Hematopoietic growth factors
Authors: R. G. Bociek, J. O. Armitage
Journal: CA A Cancer Journal for Clinicians (1996)

Although there have been significant and potentially curative advances in the treatment of many common human malignancies in the past 20 years, conventional combination chemotherapy regimens continue to have dose-limiting toxicities that place practical limitations on dose-intensity. In particular, most common chemotherapy regimens produce dose-limiting hematopoietic toxicity, a factor that until recently has constrained the ability to safely design more dose-intense treatment regimens. Inevitable periods of neutropenia and thrombocytopenia place patients at risk for potentially serious and occasionally life-threatening infectious and hemostatic complications, particularly in the setting of high-dose cancer therapy. Pharmacologic agents that accelerate hematopoietic recovery obviously have the potential to improve selected therapeutic and supportive care aspects of cancer medicine. This article will review the important clinical and scientific aspects of hematopoietic growth factors, with an emphasis on their present-day use in the care of cancer patients. Hematopoietic growth factors are a family of cytokines that interact with specific receptors on hematopoietic cells.1,2 These molecules regulate the functional activation of the specific cells with which they interact and are required for the survival, proliferation, and differentiation of hematopoietic progenitors.1 The development of recombinant DNA technology has made it possible to synthesize and purify pharmacologic doses of a variety of hematopoietic growth factors. A number of growth factors have been studied in clinical trials since the mid-1980s, including granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), erythropoietin (EPO), interleukin-3 (IL-3), and, more recently, the molecule putatively thought to represent thrombopoietin. The hematopoietic system is a hierarchical structure, with multipotent stem cells eventually becoming nonreplicative mature hematopoietic cells that circulate in the peripheral blood. The earliest hematopoietic cells are called stem cells, because of their presumed lifelong ability to replicate and produce progeny that develop and differentiate along all three hematopoietic lineages. Early hematopoietic progenitors probably contain multiple cytokine receptors, selectively losing the ability to respond to all but a few selected growth factors as they become mature committed hematopoietic cells. The human stem cell has not truly been morphologically and immunologically characterized, although certain cell-surface receptors (such as the receptor for CD34) have been used as markers of cells that currently represent our understanding of the "least committed" hematopoietic progenitors. Except for these earliest stem cells, all major hematopoietic progenitors have now been identified in culture and are known to be dependent on specific hematopoietic growth factors for differentiation along specific lineages. The major endogenous sources of hematopoietic growth factors include fibroblasts, endothelial cells, lymphocytes, monocytes, and macrophages.3–5 Some growth factors (e.g., GM-CSF) appear to have a rather broad array of action on very early hematopoietic progenitors, leading to multilineage increases in hematopoietic cell production/differentiation, while others (e.g., G-CSF) seem to act mainly on more terminally differentiated cell types, producing fairly incisive changes in specific committed populations, such as neutrophils. Granulocyte colony-stimulating factor is made predominantly by endothelial cells, monocytes, and fibroblasts.5–7 The main biologic effect of G-cSF is to cause an increase in proliferation and differentiation of neutrophils from committed progenitor cells.8–10 G-CSF potentiates the function and survival of neutrophils and in certain instances appears to produce stimulation of all hematopoietic lineages, although the clinical effect of this is negligible.9,11 The side effect most commonly reported with G-CSF is bone pain, and increases in alkaline phosphatase, lactate dehydrogenase, and urate have been observed in patients receiving this drug. Although leukemic cells appear to have surface receptors for G-CSF,12 the effect of growth factor administration on relapse has not been well studied as an endpoint in clinical trials to date and as such remains uncertain. The hematopoietic system is a hierarchical structure with multipotent stem cells eventually becoming nonreplicative mature hematopoietic cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is produced in vivo by T lymphocytes,13 monocytes,14 fibroblasts,15 and endothelial cells.16 The molecule affects a series of somewhat less-committed progenitors, including progenitors for the granulocyte, macrophage, eosinophil, and megakaryocyte lineages. The predominant effect of GM-CSF is seen on the granulocyte/macrophage lineages, leading to increases in the production of mature neutrophils and macrophages.17 GM-CSF sustains the viability of and potentiates the function of mature neutrophils,18 and it appears to have the ability to increase microbial killing through increased phagocytosis and superoxide production.19,20 The broad array of cellular interactions (including the ability to induce the production of other cellular cytokines) has made it difficult to separate out the primary activity of GM-CSF from secondary effects caused by the induction of other cytokines. This broad scope of activity likely explains the more diffuse and systemic nature of its side effect/toxicity profile, which includes fever, chills, malaise, hypersensitivity reactions, musculoskeletal effects, and several clinical manifestations of a capillary leak syndrome, including generalized edema with weight gain and pleural and pericardial effusions. Both G-CSF and GM-CSF are now routinely administered as subcutaneous injections, although in early clinical trials they were often administered as intermittent or continuous intravenous infusions. In the early 1900s, it was postulated that erythropoiesis was regulated by humoral agents that responded to changes in tissue oxygenation. In 1953 Erslev21 demonstrated that an increase in erythropoiesis could be brought about in normal rabbits by the infusion of large volumes of serum obtained from rabbits that had been rendered anemic from blood loss. The product, named erythropoietin, was eventually isolated, purified, cloned, and expressed in mammalian cells.22–24 Once pharmacologic amounts of EPO became available, clinical trials quickly followed demonstrating its efficacy in treating the anemia of chronic renal failure.25,26 EPO is produced by the liver in utero, but after birth the major source of production is the kidney.27 EPO is present in physiologic quantities in normal individuals, and its production is increased in response to decreased oxygen tension as perceived by the kidney. It interacts with specific receptors on erythroid burst-forming units and erythroid colony-forming units and is effective when administered in pharmacologic doses either intravenously or subcutaneously.28 Although several known hematopoietic growth factors have demonstrable effects on megakaryocyte and platelet production,29–31 the growth factor directly responsible for megakaryopoiesis/thrombopoiesis had until recently remained elusive. The discovery of the cytokine receptor c-Mpl and its relationship to megakaryopoiesis has led to the discovery of two cytokines that appear to be ligands for c-Mpl.32 These cytokines are thought to be lineage specific, affecting principally committed progenitors of megakaryocytes and platelets. A murine ligand for c-Mpl has been identified and cloned, and its expression appears to be restricted to primitive stem cells, megakaryocytes, and platelets.32 Intraperitoneally injected mice demonstrate an increase in megakaryocytes in both bone marrow and spleen, with a fourfold increase in circulating platelets after seven days.32 This c-Mpl ligand demonstrates only minimal effects on erythrocyte and granulocyte lineages, although an apparent synergistic effect on erythropoietin has been described.33 The c-Mpl ligand seems to induce proliferative changes in megakaryocyte precursors as well as differentiation of mature megakaryocytes and may actually expand an earlier pool of committed megakaryocyte progenitors.34 The cDNA for the human Mpl ligand has been identified, cloned, and expressed in mammalian cells and appears to encode a functional protein.35 Both the full-length and truncated forms of recombinant human Mpl ligand stimulate human megakaryopoiesis in vitro.35 Platelets produced in vitro by a ligand of the c-Mpl receptor appear to be morphologically and structurally identical to plasmaderived human platelets.36 Functional c-Mpl receptors have been demonstrated on acute myelogenous leukemia cells, and recombinant human thrombopoietin appears to stimulate the proliferation of such cell lines in vitro.37 Currently available evidence from de Sauvage et al35 suggests that their ligand is both a proliferative and maturation factor. Continued studies both in vitro and in vivo will hopefully define further the clinical benefit expected from the eventual use of this growth factor. The ability to augment hematopoietic cell cycling using hematopoietic growth factors has led to two interrelated areas of clinical investigation: namely, application of hematopoietic growth factors to facilitate more dose-intense treatments (in an attempt to improve upon primary therapeutic outcomes) and application of hematopoietic growth factors for supportive care management (in an attempt to decrease treatment-related complications). Mounting evidence, both direct and indirect, supports the concept of maintaining adequate dose-intensity in both standard-dose and high-dose chemotherapy settings.38–45 Because therapeutic progress in this area has the potential to improve the outcome in many malignancies, clinical trials involving augmented dose-intensity with hematopoietic growth factors are likely to continue to be an active area of research for years to come. Although retrospective39,46 and prospective38,41 studies have suggested improvement in response rates and treatment outcomes when dose-intensity is maintained, few randomized studies have addressed the potential benefit of increasing the dose-intensity of standard-dose chemotherapeutic regimens using hematopoietic growth factors alone (i.e., without the use of peripheral blood or marrow rescue products). The rationale for dose-intensification can be explored through the Goldie-Coldman hypothesis.47 This mathematical model proposes that tumor resistance to anticancer therapy is proportional to both the total number of malignant cells present (reflecting tumor burden) and the spontaneous mutation rate (reflecting inherent genetic instability) of each of those cells. If the Goldie-Coldman hypothesis holds true, then dose-intensification might be beneficial whether achieved through increasing total dose delivered (directly overcoming existing cellular resistance mechanisms) or by shortening the treatment interval (because both the period of regrowth and the time for development of resistant malignant cellular clones would be correspondingly shortened). This could be advantageous whether the malignant cell burden is microscopic (as in the setting of adjuvant chemotherapy) or overt (as in a newly diagnosed malignant lymphoma). Two randomized trials in small-cell lung cancer offer limited evidence in support of this concept. In one trial patients with small-cell lung cancer (n=65) were randomized to receive chemotherapy with or without G-CSF support.48 Because dose reductions were not allowed in the study, both groups of patients received essentially full doses of chemotherapeutic agents. However, there was no prescribed treatment interval, and because of more expedient myeloid recovery, patients randomized to receive G-CSF were able to receive their therapy over a shorter duration, increasing their dose-intensity. There was a suggestion of increased two-year survival in the group randomized to G-CSF (32 percent for patients randomized to G-CSF versus 15 percent in the control group), although the result was not statistically significant because of the small number of patients in the study (the 95-percent confidence intervals for the survival durations of the two groups overlap). Another prospective, randomized study of small-cell lung cancer reported improved progression-free and overall survival in patients who received combination chemotherapy/radiotherapy over a shortened period compared with controls, although in this study, the total treatment time was shortened by virtue of giving early versus late radiotherapy, not by altering the chemotherapy dose interval.49 Evidence for the importance of dose-intensity in adjuvant chemotherapy for breast cancer was first suggested by the 1981 retrospective analysis of Bonadonna and Valagussa.46 The authors found a correlation between doses actually received by patients and outcome, with patients who had received 85 percent or more of their planned total doses having improved relapse-free survival compared with patients receiving less. Current mature prospective evidence for the effect of varying dose-intensity has been largely limited to studies that have not used growth factors in their design. The CALGB 8541 trial was a randomized, prospective study of three different dose levels of adjuvant cyclophosphamide, doxorubicin, and fluorouracil for patients with unilateral stage II breast cancer.41 The regimens were designed such that the most dose-intense arm of the trial still rested within fairly conventional dose levels for all agents, and as such, hematopoietic growth factors were not used. The dose-intensity of the high-dose arm was 50 percent greater than the moderate-dose arm and 100 percent greater than the low-dose arm. The results at a median of 3.4 years revealed a significant increase in both disease-free and overall survival when comparing the low-dose arm with both the moderate- and high-dose arms, although no statistically significant difference was seen between the moderate- and high-dose arms. Nevertheless, this is one of the few prospective studies of dose-intensity that demonstrates a relationship between dose delivered and outcome. It suggests at least the existence of a threshold dose below which treatment results are likely to be inferior. It does not exclude the possibility that further escalation in dose-intensity (e.g., beyond levels achieved in this study, requiring the use of growth factors) might produce a superior outcome. NSABp B-22 was another prospective, randomized study designed to look at the effect of differences in both total dose and dose-intensity of cyclophosphamide in the adjuvant setting for patients with axillary-node-positive breast cancer.50 The chemotherapy regimen consisted of cyclophosphamide and doxorubicin. The dose of doxorubicin was held constant (60 mg/m2 per course) in all three treatment groups. The "standard" group received cyclophosphamide at a dose-intensity of 200 mg/m2/week (600 mg/m2 every three weeks for four cycles). The dose-intensity of cyclophosphamide was doubled to 400 mg/m2/week in the second group (1,200 mg/m2 every three weeks for two cycles only) and doubled as well as increased in total dose (1,200 mg/m2 every three weeks for four cycles) in the third treatment group. Despite a large number of randomized patients (over 700 per group), the results of this study revealed no significant difference in disease-free or overall survival at three years for any of the three treatment groups. An arguable criticism of this study is that the preferable drug to dose escalate is doxorubicin, because it is the drug with the higher single-agent response rates in breast cancer. The main biologic effect of G-CSF is to cause an increase in proliferation and differentiation of neutrophils from committed progenitor cells. Studies looking at the benefit of dose-intensifying chemotherapy regimens along with the use of hematopoietic growth factors (NSABP B-25, SWOG 9313, NCIC/EORTC MA-10) are currently under way or recently completed, but have not been reported. In summary, although there is limited evidence to suggest that for some tumors "more is better" (within relatively conventional dosing schedules), there are insufficient data to truly characterize any existing dose-response curve for these tumors. Because definite improvements in outcome have not been demonstrated by maximizing delivery of standard-dose chemotherapy using growth factors, their use in standard-dose chemotherapy regimens should be restricted to (1) reducing the incidence of neutropenic complications in chemotherapy regimens with a significant risk (e.g., greater than 40 percent) of neutropenic fever, as discussed later, and perhaps (2) treating patients with tumors curable with primary chemotherapy (such as germ cell malignancies and lymphomas) in whom appropriate dose-intensity cannot be maintained due to prolonged myelosuppression. However, it should be stressed that good evidence for benefit in the second instance is still lacking. In other clinical situations (e.g., adjuvant chemotherapy for breast cancer), dose reductions or delays remain reasonable options for patients who experience prolonged myelosuppression. Additional appropriately designed prospective studies will hopefully define the extent of any survival advantage conferred from using growth factors in these settings. There are now several established roles for the use of hematopoietic growth factors in high-dose therapy with progenitor cell support. The ability to treat various malignancies with potentially myeloablative doses of chemotherapy/irradiation has been made possible by the collection and reinfusion of various sources of hematopoietic progenitor cells.51–56 Use of a rescue product provides a means by which lethal marrow injury can be reversed. When nonmyeloablative regimens are used, the use of a rescue product dramatically shortens the duration of myelosuppression, reducing the risks associated with prolonged neutropenia/thrombocytopenia. Bone marrow was logically the initial source of progenitor cells used for autologous transplantation, but because not all patients were candidates for marrow harvesting (marrow involvement with tumor, prior pelvic irradiation, marrow hypocellularity), attempts were made to collect and reinfuse circulating peripheral blood progenitor cells as an alternative source of hematopoietic rescue. Initially these cells were collected without any attempt to increase their number (nonmobilized), but achieving an adequate number of cells for transplantation required numerous aphereses.51 Subsequently, chemotherapeutic agents,57 hematopoietic growth factors,58 or both59 have been used to augment the number of circulating progenitor cells for transplantation, such that an adequate number of cells can now often be collected with a single apheresis.52 Potential advantages of peripheral blood progenitor cells include ease of collection, diminished contamination with tumor,60–63 accelerated hematologic recovery,64 perhaps some degree of immunomodulatory antitumor activity,65 and more rapid immunologic recovery compared with bone marrow.64 It has recently been suggested that hematopoietic recovery may depend more on the presence of graft priming prior to harvest (bone marrow or peripheral blood collected after growth factor administration) than on the actual progenitor cell source.66 As confidence with the long-term engraftment capability of peripheral blood continues to grow, its use is likely to continue to expand because current evidence would suggest that peripheral blood collections appear to be less contaminated by tumor cells than is bone marrow. This is of increasing importance as evidence implicating infused tumor cells as a source of disease relapse accumulates.67,68 There are now several established roles for the use of hematopoietic growth factors in high-dose therapy with progenitor cell support. Numerous studies support the use of G-CSF or GM-CSF to shorten the duration of profound neutropenia after high-dose therapy using autologous bone marrow as the source of progenitor cell support.69–73 Evidence of benefit from using growth factors after infusion of allogeneic bone marrow is more limited. However, the evidence does suggest that some enhancement in neutrophil recovery is achieved with no apparent increase in graft-versus-host disease or nonengraftment.74–77 Hematopoietic growth factors have also been used to "prime" marrow prior to autologous bone marrow harvest, with some evidence (at least historically) to suggest that hematopoietic recovery is hastened over the use of unprimed bone marrow.66 The need for administration of hematopoietic growth factors to patients after infusion of peripheral blood progenitor cells, although standard practice in many transplant centers, was until recently of unproven benefit. A randomized trial performed by Klumpp et al78 demonstrated significant reductions in time to neutrophil engraftment (10.5 days versus 16 days), posttransplant hospitalization (18 days versus 24 days), and number of days of nonprophylactic antibiotic use (11 days versus 15 days) when G-CSF was used after autologous peripheral blood progenitor cell transplantation or bone marrow transplantation plus peripheral blood progenitor cell transplantation. A second randomized trial also found a significant improvement in neutrophil recovery (10 days versus 16 days) but a smaller effect on length of hospitalization (19 days versus 21 days) when given a combination of G-CSF/GM-CSF after infusion of autologous bone marrow and peripheral blood progenitor cells.79 The logical next step is to address the optimal timing for posttransplantation use of hematopoietic growth factors. Theoretically, until an adequate number of fairly mature neutrophil precursors have been generated by the rescue product, there might not be any benefit to using hematopoietic growth factors, especially for terminally acting growth factors such as G-CSF. This question has been evaluated for bone marrow as a rescue product in two retrospective analyses.80,81 The results of both of these studies, although based on small numbers of patients and using historical controls as comparison groups, suggest that delaying growth factor administration until six to eight days after bone marrow infusion (rather than starting on day one postinfusion) is not associated with any significant prolongation of time to neutrophil recovery. A similar result was reported in a sequential cohort study of 96 patients receiving autologous peripheral blood progenitor cells after high-dose chemotherapy for stage II to IV breast cancer.82 In this study, a delay in administration of G-CSF for up to seven days postinfusion did not appear to prolong time to neutrophil recovery (median, 11 days) compared with patients receiving G-CSF starting at day zero (median, 10 days). However, the group receiving no G-CSF postinfusion experienced a significant prolongation in time to neutrophil recovery (median, 15 days) compared with the cohorts receiving G-CSF. Therefore, although current data suggest more rapid myeloid recovery in patients who receive hematopoietic growth factors after rescue product infusion, the required duration and starting point for infusion are less well defined. Recently, one of the most active areas in autologous transplantation has been the use of hematopoietic growth factors for the purpose of ex vivo expansion. Hematopoietic recovery is influenced by the absolute number of granulocyte-macrophage colony-forming units infused83 and probably by the proportion of mature to immature cells that constitute the graft. The apparent improvement in engraftment time using mobilized peripheral blood progenitors (as opposed to bone marrow) is likely the result of the relative increase in more mature (postprogenitor) cells present in a mobilized graft. By producing greater numbers of postprogenitor cells, cytokine-supported ex vivo expansion could reduce even further the time to hematopoietic recovery, decrease the required number of harvested cells, and perhaps also purge grafts of tumor cells by providing a microenvironment conducive only to the growth of hematopoietic cells. The feasibility of ex vivo amplification/expansion has been well demonstrated in both short- and long-term culture systems,84–86 including the amplification of peripheral blood collected from patients with extensive prior chemotherapy exposure.87 At present, experience with reinfusion of ex vivo expanded progenitor cells is limited,88–91 but has been performed after expansion of about one tenth of a single leukapheresis product.88,89 Reinfusion of the expanded product appears to produce rapid sustained engraftment profiles similar to that of standard nonexpanded progenitor cell collections.88–90 Lastly, mobilized peripheral blood progenitor cells have recently been used in allogeneic transplantation. In studies reported to date, engraftment of neutrophils (day 10 to 12) and platelets (day 10 to 18) appears to be rapid, without an obvious increase in the incidence of acute severe graft-versus-host disease.92–95 From the donor perspective, apheresis can be accomplished without the need for central catheters by using antecubital veins, and adequate numbers of progenitor cells for transplantation can be collected after five days of G-CSF followed by two to three leukaphereses.96–98 One of the most active areas in autologous transplantation has been the use of hematopoietic growth factors for ex vivo expansion. However, it appears that more T lymphocytes and natural killer cells are contained in allogeneic progenitor cell grafts,96 and early data on small numbers of patients suggest that the use of unmanipulated (non-T-cell depleted) grafts may be associated with a higher incidence of chronic graft-versus-host disease than when allogeneic bone marrow is used.99,100 A much larger body of comparative clinical data is clearly required, and the determination of the actual T-cell subsets responsible for graft-versus-host disease as well as further work on the effects of T-cell depletion (selective or nonselective) may enhance the attractiveness of mobilized allogeneic peripheral blood as a rescue product. The generation of high-quality data comparing high-dose therapy with conventional therapy has been slow largely because many centers have focused on continuing phase II trials rather than on randomized, controlled trials. Accrual to randomized trials has been hampered in many instances by preexisting bias (on the part of both physicians and patients) in favor of high-dose therapy as the superior treatment. There are now a limited number of randomized trials that despite imperfections in design and small numbers offer the best current available evidence to support the use of high-dose therapy/stem cell transplantation in certain clinical settings. The British National Lymphoma Investigation undertook a randomized trial comparing high-dose chemotherapy with bone marrow transplantation to conventional-dose chemotherapy for patients failing primary therapy for Hodgkin's disease.40 The high-dose arm consisted of high-dose BCNU, etoposide, cytosinearabinoside, and melphalan, and the conventional arm consisted of conventional doses of the same agents. Event-free survival (53 percent versus 10 percent at three years) and progression-free survival (about 62 percent versus 10 percent) were significantly increased in the high-dose arm. A recent randomized trial compared high-dose chemotherapy/bone marrow transplantation with continuation of conventional-dose salvage chemotherapy in patients with chemosensitive relapses of intermediate- or high-grade non-Hodgkin's lymphoma.42 Patients were ineligible if at relapse they had involvement of the central nervous system or bone marrow. Eligible patients (n=215) received two cycles of induction chemotherapy with dexamethasone, Ara-C, cisplatin (DHAP) followed by an assessment of disease response. Patients with responding disease (n=109) were then randomized to continuation of conventional chemotherapy (n=54) or high-dose chemotherapy with bone marrow transplantation (n=55). The high-dose arm used a regimen of high-dose BCNU, etoposide, Ara-C, and cyclophosphamide (BEAC). Patients randomized to continuation of salvage therapy received four additional cycles of DHAP. At a median follow-up of 63 months, the results of this trial reveal significantly higher event-free survival (46 percent versus 12 percent) and overall survival (53 percent versus 32 percent) in the high-dose arm. The question of consolidative high-dose chemotherapy/transplantation for patients with high-risk non-Hodgkin's lymphoma (i.e., as consolidation of first remission therapy) has unfortunately been less well answered. A randomized trial comparing continuation of chemotherapy with use of autologous bone marrow transplantation in slowly responding patients with intermediate- or high-grade non-Hodgkin's lymphoma failed to find any benefit to transplantation, although the study had inadequate statistical power to demonstrate anything other than a very large difference in outcome between the two groups.101 The recently reported LNH87–2 trial randomized 918 patients to either consolidative conventional-dose therapy or to consolidative high-dose therapy with high-dose cyclophosphamide, BCNU, etoposide, and autologous bone marrow support.45 Although no overall benefit to transplantation could be demonstrated, the subset of patients identified by the International prognostic index102 with high-intermediate or high-risk disease (2 to 3 nega