Leukemia Research VoL 14. No. 8, pp. 721-729, 1990. Printed in Great Britain.
0145-2126/90 $3.00 + .00 Pergamon Press pie
THE ROLE OF H A E M O P O I E T I C GROWTH FACTORS IN BONE M A R R O W TRANSPLANTATION ANTHONY H. GOLDSTONE* and ASIM KHWAJAt *University College Hospital, London and tDepartment of Haematology, University College and Middlesex School of Medicine, London, U.K.
(Received for publication 20 March 1990) Abstract--The availability of recombinant haemopoietic growth factors for clinical use has led to a proliferation of trials in the setting of bone marrow transplantation. Early results from these studies, using GM-CSF and G-CSF, show that these factors are able to reduce the period of cytotoxic induced neutropaenia but have little effect on platelet recovery. Toxicity has been relatively mild unless very high doses are administered. Randomised controlled trials are in progress and will help to define the exact role of growth factors in this setting. Future prospects include the increasing availability of other growth factors for clinical use and the potential for combination growth factor therapy to provide a more optimal haemopoietic response.
Key words: Colony-stimulating factor, bone marrow transplantation, haemopoietic growth factors.
PROPERTIES AND TYPES OF HGFs
INTRODUCTION
The various HGFs may best be considered as three main groups of factors with overlapping activities (Table 1). The lineage-specific factors, which include granulocyte colony-stimulating factor (G-CSF), monocyte colony-stimulating factor (M-CSF), and erythropoietin (epo) act on mature progenitor cells during the development of specific cell lineages [1]. In addition to their effects on haemopoiesis, G-CSF and M-CSF influence the biological activities of neutrophils and monocytes respectively. The multilineage CSFs are less restricted in their activity and include granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin 3 (IL-3). GMCSF is active in granulopoiesis in vitro, supports the proliferation of macrophage colonies, promotes early erythroid colony formation (in the presence of epo) and enhances the proliferation of megakaryocytic progenitors [2]. GM-CSF is also a survival and activating factor for neutrophils, eosinophils and monocytes. IL-3 has multilineage activity and probably acts on progenitor cells at an earlier stage in the differentiation pathway than GM-CSF; it supports the proliferation of myeloid, macrophage and megakaryocyte lineages and activates mature eosinophils and monocytes. The third group of HGFs, of which the properties of interleukin 1 (IL-1) are the best characterised, act on primitive haemopoietic progenitors and render them sensitive to the later acting factors [3]. There is increasing evidence that IL-4 and IL-6 may play a similar role in haemopoiesis [4--6].
THE HUMANhaemopoietic growth factors (HGFs) are a family of glycoproteins responsible for sustaining the proliferation, differentiation and survival of immature haemopoietic progenitor cells. Autologous and allogeneic BMT is increasingly being used as salvage or first line therapy in the treatment of both solid tumours and haematological malignancies and as many of the problems associated with BMT are related to the period of bone marrow aplasia following cytotoxic therapy, there has been great interest in the use of HGFs to facilitate earlier engraftment. The genes for a number of growth factors have now been cloned and the application of recombinant DNA methodology to produce large amounts of these factors has led to an increased understanding of their role in regulating haemopoiesis and also of their potential clinical application.
Abbreviations: HGF, haemopoietic growth factor; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL-3, interleukin 3; BMT, bone marrow transplantation; ABMT, autologous bone marrow transplantation; M-CSF, monocyte colony-stimulating factor; CSF-HU, human urinary colony-stimulating factor; ANC, absolute neutrophil count. Correspondence to: Dr A. H. Goldstone, Department of Haematology, University College Hospital, Grafton Way, London WC1 6AU, U.K. 721
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A. H. GOLDSTONE and A. KHWAJA TABLE 1. PROPERTIES OF HAEMOPOIETIC GROWTH FACTORS
Protein size (kDa) Chromosomal location
Core
Factor G-CSF
17q21-22
18.6
20
M-CSF
5q33.1
26 16
70-90 40-50
Epo
7ql 1-22
18
34-39
GM-CSF
5q21-31
14-15
18-35
Biological activity
Glycosylated
Cellular source
IL-3
5q21-31
14-15
20-26
monocytes fibroblasts monocytes fibroblasts endothelial cells kidney (monocytes) T cells endothelial cells fibroblasts monocytes T cells
IL-5
5q31
13
20-30
T ceils
Progenitor cells
Mature cells
n
n
rn
rn
ery (meg)
n, m, eo,
n, m,
ery meg
eo
n, m, eo, b, ery
eo, m
meg
eo
eo
n, neutrophil; eo, eosinophil; b, basophil; ery, erythroid; m, monocyte/macrophage; meg, megakaryocyte.
TABLE 2. THE POTENTIAL USES OF H G F S IN BONE MARROW TRANSPLANTATION
1. Reduce the period of cytotoxic induced cytopaenia. 2. Enhance the harvesting of peripheral blood progenitor cells for ABMT. 3. Stimulate leukaemic cells into cycle prior to chemotherapy. 4, Augment phagocyte response against infection. 5. Enhance host anti-tumour response.
All the HGFs are active at low (pM) concentrations on target haemopoietic and mature cells which bear relatively low numbers of specific high affinity receptors [7]. In the native state they are all glycosylated. Recombinant factors derived from yeast or mammalian cell systems are also glycosylated, whereas those expressed in bacterial systems (such as E. coli) will be non-glycosylated. The degree of glycosylation may have clinical relevance with regard to efficacy and antigenicity of the recombinant molecule. The most heavily glycosylated form of GM-CSF is not as active in vitro but is cleared more slowly in vivo than the non-glycosylated form [8]. O V E R V I E W OF P O T E N T I A L USES OF HGFs IN BONE M A R R O W T R A N S P L A N T A T I O N The most obvious, and so far the most frequently investigated, role for HGFs in BMT is to reduce the period of cytopaenia induced by cytotoxic therapy. However, there are other possible areas of use (Table 2). In the non-transplant setting, perhaps the most
interesting is the potential for improving tumour response rates by permitting dose escalation of cytotoxic agents and thereby reducing the need for intensive salvage treatment regimens.
CLINICAL STUDIES OF HGFs IN BMT GM-CSF
This was the first H G F to be used in BMT after initial studies in patients with the acquired immunodeficiency syndrome [9] and myelodysplastic syndrome (MDS) [10] demonstrated a dose-dependent rise in leucocyte counts. At Duke University, 19 patients undergoing autologous BMT (ABMT) for disseminated melanoma and carcinoma of the breast received continuous intravenous (i.v.) GM-CSF for 14 days after marrow re-infusion at doses ranging from 1.2 to 19.2 ~tg/kg/day [11]. Total leucocyte and granulocyte recovery was accelerated when compared with historical controls although this did not reach statistical significance. There was a fall in the leucocyte count over a 3-4 day period on stopping GM~CSF, after which haemopoietic recovery proceeded at a rate similar to that in control patients. No consistent effect on recovery of platelet counts was seen. Patients receiving GM-CSF had fewer episodes of culture positive bacteraemia (16% vs 35%) and less of an elevation in creatinine and bilirubin levels during the post-transplant period. The authors postulated that inhibition of bacteraemia and consequent multiple organ dysfunction may have been responsible for the latter finding. Significant toxicity
Growth factors and transplantation TABLE 3. SIDE EFFECTS OF H G F TREATMENT
GM-CSF Low dose High dose
bone pain fever thrombophlebitis capillary leak syndrome central venous thrombosis
G-CSF bone pain myalgia fever
(Table 3) was observed in the group of patients receiving 19.2 ~tg/kg/day with erythroderma and a capillary leak syndrome characterised by oedema, pleuro-pericardial effusions and weight gain. This toxicity reversed with discontinuation of GM-CSF. The Seattle group have also reported results from a study involving 15 patients undergoing ABMT for lymphoid malignancies [12]. Patients in the study group received GM-CSF by daily 2 h i.v. infusions at a dose range between 15 and 240 Ixg/m2/day for 14 days and comparison was made with a historical group of patients who had not received growth factors. Patients receiving more than 60 ~tg/m2/day showed accelerated recovery of neutrophil counts (days to 0.5 × 109/1, 14 vs 25), became independent of platelet transfusions quicker (29 vs 38 days), had fewer febrile days and a reduced period of hospitalisation. Fewer culture positive bacteraemias were also noted in this study (13% vs 30%) and no significant GM-CSF associated toxicity observed. In our own centre, 12 patients with resistant Hodgkin's disease receiving high-dose chemotherapy and ABMT were treated with doses of GM-CSF between 100 and 400 ~tg/mE/day [13]. The median time taken to reach an absolute neutrophil count (ANC) of 0.5 × 109/1 in those receiving GM-CSF was 16.3 days compared with 25 days in 19 concurrent controls. No consistent effect on platelet recovery was seen and there was no difference between the two groups with regard to the rate of bacterial infection, number of febrile days or period of hospitalisation. The Minnesota group have recently reported their results of GM-CSF treatment in 25 patients having ABMT for acute lymphoblastic leukaemia (ALL) [14]. This study differed from others previously reported in that bone marrows were purged ex oivo with 4-hydroperoxycyclophosphamide and lineage specific antibodies before transplantation. The dose range of GM-CSF administered was between 16 and 256 ~tg/m2/day--patients receiving less than 60 ~tg/ m2/day did not show any response and of those receiving more than this dose, only patients reinfused
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>1.2 x 104 granulocyte-monocyte colony forming units (CFU-GM) per kg showed an accelerated haemopoietic recovery. G-CSF
The use of G-CSF in BMT has now been reported in several studies and the overall results strongly suggest a significant reduction in the period of cytotoxic induced neutropaenia. At Duke University, 15 patients undergoing ABMT for solid tumours were treated with G-CSF by continuous i.v. infusion at doses between 16 and 64 txg/kg/day for 14 days [15]. Neutrophil recovery was significantly accelerated and no significant toxicity observed. The MD Anderson group have reported a study of G-CSF in 26 patients with Hodgkin's disease, 18 by bolus and 8 by continuous infusion, at doses between 30 and 120 ~tg/kg/ day for up to 28 days post-ABMT [16]. Days to reach an ANC of 0.5 x 109/1 were 13 in the bolus dose group, 11 in the continuous infusion group and 22 in a group of historical controls. The treated group had fewer febrile days and a lower rate of gram positive infections (17% vs 39%). There was minor toxicity consisting of myalgia and bone pain in 5 of 26 patients. Sheridan and co-workers have reported the use of subcutaneously administered G-CSF at a dose of 20 ~tg/kg/day in ABMT for a range of malignant disorders [17]. As well as a shortened neutropaenic period, they were able to demonstrate both a significant reduction in antibiotic usage and a shorter duration of hospitalisation when compared with historical controls, a finding with potentially important economic implications. To date the only report of the systematic use of GCSF in allogeneic BMT has come from the Japan BMT Study Group [18]. Thirty-four patients (10 ALL, 8 acute myeloblastic leukaemia, 9 chronic myeloid leukaemia, 4 severe aplastic anaemia, 3 lymphoma) received either 200 or 400 ~tg/m2 of G-CSF for 14 days. Median time to an ANC of 0.5 × 109/1 was 17 days in the group receiving the lower dose, 15 days in those treated with the higher dose of GCSF and 27.4 days in a group of historical controls. No effect on platelet or reticulocyte counts was seen and no obvious effect on the incidence or severity of graft versus host disease (GVHD) noted. The cohort of patients is still at an early stage but to date no adverse effect on disease relapse rate has been observed. CSF-HU
This is a dimeric glycoprotein of molecular weight 84,000 derived from human urine which binds to monocytes and stimulates the formation of G-CSF [19]. The primary structure of CSF-HU contains the
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A.H. GOLDSTONEand A. KHWAJA
The intravenous administration of both G-CSF and GM-CSF has been shown to increase markedly the numbers of circulating peripheral blood C F U - G M and erythroid burst foiming units (BFU-E) [22, 23]. This has led to an interest in the use of HGFs in facilitating the harvest of peripheral blood progenitors for autotransplantation and an Italian group have recently reported their results of the use of GM-CSF in this setting [24]. GM-CSF was used to accelerate recovery from cyclophosphamide induced cytopaenia in a group of seven patients with large cell lymphoma. Patients underwent leucaphereses in the recovery phase from chemotherapy and high yields of C F U - G M (mean of nine-fold increase over seven control patients treated with identical chemotherapy without GM-CSF) were obtained. Conditioning with melphalan/total body irradiation was followed by re-infusion of both previously harvested autologous bone marrow and peripheral stem cells. Four out of seven patients received continuous GMCSF infusions (5.5 ~tg/kg/day) after the re-infusion of autologous cells. Overall results for the seven patients (results for control patients) were a median time of 9.1 (11.4) days to an ANC of 0.5 x 109/1 and 10.7 (16.9) days to reach a platelet count of 50 x 109/ 1. These differences were modest but highly significant. The fact that patients received both autologous marrow and peripheral progenitors, and that four of seven patients were treated with GM-CSF after stem cell re-infusion, makes it difficult to reach any firm conclusions about the role of GM-CSF in this setting, but the results are sufficiently encouraging to merit further evaluation.
>0.1 x 109/1 by day 28 post-BMT or the development of a sustained drop in ANC to 27 x 109/1 with > 5 0 % eosinophils. Nissen et al. report the use of GM-CSF in four patients with very severe A A , three of whom had no evidence of residual myelopoiesis [28]. Only the patient with minimal residual myelopoiesis responded with an increase in neutrophil numbers and resolution of infection. One of the main mechanisms of the development of A A is presumed to be a qualitative or quantitative defect in stem cell numbers and patients often have high circulating levels of endogenous HGFs [29]; therefore it is not surprising that GM-CSF may have little effect in patients with very severe AA. So is there a role for the use of HGFs in AA? They may be useful as interim therapy for patients who are awaiting a response to antilymphocyte globulin ( A L G ) , which may take up to 12 weeks to have a beneficial effect, and for those patients for whom a suitable donor for B M T is being sought. In view of the enhancing effect of GM-CSF on antigen presenting cells however [30], there may be an increased risk of allo-immunisation with consequent difficulties in transplantation. There may be a role for HGFs in the long term treatment of patients who have no suitable donor for B M T and who have not responded to A L G , but who still have some residual myelopoiesis. In the light of the so far disappointing lack of effect of GM-CSF on RBC or platelet transfusion requirements, future combination therapy with HGFs may prove to be more effective in this setting.
HGFs 1N E N G R A F T M E N T F A I L U R E AND A P L A S T I C A N A E M I A (AA)
EFFECTS OF HGFs ON P H A G O C Y T E FUNCTION
GM-CSF has been used by the Seattle group for graft failure after allogeneic and autologous BMT [25]. Graft failure in this study was defined as a failure to reach an absolute neutrophil count (ANC)
G-CSF and GM-CSF are both known to affect the survival and function of mature phagocytes. In-vitro studies show that GM-CSF prolongs the survival of neutrophils and eosinophils, enhances the ability of
amino acid sequence of human M-CSF in each of its monomeric subunits. CSF-HU stimulates the production of neutrophils in v i v o [20] and has been administered to 37 Japanese patients undergoing allogeneic B M T [21]. Results from this study show that granulocyte recovery to an ANC of 0.5 x 109/1 was a mean of 22 days in the treatment group and 28 days in matched historical controls. There is no data provided for platelet recovery. The authors report no detrimental effect on engraftment, incidence or grade of G V H D or leukaemic relapse. HGFs IN P E R I P H E R A L STEM C E L L BMT
Growth factors and transplantation phagocytes to kill tumour targets via antibody dependent cellular cytotoxicity ADCC [31], stimulates phagocytosis [32] and augments neutrophil superoxide production in response to physiological chemoattractants such as the bacterial peptide f-MLP, complement derived C5a and leukotriene B4 [33]. GM-CSF also stimulates the surface expression of the neutrophil cellular adhesion molecules (CAMs) recognised by the antibodies C D l l b and CD18 [34]. G-CSF enhances neutrophil ADCC, production of superoxide anions, and phagocytosis [2]. M-CSF has similar effects on monocytes [35]. Administration of GM-CSF in vivo has been shown to cause a transient leucopaenia maximal 30 min after starting an infusion [36]. Radionuclide labelling studies show this to be due to margination of neutrophils and monocytes predominantly in the pulmonary vasculature. This margination may be related to endothelial adherence mediated by upregulation of CAM expression [37]. However, as neutrophil demargination occurs at a time when CAM expression is still high, other factors must be involved in producing this phenomenon. The pulmonary sequestration of neutrophils may have important clinical consequences as symptoms and signs of consolidation have been observed in patients with pre-existing lung pathology and/or relatively high numbers of circulating granulocytes [13, 27]. It suggests that GM-CSF may best be given as a continuous infusion rather than intermittent bolus doses to avoid repeated margination and demargination. Furthermore, it may be wise not to prolong unnecessarily the administration of this growth factor once neutrophil recovery after BMT has taken place. Studies examining the effects of HGFs on the migration of neutrophils into an artificially created sterile inflammatory site ("skin window") have shown that neutrophil migration during GM-CSF infusion is significantly impaired [38, 39]. This inhibition may be due to the systemic administration of a chemokinetic substance giving a non-directional stimulus to the circulating neutrophil and impairing its subsequent response to an appropriate signal from an inflammatory focus. Such an impairment of phagocyte tissue penetration should focus the attention of investigators on the risk of exacerbating deep seated infection during GM-CSF therapy and suggests that the use of this factor as adjuvant therapy for the treatment of severe infections in patients with normal neutrophil counts may not necessarily be appropriate. A study looking at neutrophil migration during G-CSF infusions following ABMT [15] showed that, in contrast to the findings with GM-CSF, there was no significant difference in this response between patients on growth factor therapy and matched controis.
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Enhanced superoxide release from neutrophils harvested from patients receiving intravenous infusions of GM-CSF has also been reported [40]. Such augmentation of the neutrophil response could be of benefit in responding to circulating microorganisms and may be responsible for the reduced rate of culture positive bacteraemia observed in several studies [11, 12]. However, the presence of activated cells in the circulation that may have an impaired tissue migratory response could result in endothelial damage. Secretion of neutrophil secondary granules during infusions of GM-CSF has also been described [41]. These contain substances such as lactoferrin and transcobalamins which have bacteriostatic functions, and enzymes which may be involved in neutrophil migration through the vascular endothelium. Patients with congenital deficiencies of secondary granules have been described and their neutrophils show a reduced ability to migrate into inflammatory foci [42]; therefore, a non-specific release of these granules caused by GM-CSF administration may be associated with the observed impairment of neutrophil migration and be detrimental to host defence. EFFECTS OF HGFs ON IMMUNE FUNCTION GM-CSF has been shown to have direct effects on host immune function. In the murine model, it has been shown to enhance the function of antigen presenting cells by increasing the expression of class II MHC antigens and increasing IL-1 production [30]. In this study, IL-2 production by T cells in response to a mitogenic stimulus was also enhanced by exposure to GM-CSF. Administration of GM-CSF in vivo in humans with lymphoid malignancies has been reported as causing increases in circulating soluble IL-2 receptor levels as well as enhancing the production of IL-2 by peripheral blood mononuclear cells (PBMC) [43]. GM-CSF has been shown to enhance the production of both IL-lc~ and /3 and tumour necrosis factor by PBMC in vitro [44, 45] and to enhance monocyte cytotoxicity in samples taken from patients on growth factor therapy [46]. The production of these factors may be responsible for some of the toxicity seen with GM-CSF treatment at relatively high doses, but has potentially valuable anti-tumour effects in the context of minimal residual disease. EFFECTS OF HGFs ON LEUKAEMIC CELLS In-vitro studies have reported that GM-CSF is able to stimulate AML blast colony formation in methylcellulose cultures and can enhance the growth
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A . H . GOLDSTONE and A. KHWAJA
of blast progenitors in suspension, suggesting an ability to support blast self-renewal [47]. Other work has shown that GM-CSF, G-CSF and IL-3 were all able to stimulate AML blast self-renewal and terminal division with varying responses between patients [48]. The possibility of stimulating myeloid leukaemic growth in vivo is perhaps best assessed by looking at the effects of GM-CSF treatment as reported in studies in patients with pre-leukaemic states. Administration of GM-CSF in MDS has yielded variable results with regard to stimulation of blast numbers. Vadhan-Raj and colleagues reported no increase in absolute blast counts in a group of eight patients, four of whom had refractory anaemia with excess blasts in transformation (RAEBt) by the FAB classification [10]. Antin and colleagues reported a reversible increase in myeloblasts in two of seven patients with MDS [26]; the presence of RAEBt was one of the exclusion criteria in this study. More recently, Ganser has described an increase in marrow myeloblast count in four of eleven patients treated with GM-CSF [49]. These rises were only seen in patients with pre-GM-CSF treatment bone marrow blast counts greater than 14%. Negrin has reported the use of G-CSF in MDS and has found no evidence of leukaemic stimulation [50]. Stimulation of leukaemic cell growth and proliferation by HGFs may be used to the potential benefit of the patient to synchronize the entry of malignant cells into the cell cycle and render them more sensitive to cycle-specific cytotoxic therapy. Invitro work using leukaemic blast cell cultures shows that incubation with cytosine arabinoside (Ara-C) after treatment with HGFs increases cell kill [51,52]. Approaches combining HGF treatment with cytotoxic therapy may have a place in the treatment of myeloid leukaemias with and without BMT and merit further evaluation.
THE EFFECTS OF HGFs ON NONHAEMOPOIETIC CELLS As ABMT is now often used for the treatment of non-haemopoietic malignancies, studies examining the effects of HGFs on tumour cells of non-haematological origin have increasing relevance. It has been reported that GM-CSF receptors are present on certain small cell carcinoma lines and that incubation with GM-CSF enhances their proliferation [53]. More recently, Salmon has described no significant inhibitory or stimulatory effect of GM-CSF on the growth of cells taken from tumour biopsies of various cell types [54].
THE EFFECTS OF HGFs ON ENGRAFTMENT Theoretical concern about HGF treatment leading to stem cell "exhaustion" in the post-transplant period has largely been negated by the results of the clinical trials as reported so far, with no evidence of late graft failure. Blazar has described the results of the use of recombinant murine GM-CSF (rmuGMCSF) in a murine model of major histocompatibility complex mismatched allogeneic BMT [55]. In a system where T-cell depletion of donor marrow leads to a low incidence of GVHD and a high rate of engraftment failure, it was demonstrated that donor graft incubation ex vivo with, or a single injection in vivo on the day of marrow infusion of, rmuGM-CSF were both effective means of promoting engraftment. This effect was only seen in experiments where animals had a very low rate of engraftment; where the engraftment rate in control animals was relatively high, no further enhancement was seen with the use of rmuGM-CSF. The mechanism by which rmuGMCSF promoted engraftment is not clear. The only dose at which any positive effect was seen was several fold higher than that required for bone marrow receptor saturation--it is possible that the effect of GMCSF may have been an indirect one, perhaps mediated by the release of other cytokines. In another study, it was demonstrated that GM-CSF down-regulated the expression of IL-2 receptor (IL2-R) on human monocyte/macrophages via a prostaglandin E dependent mechanism [56]. This effect may be relevant to the transplant situation as it has been demonstrated that IL-2-R positive macrophages and T cells may have a significant role in mediating allograft rejection and GVHD.
FUTURE APPROACHES IN THE USE OF HGFs The effects of other HGFs have been studied in animals, both singly and in combination, and in the case of IL-3 in preliminary human trials. IL -3
Overall data from animal work suggests that administration of IL-3 on its own has little haemopoietic effect [57]. However, combination treatment with IL-3 followed by GM-CSF has been reported as causing marked increases in the numbers of circulating neutrophils, monocyte, reticulocytes and platelets in non-human primates [57]. A recent report describes the use of subcutaneous IL-3 in patients with advanced neoplasms [58]. Fourteen patients have received IL-3 for 15 days at doses between 60 and 500 ~tg/m2---of the nine evaluable patients, all have shown increases in neutrophil,
Growth factors and transplantation eosinophil, monocyte, reticulocyte and platelet numbers. These increases occurred in the second week of treatment, as might be expected on theoretical grounds, and continued for 1-2 weeks after the end of IL-3 administration. There was a mean increase of 179 x 109/1 in platelet numbers and two patients who had "prolonged thrombocytopaenia" with counts of 3 × 109/1 and 22.5 × 109/1 increased to 104 × 109/1 and 184 × 109/1 respectively. These initial results are extremely encouraging and trials of IL-3 in the setting of BMT are awaited with interest.
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radiotherapy while keeping myelotoxicity at acceptable levels; improvement in tumour responses to such dose escalation has the potential for reducing the requirement for later more intensive salvage regimens requiring BMT. We are still in the early stages of discovering the true potential of the clinical use of HGFs; it is imperative that investigation of their efficacy should be conducted in well designed trials, and that conclusions about their definitive role in cancer treatment and BMT be based on objective clinical evidence of patient benefit and not merely on observed manipulations of the blood count.
IL-1
In mice, IL-1 given before sublethal total body irradiation has been reported to prevent severe myelosuppression [59]--the mechanism for this is obscure and difficult to evaluate in vivo, as IL-1 has multiple effects, including the stimulation of production of other growth factors.
CONCLUSIONS The results of preliminary clinical trials with HGFs show that treatment with these factors can reduce the period of neutropaenia following BMT. Disappointingly, there has so far been no clear evidence of acceleration of platelet regeneration in trials with GM-CSF, as might have been predicted by its activities in vitro. Effective reduction in the post-transplant thrombocytopaenic period may have to await the isolation of "thrombopoietin" [60] or may be effected by combination growth factor therapy, for example with IL-3 and GM-CSF. Whether treatment with HGFs will lead to a reduction in treatment related morbidity and mortality associated with BMT will become clearer with the publication of the results of current randomised trials. Death due to infection during BMT is now an event with a relatively low frequency [61] and it may prove difficult to demonstrate a significant reduction in this parameter as a consequence of H G F use. If the use of growth factors can be shown to reduce the use of antimicrobials and blood products and decrease the length of inpatient stay then this will have obvious economic advantages as well as increasing patient acceptability of a procedure such as BMT. Perhaps the most interesting potential application of HGFs is in allowing cytotoxic dose escalation in the non-transplant setting. There is good evidence in the treatment of several tumour types that the best responses are seen with the early administration of maximal tolerated doses of cytotoxics [62]. Concomitant H G F administration could thus be used to allow an increase in the dose intensity of chemo/
REFERENCES 1. Sieff C. A. (1987) Hematopoietic growth factors. J. clin. Invest. 79, 1549. 2. Clark S. C. & Kamen R. (1987) The human hematopoietic colony-stimulating factors. Science 236, 1229. 3. Bagby G. C. (1989) Interleukin-1 and hematopoiesis. Bood Rev 3, 152. 4. Rennick D., Yang G., Muller-Sieburg C., Smith C., Arai N., Takabe Y. & Gemmell L. (1987) Interleukin 4 (B-cell stimulatory factor 1) can enhance or antagonize the factor-dependent growth of hemopoietic progenitor cells. Proc. nam. Acad. Sci. U.S.A. 84, 6889. 5. Caracciolo D., Clark S. C. & Rovera G. (1989) Human interleukin-6 supports granulocytic differentiation of hematopoietic progenitor cells and acts synergistically with GM-CSF. Blood 73, 666. 6. Bot F. J., Van Eijk L., Broeders L., Aarden L. A. & Lowenberg B. (1989) Interleukin-6 synergizes with MCSF in the formation of macrophage colonies from purified human marrow progenitor cells. Blood 73, 435. 7. Nicola N. A. (1989) Hemopoietic cell growth factors and their receptors. Ann. Reo. Biochem. 58, 45. 8. Sieff C. A. (1988) Haemopoietic growth factors: in vitro and in vivo studies. In Recent Advances in Haematology 5 (Hoffbrand A. V., Ed.), p. 1. Churchill Livingstone, London. 9. Groopman J. E., Mitsuyasu R. T., DeLeo M. J., Oette D. H. & Golde D. W. (1987) Effect of recombinant human granulocyte-macrophage colony-stimulating factor on myeiopoiesis in the acquired immunodeficiency syndrome. New Engl. J. Med. 317, 593. 10. Vadhan-Raj S., Keating M., LeMaistre A., Hittelman W. N., McCredie K., Trujillo J. M., Broxmeyer H. E., Henney C. & Gutterman J. U. (1987) Effects of recombinant human granulocyte-macrophage colonystimulating factor in patients with myelodysplastic syndromes, New Engl. J. Med. 317, 1545. 11. Brandt S. J., Peters W. P., Atwater S. K., Kurtzberg J., Borowitz M. J., Jones R. B., Shpall E. J., Bast R. C., Gilbert C. J. & Oette D. H. (1988) Effects of recombinant human granulocyte-macrophage colonystimulating factor on hematopoietic reconstitution after high-dose chemotherapy and autologous bone marrow transplantation. New Engl. J. Med. 318, 869. 12. Nemunaitis J., Singer J. W., Buckner C. D., Hill R., Storb R., Thomas E. D. & Appelbaum F. R. (1988)
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Use of recombinant human granulocyte-macrophage colony-stimulating factor in autologous bone marrow transplantation for lymphoid malignancies. Blood 72, 834. 13. Devereux S., Linch D. C., Gribben J. G., McMillan A., Patterson K. & Goldstone A. H. (1989) GM-CSF accelerates neutrophil recovery after autologous bone marrow transplantation for Hodgkin's disease. Bone Marrow Transplant. 4, 49. 14. Blazar B. R., Kersey J. H., McGlave P. B., Vallera D. A., Lasky L. C., Haake R. J., Bostrom B., Weisdorf D. R., Epstein C. & Ramsay N, K. C. (1989) In vivo administration of recombinant human granulocytemacrophage colony-stimulating factor in acute lymphoblastic leukaemia patients receiving purged autografts. Blood 73, 849. 15. Peters W. P., Kurtzberg J., Atwater S., Borowitz M., Gilbert C., Rao M., Currie M., Shogan J., Jones R. B., Shpall E. J. & Souza L. (1988) Comparative effects of rHuG-CSF and rHuGM-CSF on hematopoietic reconstitution and granulocyte function following highdose chemotherapy and autologous bone marrow transplantation. Blood 72, (suppl. 1) 130a (abstr). 16. Spitzer G., Jagannath S., Taylor K. Dicke K., Horowitz L., Tucker S., Fogel B. & Souza L. (1989) Studies of recombinant human granulocyte colony-stimulating factor after high dose cyclophosphamide, carmustine and etoposide therapy with autologous bone marrow transplantation in relapsed Hodgkin's disease. Expl Haemat. 17, 517 (abstr). 17. Sheridan W. P., Morstyn G., Wolf M., Dodds A., Lusk J., Maher D., Layton J. E., Green M. D., Souza L. & Fox R. M. (1989) Granulocyte colony-stimulating factor and neutrophil recovery after high-dose chemotherapy and autologous bone marrow transplantation. Lancet ii, 891. 18. Masaoka T., Shibata H., Takaku F., Kodera Y. & Kato S. (1989) Recombinant human granulocyte colony-stimulating factor for allogeneic bone marrow transplantation. Expl Haemat. 17, 521 (abstr). 19. Motoyoshi K., Yoshida K., Hatake K., Saito M., Miura Y., Yanai N., Yamada M., Kawashima T., Wong G. G., Temple P. A., Leary A. C., Witek-Giannoti J. S., Fujisawa M., Yuo A., Okabe T. & Takaku F. (1989) Recombinant and native human urinary colony-stimulating factor directly augments granulocytic and granulocyte-macrophage colony-stimulating factor production of human peripheral blood monocytes. Expl Haemat 17, 68. 20. Komiyama A., Ishiguro A., Kubo T., Matsuoka T., Yasukohchi S., Yasui K., Yanagisawa M., Yamada S., Yamazaki M. & Akabane T. (1988) Increases in neutrophil counts by purified human urinary colonystimulating factor in chronic neutropaenia of childhood. Blood 71, 41. 21. Masaoka T., Motoyoshi K., Takaku F., Kato S., Harada M., Kodera Y., Kanamaru A., Moriyama Y., Ohno R., Ohira M., Shibata H. & Inoue T. (1988) Administration of human urinary colony-stimulating factor after bone marrow transplantation. Bone Marrow Transplant. 3, 121. 22. Duhrsen U., Villeval J-L., Boyd J., Kannourakis G., Morstyn G. & Metcalf D. (1988) Effects of recombinant human granulocyte colony-stimulating factor on hematopoietic progenitor cells in cancer patients. Blood 72, 2074.
23. Socinski M. A., Cannistra S. A., Elias A., Antman K. H., Schnipper L. & Griffin J. D. (1988) Granulocytemacrophage colony-stimulating factor expands the circulating haemopoietic progenitor cell compartment in man. Lancet i, 1194. 24. Gianni A. M., Siena S., Bregni M., Tarella C., Stern A. C., Pileri A. & Bonadonna G. (1989) Granulocytemacrophage colony-stimulating factor to harvest circulating haemopoietic stem cells for autotransplantation. Lancet ii, 580. 25. Nemunaitis J., Singer J. W., Epstein C., Buckner C. D., Storb R., Hill R., Thomas E. D. & Appelbaum F. R. (1989) The use of rhGM-CSF for graft failure in patients after autologous or allogeneic bone marrow transplantation. Expl Haemat. 17, 388 (abstr). 26. Antin J. H., Smith B. R., Holmes W. & Rosenthal D. S. (1988) Phase I/II study of recombinant human granulocyte-macrophage colony-stimulating factor in aplastic anemia and myelodysplastic syndrome. Blood 72, 705. 27. Champlin R. E., Nimer S. D., Ireland P., Oette D. H. & Golde D. W. (1989) Treatment of refractory aplastic anemia with recombinant human granulocyte-macrophage colony-stimulating factor. Blood 73, 694. 28. Nissen C., Tichelli A., Gratwohl A., Speck B., Milne A., Gordon-Smith E. C. & Schaedlin J. (1988) Failure of recombinant human granulocyte-rnacrophage colony-stimulating factor in aplastic anemia patients with very severe neutropenia. Blood 72, 2045. 29. Nissen-Druey C. (1989) Pathophysioiogy of aplastic anaemia. In Clinical Haematology 2 (Gordon-Smith E. C., Ed.), p. 37. Bailliere Tindall, London. 30. Morrissey P. J., Bressler L., Park L. S., Alpert A. & Gillis S. (1987) Granulocyte-macrophage colonystimulating factor augments the primary antibody response by enhancing the function of antigen-pesenting cells. J. lmmun. 139, 1113. 31. Lopez A. F., Williamson D. J., Gamble J. R., Begley C. G., Harlan J. M., Klebanoff S. J., Waltersdorph A., Wong G., Clark S. C. & Vadas M. A. (1986) Recombinant human granulocyte-macrophage colonystimulating factor stimulates in vitro mature human neutrophil and eosinophil function, surface receptor expression, and survival. J. clin. Invest. 78, 1220. 32. Fleischmann J., Golde D. W., Weisbart R. H. & Gasson J. C. (1986) Granulocyte-macrophage colonystimulating factor enhances phagocytosis of bacteria by human neutrophils. Blood 68, 708. 33. Weisbart R. H., Kwan L., Golde D. W. & Gasson J. C. (1987) Human GM-CSF primes neutrophils for enhanced oxidative metabolism in response to the major physiological chemoattractants. Blood 69, 18. 34. Arnaout M. A., Wang E. A., Clark S. C. & Sieff C. A. (1986) Human recombinant granulocyte-macrophage colony-stimulating factor increases cell-to-cell adhesion and surface expression of adhesion-promoting glycoproteins on mature granulocytes. J. olin. Invest. 78, 597. 35. Rettenmier C. W. & Sherr C. J. (1989) The mononuclear phagocyte colony-stimulating factor (CSF-1, M-CSF). In Hematology/Oncology Clinics of North America 3 (Golde, D. W., Ed.), p. 479. W. B. Saunders, Philadelphia. 36. Devereux S., Linch D. C., Campos Costa D., Spittle M. F. & Jelliffe A. M. (1987) Transient leucopaenia induced by granulocyte-macrophage colony-stimulating factor. Lancet ii, 1523.
Growth factors and transplantation 37. Devereux S., Bull H. A., Campos Costa D., Saib R. & Linch D. C. (1989) Granulocyte-macrophage colony-stimulating factor induced changes in cellular adhesion molecule expression and adhesion to endothelium: in vitro and in vivo studies in man. Br. J. Haemat. 71, 323. 38. Addison I. E., Johnson B., Devereux S., Goldstone A. H. & Linch D. C. (1989) Granulocyte-macrophage colony-stimulating factor may inhibit neutrophil migration in oivo. Clin. exp. Immun. 76, 149. 39. Peters W. P., Stuart A., Affronti M. L., Kim C. S. & Coleman R. E. (1988) Neutrophil migration is defective during recombinant granulocyte-macrophage colonystimulating factor infusion after autologous bone marrow transplantation in humans. Blood 72, 1310. 40. Sullivan R., Fredette J. P., Socinski M., Elias A., Antman K., Schnipper L. & Griffin J. D. (1989) Enhancement of superoxide anion release by granulocytes harvested from patients receiving granulocyte-macrophage colony-stimulating factor. Br. J. Haemat. 71, 475. 41. Devereux S., Porter J. B., Hoyes K. P., Abeysinghe R. D., Saib R. & Linch D. C. (1989) Secretion of neutrophil secondary granules occurs during granulocyte-macrophage colony-stimulating factor induced margination. Br. J. Haemat. (in press). 42. Gallin J., Fletcher M., Seligmann B., Hoffstein S. & Cehrs K. (1982) Human neutrophil specific granule deficiency: a model to assess the role of neutrophil specific granules in the evolution of the inflammatory response. Blood 59, 1317. 43. Ho A. D., Grossman M., Mannel D. N., Haas R., Knauf W., Trumper L., Schulz G. & Hunstein W. (1988) In vivo administration of GM-CSF induces elevation of soluble interleukin-2 receptor and CD8 antigens in peripheral blood. Blood 72, (suppl. 1) 121a (abstr). 44. Cannistra S. A., Vellenga E., Groshek P., Rambaldi A. & Griffin J. D. (1988) Human granulocyte-monocyte colony-stimulating factor and interleukin 3 stimulate monocyte cytotoxicity through a tumor necrosis factor-dependent mechanism. Blood 71, 672. 45. Sisson S. D. & Dinarello C. A. (1988) Production of interleukin-1 a, interleukin-1 b and tumor necrosis factor by human mononuclear cells stimulated with granulocyte-macrophage colony-stimulating factor. Blood 72, 1368. 46. Wing E. J., Magee D. M., Whiteside T. L., Kaplan S. S. & Shadduck R. K. (1989) Recombinant human granuiocyte/macrophage colony-stimulating factor enhances monocyte cytotoxicity and secretion of tumor necrosis factor oc and interferon in cancer patients. Blood 73, 643. 47. Hoang T., Nara N., Wong G., Clark S., Minden M. D. & McCulloch E. A. (1986) Effects of recombinant GM-CSF on the blast cells of acute myeloblastic leukaemia. Blood 68, 313. 48. Miyauchi J., Kelleher C. A., Yang Y-C., Wong G. G., Clark S. C., Minden M. D., Minkin S. & McCulloch E. A. (1987) The effects of three recombinant growth factors, IL-3, GM-CSF, and G-CSF, on the blast cells of acute myeloblastic leukaemia maintained in shortterm suspension culture. Blood 70, 657.
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49. Ganser A., Volkers B., Greher J., Ottman O. G., Walther F., Becher R., Bergmann L., Schulz G. & Hoelzer D. (1989) Recombinant human granuiocytemacrophage colony-stimulating factor patientsin with myelodysplastic syndromes---a phase I/II trial. Blood 73, 31. 50. Negrin R. S., Haeuber D. H., Nagler A , Olds L. C., Donlon T., Souza L. M. & Greenberg P. L. (1989) Treatment of myelodysplastic syndromes with recombinant human granulocyte colony-stimulating factor. Ann. Intern. Med. 110, 976. 51. De Witte T., Muus P., Haanen C., Van der Lely N., Koekman E., Van der Locht A., Blankenborg G. & Wessels J. (1988) GM-CSF enhances sensitivity of leukemic clonogenic cells to long-term low dose cytosine arabinoside with sparing of the normal clonogenic cells. Behring Inst. Mitt. 83, 301. 52. Tafuri A., Hegewisch S., Souza L. & Andreef M. (1988) Stimulation of leukemic blast cells in vitro by colony-stimulating factors (G-CSF, GM-CSF) and interleukin 3: evidence of recruitment and increased cell killing with cytosine arabinoside. Blood 72, (suppl. 1) 105a (abstr). 53. Baldwin G. C., Gasson J. C., Kaufman S. E., Quan S. G., Williams R. E., Avalos B. R., Gazdar A. F., Golde D. W. & DiPersio J. F. (1989) Nonhematopoietic tumor ceils express functional GM-CSF receptors. Blood 73, 1033. 54. Salmon S. E. & Liu R. (1989) Effects of granulocytemacrophage colony-stimulating factor on in vitro growth of human solid tumors. J. clin. Oncol. 7, 1346. 55. Blazar B. R., Widmer M. B., Soderling C. C. B., Urdal D. L., Gillis S., Robison L. L. & Vallera D. A. (1988) Augmentation of donor bone marrow engraftment in histoincompatible murine recipients by granulocytemacrophage colony-stimulating factor. Blood 71,320. 56. Hancock W. W., Pleau M. E. & Kobzik L. (1988) Recombinant granulocyte-macrophage colony-stimulating factor down-regulates expression of IL-2 receptor on human mononuclear phagocytes by induction of prostaglandin E. J. Imrnun. 140, 3021. 57. Krumwieh D., Weinmann E. & Seiler F. R. (1988) Human recombinant IL-3 and GM-CSF in hematopoiesis of normal cynomolgus monkeys. Behring Inst. Mitt. 83, 250. 58. Ganser A., Lindemann A., Ottman O. G., Hermann F., Schulz G., Mertelsmann R. & Hoelzer D. (1989) Effect of recombinant human interleukin-3 in vioo-a phase 1 trial. Expl Haemat. 17, 40 (abstr). 59. Neta R., Douches S. & Oppenheim J. J. (1986) Interleukin 1 is a radioprotector. J. Immun. 136, 2483. 60. McDonald T. P., Clift R. E. & Cottrell M. B. (1989) A four-step procedure for the purification of thrombopoietin. Expl Haemat. 17, 865. 61. Goldstone A. H. & McMillan A. K. (1989) Bone marrow transplantation for non-Hodgkin's lymphoma in Europe--the current position. In European School of Oncology Monograph (in press). 62. DeVita V. T. (1985) In Principles and Practise of Oncology. 2nd edn., p. 256. Lippincott, Philadelphia.
0145-2126/90 $3.00 + .00 Pergamon Press pie
THE ROLE OF H A E M O P O I E T I C GROWTH FACTORS IN BONE M A R R O W TRANSPLANTATION ANTHONY H. GOLDSTONE* and ASIM KHWAJAt *University College Hospital, London and tDepartment of Haematology, University College and Middlesex School of Medicine, London, U.K.
(Received for publication 20 March 1990) Abstract--The availability of recombinant haemopoietic growth factors for clinical use has led to a proliferation of trials in the setting of bone marrow transplantation. Early results from these studies, using GM-CSF and G-CSF, show that these factors are able to reduce the period of cytotoxic induced neutropaenia but have little effect on platelet recovery. Toxicity has been relatively mild unless very high doses are administered. Randomised controlled trials are in progress and will help to define the exact role of growth factors in this setting. Future prospects include the increasing availability of other growth factors for clinical use and the potential for combination growth factor therapy to provide a more optimal haemopoietic response.
Key words: Colony-stimulating factor, bone marrow transplantation, haemopoietic growth factors.
PROPERTIES AND TYPES OF HGFs
INTRODUCTION
The various HGFs may best be considered as three main groups of factors with overlapping activities (Table 1). The lineage-specific factors, which include granulocyte colony-stimulating factor (G-CSF), monocyte colony-stimulating factor (M-CSF), and erythropoietin (epo) act on mature progenitor cells during the development of specific cell lineages [1]. In addition to their effects on haemopoiesis, G-CSF and M-CSF influence the biological activities of neutrophils and monocytes respectively. The multilineage CSFs are less restricted in their activity and include granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin 3 (IL-3). GMCSF is active in granulopoiesis in vitro, supports the proliferation of macrophage colonies, promotes early erythroid colony formation (in the presence of epo) and enhances the proliferation of megakaryocytic progenitors [2]. GM-CSF is also a survival and activating factor for neutrophils, eosinophils and monocytes. IL-3 has multilineage activity and probably acts on progenitor cells at an earlier stage in the differentiation pathway than GM-CSF; it supports the proliferation of myeloid, macrophage and megakaryocyte lineages and activates mature eosinophils and monocytes. The third group of HGFs, of which the properties of interleukin 1 (IL-1) are the best characterised, act on primitive haemopoietic progenitors and render them sensitive to the later acting factors [3]. There is increasing evidence that IL-4 and IL-6 may play a similar role in haemopoiesis [4--6].
THE HUMANhaemopoietic growth factors (HGFs) are a family of glycoproteins responsible for sustaining the proliferation, differentiation and survival of immature haemopoietic progenitor cells. Autologous and allogeneic BMT is increasingly being used as salvage or first line therapy in the treatment of both solid tumours and haematological malignancies and as many of the problems associated with BMT are related to the period of bone marrow aplasia following cytotoxic therapy, there has been great interest in the use of HGFs to facilitate earlier engraftment. The genes for a number of growth factors have now been cloned and the application of recombinant DNA methodology to produce large amounts of these factors has led to an increased understanding of their role in regulating haemopoiesis and also of their potential clinical application.
Abbreviations: HGF, haemopoietic growth factor; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL-3, interleukin 3; BMT, bone marrow transplantation; ABMT, autologous bone marrow transplantation; M-CSF, monocyte colony-stimulating factor; CSF-HU, human urinary colony-stimulating factor; ANC, absolute neutrophil count. Correspondence to: Dr A. H. Goldstone, Department of Haematology, University College Hospital, Grafton Way, London WC1 6AU, U.K. 721
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A. H. GOLDSTONE and A. KHWAJA TABLE 1. PROPERTIES OF HAEMOPOIETIC GROWTH FACTORS
Protein size (kDa) Chromosomal location
Core
Factor G-CSF
17q21-22
18.6
20
M-CSF
5q33.1
26 16
70-90 40-50
Epo
7ql 1-22
18
34-39
GM-CSF
5q21-31
14-15
18-35
Biological activity
Glycosylated
Cellular source
IL-3
5q21-31
14-15
20-26
monocytes fibroblasts monocytes fibroblasts endothelial cells kidney (monocytes) T cells endothelial cells fibroblasts monocytes T cells
IL-5
5q31
13
20-30
T ceils
Progenitor cells
Mature cells
n
n
rn
rn
ery (meg)
n, m, eo,
n, m,
ery meg
eo
n, m, eo, b, ery
eo, m
meg
eo
eo
n, neutrophil; eo, eosinophil; b, basophil; ery, erythroid; m, monocyte/macrophage; meg, megakaryocyte.
TABLE 2. THE POTENTIAL USES OF H G F S IN BONE MARROW TRANSPLANTATION
1. Reduce the period of cytotoxic induced cytopaenia. 2. Enhance the harvesting of peripheral blood progenitor cells for ABMT. 3. Stimulate leukaemic cells into cycle prior to chemotherapy. 4, Augment phagocyte response against infection. 5. Enhance host anti-tumour response.
All the HGFs are active at low (pM) concentrations on target haemopoietic and mature cells which bear relatively low numbers of specific high affinity receptors [7]. In the native state they are all glycosylated. Recombinant factors derived from yeast or mammalian cell systems are also glycosylated, whereas those expressed in bacterial systems (such as E. coli) will be non-glycosylated. The degree of glycosylation may have clinical relevance with regard to efficacy and antigenicity of the recombinant molecule. The most heavily glycosylated form of GM-CSF is not as active in vitro but is cleared more slowly in vivo than the non-glycosylated form [8]. O V E R V I E W OF P O T E N T I A L USES OF HGFs IN BONE M A R R O W T R A N S P L A N T A T I O N The most obvious, and so far the most frequently investigated, role for HGFs in BMT is to reduce the period of cytopaenia induced by cytotoxic therapy. However, there are other possible areas of use (Table 2). In the non-transplant setting, perhaps the most
interesting is the potential for improving tumour response rates by permitting dose escalation of cytotoxic agents and thereby reducing the need for intensive salvage treatment regimens.
CLINICAL STUDIES OF HGFs IN BMT GM-CSF
This was the first H G F to be used in BMT after initial studies in patients with the acquired immunodeficiency syndrome [9] and myelodysplastic syndrome (MDS) [10] demonstrated a dose-dependent rise in leucocyte counts. At Duke University, 19 patients undergoing autologous BMT (ABMT) for disseminated melanoma and carcinoma of the breast received continuous intravenous (i.v.) GM-CSF for 14 days after marrow re-infusion at doses ranging from 1.2 to 19.2 ~tg/kg/day [11]. Total leucocyte and granulocyte recovery was accelerated when compared with historical controls although this did not reach statistical significance. There was a fall in the leucocyte count over a 3-4 day period on stopping GM~CSF, after which haemopoietic recovery proceeded at a rate similar to that in control patients. No consistent effect on recovery of platelet counts was seen. Patients receiving GM-CSF had fewer episodes of culture positive bacteraemia (16% vs 35%) and less of an elevation in creatinine and bilirubin levels during the post-transplant period. The authors postulated that inhibition of bacteraemia and consequent multiple organ dysfunction may have been responsible for the latter finding. Significant toxicity
Growth factors and transplantation TABLE 3. SIDE EFFECTS OF H G F TREATMENT
GM-CSF Low dose High dose
bone pain fever thrombophlebitis capillary leak syndrome central venous thrombosis
G-CSF bone pain myalgia fever
(Table 3) was observed in the group of patients receiving 19.2 ~tg/kg/day with erythroderma and a capillary leak syndrome characterised by oedema, pleuro-pericardial effusions and weight gain. This toxicity reversed with discontinuation of GM-CSF. The Seattle group have also reported results from a study involving 15 patients undergoing ABMT for lymphoid malignancies [12]. Patients in the study group received GM-CSF by daily 2 h i.v. infusions at a dose range between 15 and 240 Ixg/m2/day for 14 days and comparison was made with a historical group of patients who had not received growth factors. Patients receiving more than 60 ~tg/m2/day showed accelerated recovery of neutrophil counts (days to 0.5 × 109/1, 14 vs 25), became independent of platelet transfusions quicker (29 vs 38 days), had fewer febrile days and a reduced period of hospitalisation. Fewer culture positive bacteraemias were also noted in this study (13% vs 30%) and no significant GM-CSF associated toxicity observed. In our own centre, 12 patients with resistant Hodgkin's disease receiving high-dose chemotherapy and ABMT were treated with doses of GM-CSF between 100 and 400 ~tg/mE/day [13]. The median time taken to reach an absolute neutrophil count (ANC) of 0.5 × 109/1 in those receiving GM-CSF was 16.3 days compared with 25 days in 19 concurrent controls. No consistent effect on platelet recovery was seen and there was no difference between the two groups with regard to the rate of bacterial infection, number of febrile days or period of hospitalisation. The Minnesota group have recently reported their results of GM-CSF treatment in 25 patients having ABMT for acute lymphoblastic leukaemia (ALL) [14]. This study differed from others previously reported in that bone marrows were purged ex oivo with 4-hydroperoxycyclophosphamide and lineage specific antibodies before transplantation. The dose range of GM-CSF administered was between 16 and 256 ~tg/m2/day--patients receiving less than 60 ~tg/ m2/day did not show any response and of those receiving more than this dose, only patients reinfused
723
>1.2 x 104 granulocyte-monocyte colony forming units (CFU-GM) per kg showed an accelerated haemopoietic recovery. G-CSF
The use of G-CSF in BMT has now been reported in several studies and the overall results strongly suggest a significant reduction in the period of cytotoxic induced neutropaenia. At Duke University, 15 patients undergoing ABMT for solid tumours were treated with G-CSF by continuous i.v. infusion at doses between 16 and 64 txg/kg/day for 14 days [15]. Neutrophil recovery was significantly accelerated and no significant toxicity observed. The MD Anderson group have reported a study of G-CSF in 26 patients with Hodgkin's disease, 18 by bolus and 8 by continuous infusion, at doses between 30 and 120 ~tg/kg/ day for up to 28 days post-ABMT [16]. Days to reach an ANC of 0.5 x 109/1 were 13 in the bolus dose group, 11 in the continuous infusion group and 22 in a group of historical controls. The treated group had fewer febrile days and a lower rate of gram positive infections (17% vs 39%). There was minor toxicity consisting of myalgia and bone pain in 5 of 26 patients. Sheridan and co-workers have reported the use of subcutaneously administered G-CSF at a dose of 20 ~tg/kg/day in ABMT for a range of malignant disorders [17]. As well as a shortened neutropaenic period, they were able to demonstrate both a significant reduction in antibiotic usage and a shorter duration of hospitalisation when compared with historical controls, a finding with potentially important economic implications. To date the only report of the systematic use of GCSF in allogeneic BMT has come from the Japan BMT Study Group [18]. Thirty-four patients (10 ALL, 8 acute myeloblastic leukaemia, 9 chronic myeloid leukaemia, 4 severe aplastic anaemia, 3 lymphoma) received either 200 or 400 ~tg/m2 of G-CSF for 14 days. Median time to an ANC of 0.5 × 109/1 was 17 days in the group receiving the lower dose, 15 days in those treated with the higher dose of GCSF and 27.4 days in a group of historical controls. No effect on platelet or reticulocyte counts was seen and no obvious effect on the incidence or severity of graft versus host disease (GVHD) noted. The cohort of patients is still at an early stage but to date no adverse effect on disease relapse rate has been observed. CSF-HU
This is a dimeric glycoprotein of molecular weight 84,000 derived from human urine which binds to monocytes and stimulates the formation of G-CSF [19]. The primary structure of CSF-HU contains the
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A.H. GOLDSTONEand A. KHWAJA
The intravenous administration of both G-CSF and GM-CSF has been shown to increase markedly the numbers of circulating peripheral blood C F U - G M and erythroid burst foiming units (BFU-E) [22, 23]. This has led to an interest in the use of HGFs in facilitating the harvest of peripheral blood progenitors for autotransplantation and an Italian group have recently reported their results of the use of GM-CSF in this setting [24]. GM-CSF was used to accelerate recovery from cyclophosphamide induced cytopaenia in a group of seven patients with large cell lymphoma. Patients underwent leucaphereses in the recovery phase from chemotherapy and high yields of C F U - G M (mean of nine-fold increase over seven control patients treated with identical chemotherapy without GM-CSF) were obtained. Conditioning with melphalan/total body irradiation was followed by re-infusion of both previously harvested autologous bone marrow and peripheral stem cells. Four out of seven patients received continuous GMCSF infusions (5.5 ~tg/kg/day) after the re-infusion of autologous cells. Overall results for the seven patients (results for control patients) were a median time of 9.1 (11.4) days to an ANC of 0.5 x 109/1 and 10.7 (16.9) days to reach a platelet count of 50 x 109/ 1. These differences were modest but highly significant. The fact that patients received both autologous marrow and peripheral progenitors, and that four of seven patients were treated with GM-CSF after stem cell re-infusion, makes it difficult to reach any firm conclusions about the role of GM-CSF in this setting, but the results are sufficiently encouraging to merit further evaluation.
>0.1 x 109/1 by day 28 post-BMT or the development of a sustained drop in ANC to 27 x 109/1 with > 5 0 % eosinophils. Nissen et al. report the use of GM-CSF in four patients with very severe A A , three of whom had no evidence of residual myelopoiesis [28]. Only the patient with minimal residual myelopoiesis responded with an increase in neutrophil numbers and resolution of infection. One of the main mechanisms of the development of A A is presumed to be a qualitative or quantitative defect in stem cell numbers and patients often have high circulating levels of endogenous HGFs [29]; therefore it is not surprising that GM-CSF may have little effect in patients with very severe AA. So is there a role for the use of HGFs in AA? They may be useful as interim therapy for patients who are awaiting a response to antilymphocyte globulin ( A L G ) , which may take up to 12 weeks to have a beneficial effect, and for those patients for whom a suitable donor for B M T is being sought. In view of the enhancing effect of GM-CSF on antigen presenting cells however [30], there may be an increased risk of allo-immunisation with consequent difficulties in transplantation. There may be a role for HGFs in the long term treatment of patients who have no suitable donor for B M T and who have not responded to A L G , but who still have some residual myelopoiesis. In the light of the so far disappointing lack of effect of GM-CSF on RBC or platelet transfusion requirements, future combination therapy with HGFs may prove to be more effective in this setting.
HGFs 1N E N G R A F T M E N T F A I L U R E AND A P L A S T I C A N A E M I A (AA)
EFFECTS OF HGFs ON P H A G O C Y T E FUNCTION
GM-CSF has been used by the Seattle group for graft failure after allogeneic and autologous BMT [25]. Graft failure in this study was defined as a failure to reach an absolute neutrophil count (ANC)
G-CSF and GM-CSF are both known to affect the survival and function of mature phagocytes. In-vitro studies show that GM-CSF prolongs the survival of neutrophils and eosinophils, enhances the ability of
amino acid sequence of human M-CSF in each of its monomeric subunits. CSF-HU stimulates the production of neutrophils in v i v o [20] and has been administered to 37 Japanese patients undergoing allogeneic B M T [21]. Results from this study show that granulocyte recovery to an ANC of 0.5 x 109/1 was a mean of 22 days in the treatment group and 28 days in matched historical controls. There is no data provided for platelet recovery. The authors report no detrimental effect on engraftment, incidence or grade of G V H D or leukaemic relapse. HGFs IN P E R I P H E R A L STEM C E L L BMT
Growth factors and transplantation phagocytes to kill tumour targets via antibody dependent cellular cytotoxicity ADCC [31], stimulates phagocytosis [32] and augments neutrophil superoxide production in response to physiological chemoattractants such as the bacterial peptide f-MLP, complement derived C5a and leukotriene B4 [33]. GM-CSF also stimulates the surface expression of the neutrophil cellular adhesion molecules (CAMs) recognised by the antibodies C D l l b and CD18 [34]. G-CSF enhances neutrophil ADCC, production of superoxide anions, and phagocytosis [2]. M-CSF has similar effects on monocytes [35]. Administration of GM-CSF in vivo has been shown to cause a transient leucopaenia maximal 30 min after starting an infusion [36]. Radionuclide labelling studies show this to be due to margination of neutrophils and monocytes predominantly in the pulmonary vasculature. This margination may be related to endothelial adherence mediated by upregulation of CAM expression [37]. However, as neutrophil demargination occurs at a time when CAM expression is still high, other factors must be involved in producing this phenomenon. The pulmonary sequestration of neutrophils may have important clinical consequences as symptoms and signs of consolidation have been observed in patients with pre-existing lung pathology and/or relatively high numbers of circulating granulocytes [13, 27]. It suggests that GM-CSF may best be given as a continuous infusion rather than intermittent bolus doses to avoid repeated margination and demargination. Furthermore, it may be wise not to prolong unnecessarily the administration of this growth factor once neutrophil recovery after BMT has taken place. Studies examining the effects of HGFs on the migration of neutrophils into an artificially created sterile inflammatory site ("skin window") have shown that neutrophil migration during GM-CSF infusion is significantly impaired [38, 39]. This inhibition may be due to the systemic administration of a chemokinetic substance giving a non-directional stimulus to the circulating neutrophil and impairing its subsequent response to an appropriate signal from an inflammatory focus. Such an impairment of phagocyte tissue penetration should focus the attention of investigators on the risk of exacerbating deep seated infection during GM-CSF therapy and suggests that the use of this factor as adjuvant therapy for the treatment of severe infections in patients with normal neutrophil counts may not necessarily be appropriate. A study looking at neutrophil migration during G-CSF infusions following ABMT [15] showed that, in contrast to the findings with GM-CSF, there was no significant difference in this response between patients on growth factor therapy and matched controis.
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Enhanced superoxide release from neutrophils harvested from patients receiving intravenous infusions of GM-CSF has also been reported [40]. Such augmentation of the neutrophil response could be of benefit in responding to circulating microorganisms and may be responsible for the reduced rate of culture positive bacteraemia observed in several studies [11, 12]. However, the presence of activated cells in the circulation that may have an impaired tissue migratory response could result in endothelial damage. Secretion of neutrophil secondary granules during infusions of GM-CSF has also been described [41]. These contain substances such as lactoferrin and transcobalamins which have bacteriostatic functions, and enzymes which may be involved in neutrophil migration through the vascular endothelium. Patients with congenital deficiencies of secondary granules have been described and their neutrophils show a reduced ability to migrate into inflammatory foci [42]; therefore, a non-specific release of these granules caused by GM-CSF administration may be associated with the observed impairment of neutrophil migration and be detrimental to host defence. EFFECTS OF HGFs ON IMMUNE FUNCTION GM-CSF has been shown to have direct effects on host immune function. In the murine model, it has been shown to enhance the function of antigen presenting cells by increasing the expression of class II MHC antigens and increasing IL-1 production [30]. In this study, IL-2 production by T cells in response to a mitogenic stimulus was also enhanced by exposure to GM-CSF. Administration of GM-CSF in vivo in humans with lymphoid malignancies has been reported as causing increases in circulating soluble IL-2 receptor levels as well as enhancing the production of IL-2 by peripheral blood mononuclear cells (PBMC) [43]. GM-CSF has been shown to enhance the production of both IL-lc~ and /3 and tumour necrosis factor by PBMC in vitro [44, 45] and to enhance monocyte cytotoxicity in samples taken from patients on growth factor therapy [46]. The production of these factors may be responsible for some of the toxicity seen with GM-CSF treatment at relatively high doses, but has potentially valuable anti-tumour effects in the context of minimal residual disease. EFFECTS OF HGFs ON LEUKAEMIC CELLS In-vitro studies have reported that GM-CSF is able to stimulate AML blast colony formation in methylcellulose cultures and can enhance the growth
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A . H . GOLDSTONE and A. KHWAJA
of blast progenitors in suspension, suggesting an ability to support blast self-renewal [47]. Other work has shown that GM-CSF, G-CSF and IL-3 were all able to stimulate AML blast self-renewal and terminal division with varying responses between patients [48]. The possibility of stimulating myeloid leukaemic growth in vivo is perhaps best assessed by looking at the effects of GM-CSF treatment as reported in studies in patients with pre-leukaemic states. Administration of GM-CSF in MDS has yielded variable results with regard to stimulation of blast numbers. Vadhan-Raj and colleagues reported no increase in absolute blast counts in a group of eight patients, four of whom had refractory anaemia with excess blasts in transformation (RAEBt) by the FAB classification [10]. Antin and colleagues reported a reversible increase in myeloblasts in two of seven patients with MDS [26]; the presence of RAEBt was one of the exclusion criteria in this study. More recently, Ganser has described an increase in marrow myeloblast count in four of eleven patients treated with GM-CSF [49]. These rises were only seen in patients with pre-GM-CSF treatment bone marrow blast counts greater than 14%. Negrin has reported the use of G-CSF in MDS and has found no evidence of leukaemic stimulation [50]. Stimulation of leukaemic cell growth and proliferation by HGFs may be used to the potential benefit of the patient to synchronize the entry of malignant cells into the cell cycle and render them more sensitive to cycle-specific cytotoxic therapy. Invitro work using leukaemic blast cell cultures shows that incubation with cytosine arabinoside (Ara-C) after treatment with HGFs increases cell kill [51,52]. Approaches combining HGF treatment with cytotoxic therapy may have a place in the treatment of myeloid leukaemias with and without BMT and merit further evaluation.
THE EFFECTS OF HGFs ON NONHAEMOPOIETIC CELLS As ABMT is now often used for the treatment of non-haemopoietic malignancies, studies examining the effects of HGFs on tumour cells of non-haematological origin have increasing relevance. It has been reported that GM-CSF receptors are present on certain small cell carcinoma lines and that incubation with GM-CSF enhances their proliferation [53]. More recently, Salmon has described no significant inhibitory or stimulatory effect of GM-CSF on the growth of cells taken from tumour biopsies of various cell types [54].
THE EFFECTS OF HGFs ON ENGRAFTMENT Theoretical concern about HGF treatment leading to stem cell "exhaustion" in the post-transplant period has largely been negated by the results of the clinical trials as reported so far, with no evidence of late graft failure. Blazar has described the results of the use of recombinant murine GM-CSF (rmuGMCSF) in a murine model of major histocompatibility complex mismatched allogeneic BMT [55]. In a system where T-cell depletion of donor marrow leads to a low incidence of GVHD and a high rate of engraftment failure, it was demonstrated that donor graft incubation ex vivo with, or a single injection in vivo on the day of marrow infusion of, rmuGM-CSF were both effective means of promoting engraftment. This effect was only seen in experiments where animals had a very low rate of engraftment; where the engraftment rate in control animals was relatively high, no further enhancement was seen with the use of rmuGM-CSF. The mechanism by which rmuGMCSF promoted engraftment is not clear. The only dose at which any positive effect was seen was several fold higher than that required for bone marrow receptor saturation--it is possible that the effect of GMCSF may have been an indirect one, perhaps mediated by the release of other cytokines. In another study, it was demonstrated that GM-CSF down-regulated the expression of IL-2 receptor (IL2-R) on human monocyte/macrophages via a prostaglandin E dependent mechanism [56]. This effect may be relevant to the transplant situation as it has been demonstrated that IL-2-R positive macrophages and T cells may have a significant role in mediating allograft rejection and GVHD.
FUTURE APPROACHES IN THE USE OF HGFs The effects of other HGFs have been studied in animals, both singly and in combination, and in the case of IL-3 in preliminary human trials. IL -3
Overall data from animal work suggests that administration of IL-3 on its own has little haemopoietic effect [57]. However, combination treatment with IL-3 followed by GM-CSF has been reported as causing marked increases in the numbers of circulating neutrophils, monocyte, reticulocytes and platelets in non-human primates [57]. A recent report describes the use of subcutaneous IL-3 in patients with advanced neoplasms [58]. Fourteen patients have received IL-3 for 15 days at doses between 60 and 500 ~tg/m2---of the nine evaluable patients, all have shown increases in neutrophil,
Growth factors and transplantation eosinophil, monocyte, reticulocyte and platelet numbers. These increases occurred in the second week of treatment, as might be expected on theoretical grounds, and continued for 1-2 weeks after the end of IL-3 administration. There was a mean increase of 179 x 109/1 in platelet numbers and two patients who had "prolonged thrombocytopaenia" with counts of 3 × 109/1 and 22.5 × 109/1 increased to 104 × 109/1 and 184 × 109/1 respectively. These initial results are extremely encouraging and trials of IL-3 in the setting of BMT are awaited with interest.
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radiotherapy while keeping myelotoxicity at acceptable levels; improvement in tumour responses to such dose escalation has the potential for reducing the requirement for later more intensive salvage regimens requiring BMT. We are still in the early stages of discovering the true potential of the clinical use of HGFs; it is imperative that investigation of their efficacy should be conducted in well designed trials, and that conclusions about their definitive role in cancer treatment and BMT be based on objective clinical evidence of patient benefit and not merely on observed manipulations of the blood count.
IL-1
In mice, IL-1 given before sublethal total body irradiation has been reported to prevent severe myelosuppression [59]--the mechanism for this is obscure and difficult to evaluate in vivo, as IL-1 has multiple effects, including the stimulation of production of other growth factors.
CONCLUSIONS The results of preliminary clinical trials with HGFs show that treatment with these factors can reduce the period of neutropaenia following BMT. Disappointingly, there has so far been no clear evidence of acceleration of platelet regeneration in trials with GM-CSF, as might have been predicted by its activities in vitro. Effective reduction in the post-transplant thrombocytopaenic period may have to await the isolation of "thrombopoietin" [60] or may be effected by combination growth factor therapy, for example with IL-3 and GM-CSF. Whether treatment with HGFs will lead to a reduction in treatment related morbidity and mortality associated with BMT will become clearer with the publication of the results of current randomised trials. Death due to infection during BMT is now an event with a relatively low frequency [61] and it may prove difficult to demonstrate a significant reduction in this parameter as a consequence of H G F use. If the use of growth factors can be shown to reduce the use of antimicrobials and blood products and decrease the length of inpatient stay then this will have obvious economic advantages as well as increasing patient acceptability of a procedure such as BMT. Perhaps the most interesting potential application of HGFs is in allowing cytotoxic dose escalation in the non-transplant setting. There is good evidence in the treatment of several tumour types that the best responses are seen with the early administration of maximal tolerated doses of cytotoxics [62]. Concomitant H G F administration could thus be used to allow an increase in the dose intensity of chemo/
REFERENCES 1. Sieff C. A. (1987) Hematopoietic growth factors. J. clin. Invest. 79, 1549. 2. Clark S. C. & Kamen R. (1987) The human hematopoietic colony-stimulating factors. Science 236, 1229. 3. Bagby G. C. (1989) Interleukin-1 and hematopoiesis. Bood Rev 3, 152. 4. Rennick D., Yang G., Muller-Sieburg C., Smith C., Arai N., Takabe Y. & Gemmell L. (1987) Interleukin 4 (B-cell stimulatory factor 1) can enhance or antagonize the factor-dependent growth of hemopoietic progenitor cells. Proc. nam. Acad. Sci. U.S.A. 84, 6889. 5. Caracciolo D., Clark S. C. & Rovera G. (1989) Human interleukin-6 supports granulocytic differentiation of hematopoietic progenitor cells and acts synergistically with GM-CSF. Blood 73, 666. 6. Bot F. J., Van Eijk L., Broeders L., Aarden L. A. & Lowenberg B. (1989) Interleukin-6 synergizes with MCSF in the formation of macrophage colonies from purified human marrow progenitor cells. Blood 73, 435. 7. Nicola N. A. (1989) Hemopoietic cell growth factors and their receptors. Ann. Reo. Biochem. 58, 45. 8. Sieff C. A. (1988) Haemopoietic growth factors: in vitro and in vivo studies. In Recent Advances in Haematology 5 (Hoffbrand A. V., Ed.), p. 1. Churchill Livingstone, London. 9. Groopman J. E., Mitsuyasu R. T., DeLeo M. J., Oette D. H. & Golde D. W. (1987) Effect of recombinant human granulocyte-macrophage colony-stimulating factor on myeiopoiesis in the acquired immunodeficiency syndrome. New Engl. J. Med. 317, 593. 10. Vadhan-Raj S., Keating M., LeMaistre A., Hittelman W. N., McCredie K., Trujillo J. M., Broxmeyer H. E., Henney C. & Gutterman J. U. (1987) Effects of recombinant human granulocyte-macrophage colonystimulating factor in patients with myelodysplastic syndromes, New Engl. J. Med. 317, 1545. 11. Brandt S. J., Peters W. P., Atwater S. K., Kurtzberg J., Borowitz M. J., Jones R. B., Shpall E. J., Bast R. C., Gilbert C. J. & Oette D. H. (1988) Effects of recombinant human granulocyte-macrophage colonystimulating factor on hematopoietic reconstitution after high-dose chemotherapy and autologous bone marrow transplantation. New Engl. J. Med. 318, 869. 12. Nemunaitis J., Singer J. W., Buckner C. D., Hill R., Storb R., Thomas E. D. & Appelbaum F. R. (1988)
728
A.H. GOLDSTONEand A. KHWAJA
Use of recombinant human granulocyte-macrophage colony-stimulating factor in autologous bone marrow transplantation for lymphoid malignancies. Blood 72, 834. 13. Devereux S., Linch D. C., Gribben J. G., McMillan A., Patterson K. & Goldstone A. H. (1989) GM-CSF accelerates neutrophil recovery after autologous bone marrow transplantation for Hodgkin's disease. Bone Marrow Transplant. 4, 49. 14. Blazar B. R., Kersey J. H., McGlave P. B., Vallera D. A., Lasky L. C., Haake R. J., Bostrom B., Weisdorf D. R., Epstein C. & Ramsay N, K. C. (1989) In vivo administration of recombinant human granulocytemacrophage colony-stimulating factor in acute lymphoblastic leukaemia patients receiving purged autografts. Blood 73, 849. 15. Peters W. P., Kurtzberg J., Atwater S., Borowitz M., Gilbert C., Rao M., Currie M., Shogan J., Jones R. B., Shpall E. J. & Souza L. (1988) Comparative effects of rHuG-CSF and rHuGM-CSF on hematopoietic reconstitution and granulocyte function following highdose chemotherapy and autologous bone marrow transplantation. Blood 72, (suppl. 1) 130a (abstr). 16. Spitzer G., Jagannath S., Taylor K. Dicke K., Horowitz L., Tucker S., Fogel B. & Souza L. (1989) Studies of recombinant human granulocyte colony-stimulating factor after high dose cyclophosphamide, carmustine and etoposide therapy with autologous bone marrow transplantation in relapsed Hodgkin's disease. Expl Haemat. 17, 517 (abstr). 17. Sheridan W. P., Morstyn G., Wolf M., Dodds A., Lusk J., Maher D., Layton J. E., Green M. D., Souza L. & Fox R. M. (1989) Granulocyte colony-stimulating factor and neutrophil recovery after high-dose chemotherapy and autologous bone marrow transplantation. Lancet ii, 891. 18. Masaoka T., Shibata H., Takaku F., Kodera Y. & Kato S. (1989) Recombinant human granulocyte colony-stimulating factor for allogeneic bone marrow transplantation. Expl Haemat. 17, 521 (abstr). 19. Motoyoshi K., Yoshida K., Hatake K., Saito M., Miura Y., Yanai N., Yamada M., Kawashima T., Wong G. G., Temple P. A., Leary A. C., Witek-Giannoti J. S., Fujisawa M., Yuo A., Okabe T. & Takaku F. (1989) Recombinant and native human urinary colony-stimulating factor directly augments granulocytic and granulocyte-macrophage colony-stimulating factor production of human peripheral blood monocytes. Expl Haemat 17, 68. 20. Komiyama A., Ishiguro A., Kubo T., Matsuoka T., Yasukohchi S., Yasui K., Yanagisawa M., Yamada S., Yamazaki M. & Akabane T. (1988) Increases in neutrophil counts by purified human urinary colonystimulating factor in chronic neutropaenia of childhood. Blood 71, 41. 21. Masaoka T., Motoyoshi K., Takaku F., Kato S., Harada M., Kodera Y., Kanamaru A., Moriyama Y., Ohno R., Ohira M., Shibata H. & Inoue T. (1988) Administration of human urinary colony-stimulating factor after bone marrow transplantation. Bone Marrow Transplant. 3, 121. 22. Duhrsen U., Villeval J-L., Boyd J., Kannourakis G., Morstyn G. & Metcalf D. (1988) Effects of recombinant human granulocyte colony-stimulating factor on hematopoietic progenitor cells in cancer patients. Blood 72, 2074.
23. Socinski M. A., Cannistra S. A., Elias A., Antman K. H., Schnipper L. & Griffin J. D. (1988) Granulocytemacrophage colony-stimulating factor expands the circulating haemopoietic progenitor cell compartment in man. Lancet i, 1194. 24. Gianni A. M., Siena S., Bregni M., Tarella C., Stern A. C., Pileri A. & Bonadonna G. (1989) Granulocytemacrophage colony-stimulating factor to harvest circulating haemopoietic stem cells for autotransplantation. Lancet ii, 580. 25. Nemunaitis J., Singer J. W., Epstein C., Buckner C. D., Storb R., Hill R., Thomas E. D. & Appelbaum F. R. (1989) The use of rhGM-CSF for graft failure in patients after autologous or allogeneic bone marrow transplantation. Expl Haemat. 17, 388 (abstr). 26. Antin J. H., Smith B. R., Holmes W. & Rosenthal D. S. (1988) Phase I/II study of recombinant human granulocyte-macrophage colony-stimulating factor in aplastic anemia and myelodysplastic syndrome. Blood 72, 705. 27. Champlin R. E., Nimer S. D., Ireland P., Oette D. H. & Golde D. W. (1989) Treatment of refractory aplastic anemia with recombinant human granulocyte-macrophage colony-stimulating factor. Blood 73, 694. 28. Nissen C., Tichelli A., Gratwohl A., Speck B., Milne A., Gordon-Smith E. C. & Schaedlin J. (1988) Failure of recombinant human granulocyte-rnacrophage colony-stimulating factor in aplastic anemia patients with very severe neutropenia. Blood 72, 2045. 29. Nissen-Druey C. (1989) Pathophysioiogy of aplastic anaemia. In Clinical Haematology 2 (Gordon-Smith E. C., Ed.), p. 37. Bailliere Tindall, London. 30. Morrissey P. J., Bressler L., Park L. S., Alpert A. & Gillis S. (1987) Granulocyte-macrophage colonystimulating factor augments the primary antibody response by enhancing the function of antigen-pesenting cells. J. lmmun. 139, 1113. 31. Lopez A. F., Williamson D. J., Gamble J. R., Begley C. G., Harlan J. M., Klebanoff S. J., Waltersdorph A., Wong G., Clark S. C. & Vadas M. A. (1986) Recombinant human granulocyte-macrophage colonystimulating factor stimulates in vitro mature human neutrophil and eosinophil function, surface receptor expression, and survival. J. clin. Invest. 78, 1220. 32. Fleischmann J., Golde D. W., Weisbart R. H. & Gasson J. C. (1986) Granulocyte-macrophage colonystimulating factor enhances phagocytosis of bacteria by human neutrophils. Blood 68, 708. 33. Weisbart R. H., Kwan L., Golde D. W. & Gasson J. C. (1987) Human GM-CSF primes neutrophils for enhanced oxidative metabolism in response to the major physiological chemoattractants. Blood 69, 18. 34. Arnaout M. A., Wang E. A., Clark S. C. & Sieff C. A. (1986) Human recombinant granulocyte-macrophage colony-stimulating factor increases cell-to-cell adhesion and surface expression of adhesion-promoting glycoproteins on mature granulocytes. J. olin. Invest. 78, 597. 35. Rettenmier C. W. & Sherr C. J. (1989) The mononuclear phagocyte colony-stimulating factor (CSF-1, M-CSF). In Hematology/Oncology Clinics of North America 3 (Golde, D. W., Ed.), p. 479. W. B. Saunders, Philadelphia. 36. Devereux S., Linch D. C., Campos Costa D., Spittle M. F. & Jelliffe A. M. (1987) Transient leucopaenia induced by granulocyte-macrophage colony-stimulating factor. Lancet ii, 1523.
Growth factors and transplantation 37. Devereux S., Bull H. A., Campos Costa D., Saib R. & Linch D. C. (1989) Granulocyte-macrophage colony-stimulating factor induced changes in cellular adhesion molecule expression and adhesion to endothelium: in vitro and in vivo studies in man. Br. J. Haemat. 71, 323. 38. Addison I. E., Johnson B., Devereux S., Goldstone A. H. & Linch D. C. (1989) Granulocyte-macrophage colony-stimulating factor may inhibit neutrophil migration in oivo. Clin. exp. Immun. 76, 149. 39. Peters W. P., Stuart A., Affronti M. L., Kim C. S. & Coleman R. E. (1988) Neutrophil migration is defective during recombinant granulocyte-macrophage colonystimulating factor infusion after autologous bone marrow transplantation in humans. Blood 72, 1310. 40. Sullivan R., Fredette J. P., Socinski M., Elias A., Antman K., Schnipper L. & Griffin J. D. (1989) Enhancement of superoxide anion release by granulocytes harvested from patients receiving granulocyte-macrophage colony-stimulating factor. Br. J. Haemat. 71, 475. 41. Devereux S., Porter J. B., Hoyes K. P., Abeysinghe R. D., Saib R. & Linch D. C. (1989) Secretion of neutrophil secondary granules occurs during granulocyte-macrophage colony-stimulating factor induced margination. Br. J. Haemat. (in press). 42. Gallin J., Fletcher M., Seligmann B., Hoffstein S. & Cehrs K. (1982) Human neutrophil specific granule deficiency: a model to assess the role of neutrophil specific granules in the evolution of the inflammatory response. Blood 59, 1317. 43. Ho A. D., Grossman M., Mannel D. N., Haas R., Knauf W., Trumper L., Schulz G. & Hunstein W. (1988) In vivo administration of GM-CSF induces elevation of soluble interleukin-2 receptor and CD8 antigens in peripheral blood. Blood 72, (suppl. 1) 121a (abstr). 44. Cannistra S. A., Vellenga E., Groshek P., Rambaldi A. & Griffin J. D. (1988) Human granulocyte-monocyte colony-stimulating factor and interleukin 3 stimulate monocyte cytotoxicity through a tumor necrosis factor-dependent mechanism. Blood 71, 672. 45. Sisson S. D. & Dinarello C. A. (1988) Production of interleukin-1 a, interleukin-1 b and tumor necrosis factor by human mononuclear cells stimulated with granulocyte-macrophage colony-stimulating factor. Blood 72, 1368. 46. Wing E. J., Magee D. M., Whiteside T. L., Kaplan S. S. & Shadduck R. K. (1989) Recombinant human granuiocyte/macrophage colony-stimulating factor enhances monocyte cytotoxicity and secretion of tumor necrosis factor oc and interferon in cancer patients. Blood 73, 643. 47. Hoang T., Nara N., Wong G., Clark S., Minden M. D. & McCulloch E. A. (1986) Effects of recombinant GM-CSF on the blast cells of acute myeloblastic leukaemia. Blood 68, 313. 48. Miyauchi J., Kelleher C. A., Yang Y-C., Wong G. G., Clark S. C., Minden M. D., Minkin S. & McCulloch E. A. (1987) The effects of three recombinant growth factors, IL-3, GM-CSF, and G-CSF, on the blast cells of acute myeloblastic leukaemia maintained in shortterm suspension culture. Blood 70, 657.
729
49. Ganser A., Volkers B., Greher J., Ottman O. G., Walther F., Becher R., Bergmann L., Schulz G. & Hoelzer D. (1989) Recombinant human granuiocytemacrophage colony-stimulating factor patientsin with myelodysplastic syndromes---a phase I/II trial. Blood 73, 31. 50. Negrin R. S., Haeuber D. H., Nagler A , Olds L. C., Donlon T., Souza L. M. & Greenberg P. L. (1989) Treatment of myelodysplastic syndromes with recombinant human granulocyte colony-stimulating factor. Ann. Intern. Med. 110, 976. 51. De Witte T., Muus P., Haanen C., Van der Lely N., Koekman E., Van der Locht A., Blankenborg G. & Wessels J. (1988) GM-CSF enhances sensitivity of leukemic clonogenic cells to long-term low dose cytosine arabinoside with sparing of the normal clonogenic cells. Behring Inst. Mitt. 83, 301. 52. Tafuri A., Hegewisch S., Souza L. & Andreef M. (1988) Stimulation of leukemic blast cells in vitro by colony-stimulating factors (G-CSF, GM-CSF) and interleukin 3: evidence of recruitment and increased cell killing with cytosine arabinoside. Blood 72, (suppl. 1) 105a (abstr). 53. Baldwin G. C., Gasson J. C., Kaufman S. E., Quan S. G., Williams R. E., Avalos B. R., Gazdar A. F., Golde D. W. & DiPersio J. F. (1989) Nonhematopoietic tumor ceils express functional GM-CSF receptors. Blood 73, 1033. 54. Salmon S. E. & Liu R. (1989) Effects of granulocytemacrophage colony-stimulating factor on in vitro growth of human solid tumors. J. clin. Oncol. 7, 1346. 55. Blazar B. R., Widmer M. B., Soderling C. C. B., Urdal D. L., Gillis S., Robison L. L. & Vallera D. A. (1988) Augmentation of donor bone marrow engraftment in histoincompatible murine recipients by granulocytemacrophage colony-stimulating factor. Blood 71,320. 56. Hancock W. W., Pleau M. E. & Kobzik L. (1988) Recombinant granulocyte-macrophage colony-stimulating factor down-regulates expression of IL-2 receptor on human mononuclear phagocytes by induction of prostaglandin E. J. Imrnun. 140, 3021. 57. Krumwieh D., Weinmann E. & Seiler F. R. (1988) Human recombinant IL-3 and GM-CSF in hematopoiesis of normal cynomolgus monkeys. Behring Inst. Mitt. 83, 250. 58. Ganser A., Lindemann A., Ottman O. G., Hermann F., Schulz G., Mertelsmann R. & Hoelzer D. (1989) Effect of recombinant human interleukin-3 in vioo-a phase 1 trial. Expl Haemat. 17, 40 (abstr). 59. Neta R., Douches S. & Oppenheim J. J. (1986) Interleukin 1 is a radioprotector. J. Immun. 136, 2483. 60. McDonald T. P., Clift R. E. & Cottrell M. B. (1989) A four-step procedure for the purification of thrombopoietin. Expl Haemat. 17, 865. 61. Goldstone A. H. & McMillan A. K. (1989) Bone marrow transplantation for non-Hodgkin's lymphoma in Europe--the current position. In European School of Oncology Monograph (in press). 62. DeVita V. T. (1985) In Principles and Practise of Oncology. 2nd edn., p. 256. Lippincott, Philadelphia.
