William P. Petros
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
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
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.
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.
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
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.
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.
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.
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
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
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
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
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
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.
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.
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
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