following single high-dose cyclophosphamide with and without recombinant human granulocyte-macrophage colony-stimulating factor. Bone Marrow Transplant 1993; 12:49–55.

148. Elias AD, Ayash L, Anderson KC, et al. Mobilization of peripheral blood progenitor cells by chemotherapy and granulocyte-macrophage colony-stimulating factor for hematopoietic support after high-dose intensification for breast cancer. Blood 1992;79:3036–3044.

149. Huan SD, Hester J, Spitzer G, et al. Influence of mobilized peripheral blood cells on the hematopoietic recovery by autologous marrow and recombinant human granulocyte-macrophage colony-stimulating factor after high-dose cyclophosphamide, etoposide, and cisplatin. Blood 1992; 79:3388–3393.

150. Legros M, Fleury J, Bay JO, et al. RhGM-CSF vs placebo following rhGM-CSF-mobilized PBPC transplantation: a phase III double-blind randomized trial. Bone Marrow Transplant 1997; 19:209–213.

151. Kritz A, Crown JP, Motzer RJ, et al. Beneficial impact of peripheral blood progenitor cells in patients with metastatic breast cancer treated with high-dose chemotherapy plus granulocyte-macrophage colony-stimulating factor: a randomized trial. Cancer 1993;71:2515–2521.

152. Ballestrero A, Ferrando F, Garuti A, et al. Comparative effects of three cytokine regimens after high-dose cyclophosphamide: granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and sequential interleukin-3 and GM-CSF. J Clin Oncol 1999; 17:1296–1303.

153. Caballero MD, Vazquez L, Barragan JM, et al. Randomized study of filgrastim versus molgramostim after peripheral stem cell transplant in breast cancer. Haematologica 1998; 83:514–518.

154. Lieschke GJ, Cebon J, Morstyn G. Characterization of the clinical effects after the first dose of bacterially synthesized recombinant human granulocyte-macrophage colony-stimulating factor. Blood 1989; 74:2634–2643.

155. Shaffer DW, Smith LS, Burris HA, et al. A randomized phase I trial of chronic oral etoposide with or without granulocyte-macrophage colony-stimulating factor in patients with advanced malignancies. Cancer Res 1993;53:5929–5933.

156. Bunn PA, Crowley J, Kelly K, et al. Chemoradiotherapy with or without granulocyte-macrophage colony-stimulating factor in the treatment of limited-stage small-cell lung cancer: a prospective phase III randomized study of the Southwest Oncology Group. J Clin Oncol 1995;13:1632–1641.

157. Lyman GH, Dale DC, Crawford J. Incidence and predictors of low dose-intensity in adjuvant breast cancer chemotherapy: a nationwide study of community practices.J Clin Oncol 2003; 21:4524–4531.

158. Gordon MS, McCaskill-Stevens WJ, Battiato LA, et al. A phase I trial of recombinant human interleukin-11 (Neumega rhIL-11 growth factor) in women with breast cancer receiving chemotherapy. Blood 1996; 87:3615–3624.

159. Isaacs C, Robert NJ, Bailey A, et al. Randomized placebo-controlled study of recombinant human interleukin-11 to prevent chemotherapy-induced thrombocytopenia in patients with breast cancer receiving dose-intensive cyclophosphamide and doxorubicin. J Clin Oncol 1997;15:3368–3377.

160. Tepler I, Elias L, Smith JW, et al. A randomized placebo-controlled trial of recombinant human interleukin-11 in cancer patients with severe thrombocytopenia due to chemotherapy. Blood 1996;87:3607–3614.

161. Hussein A, Vredenburgh J, Elkordy M, et al. Randomized, placebo-controlled study of recombinant human interleukin eleven (Neumega rhIL-11 growth factor) in patients with breast cancer following high-dose chemotherapy with autologous hematopoietic progenitor cell support. Exp Hemetol 1996; 24:634a.

162. Choi ES, Hokom MM, Chen JL, et al. The role of MGDF in terminal stages of thrombopoiesis. Br J Haematol 1996; 95:227–233.

163. Basser RL, Rasko JE, Clarke K, et al. Thrombopoietic effects of pegylated recombinant human megakaryocyte growth and development factor in patients with advanced cancer. Lancet 1996;348;1270–1281.

164. Vadhan-Raj S, Murray LJ, Bueso-Ramos C, et al. Stimulation of megakaryocyte and platelet production by a single dose of recombinant human thrombopoietin in patients with cancer. Ann Intern Med 1997;126:673–681.

165. Fanucchi M, Glaspy J, Crawford J, et al. Effects of polyethylene glycol-conjugated recombinant human megakaryocyte growth and development factor on platelet counts after chemotherapy for lung cancer. N Engl J Med 1997;336:404–409.

166. Basser RL, Rasko JEJ, Clarke K, et al. Randomized, blinded, placebo-controlled phase I trial of pegylated recombinant human megakaryocyte growth and development factor with filgrastim after dose-intensive chemotherapy in patients with advanced cancer. Blood 1997;89:3118–3128.

167. Vadhan-Raj S, Verschraegen CF, Bueso-Ramos C, et al. Recombinant human thrombopoietin attenuates carboplatin-induced severe thrombocytopenia and the need for platelet transfusions in patients with gynecologic cancer. Ann Intern Med 2000;132:364–368.

168. Schiffer CA, Miller K, Larson RA, et al. A double-blind, placebo-controlled trial of pegylated recombinant human megakaryocyte growth and development factor as an adjunct to induction and consolidation therapy for patients with acute myeloid leukemia. Blood 2000; 95:2530–2535.

169. Schuster MW, Beveridge R, Frei-Lahr D, et al. The effects of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on platelet recovery in breast cancer patients undergoing autologous bone marrow transplantation. Exp Hematol 2002;30:1044–1050.

170. Ragnhammar P, Friesen H-J, Frodin J-E, et al. Induction of anti-recombinant human granulocyte-macrophage colony-stimulating factor (Escherichia coli-derived) antibodies and clinical effects in nonimmunocompromised patients. Blood 1994; 84:4078–4087.

171. Miller LL, Korn EL, Stevens DS, et al. Abrogation of the hematological and biological activities of the interleukin-3/granulocyte-macrophage colony-stimulating factor fusion protein PIXY321 by neutralizing anti-PIXY321 antibodies in cancer patients receiving high-dose carboplatin. Blood 1999; 93: 3250–3258.

172. Bussel, JB, George JN, Kuter DJ, et al. An open-label, dose-finding study evaluating the safety and platelet response of a novel thrombopoietic protein (AMG 531) in thrombocytopenic adult patients with immune thrombocytopenic purpura [abstract]. Blood 2003;102;293.

173. Petros WP, Rabinowitz J, Stuart A, Peters WP. Clinical pharmacology of filgrastim following high-dose chemotherapy and autologous bone marrow transplantation. Clin Cancer Res 1997;3:705–11.

174. Spitzer G, Adkins D, Mathews M, et al. Randomized comparison of G-CSF+GM-CSF vs G-CSF alone for mobilization of peripheral blood stem cells: effects on hematopoietic recovery after high-dose chemotherapy. Bone Marrow Transplant. 1997;20:921–30.