Albert-Ludwigs-University Medical Center Department of Hematology/Oncology Hugstetter Str 55, 7800 Freiburg, Germany

INTRODUCTION

Since the late 1940s systemic cancer therapy has focussed on the development of cytotoxic agents. In recent years, endogenous defense mechanisms regulated by a group of peptide hormones termed cytokines and capable of directly or indirectly suppressing and eliminating cancer cells, are attracting increasing numbers of investigators. Improved understanding of host biology, immunology, and tumor pathophysiology raises the possibility of introducing new treatment modalities for cancer patients: stimulation of host defense mechanisms, including specific and nonspecific immunological approaches, as well as strategies aimed directly at altering tumor growth and differentiation by therapeutically influencing pathophysiological mechanisms. In addition, amelioration of cancer and cancer treatment-associated morbidity offers new possibilities of improving the quality of life for many patients, as well as options for increased dose-intensity regimens, with the potential of achieving higher remission and cure rates in some patient groups. In this article I will focus on insights gained into the in vivo biology of human cytokines, the possible role of cytokines in the pathophysiology of leukemia and on the experience gained with the use of hematopoietins in cancer therapy. Hematopoietins belong to the family of cytokines which are polypeptide hormones secreted by activated cells. Soluble factors regulating immunity have been postulated and investigated for many years ( e.g. I, 2). With the advent of modern biochemical and molecular techniques a much clearer picture has emerged. In most instances, cytokines provide short range communications between cells by influencing their survival, selfrenewal, proliferation, differentiation, and state of activation. These hormone-like agents are produced by multiple cell types, bind to specific receptors, and have pleiotropic, overlapping and frequently synergistic or additive activities. The majority of cytokines have the capacity of inducing the release of other cytokines in responsive cells making a dissection of in vivo activities of individual cytokines increasingly complex. The communication between cells by cytokines could be compared to a language where cytokines represent the letters of the alphabet and where individual letters are mostly meaningless while "words", i.e. multiple cytokines in correct sequence and concentration, provide the desired information. The cytokine family of genes is not only involved in regulating cellular interactions in the mature organism but also play an important role during embryogenesis [3]. Conversely, another set of developmental genes, the homeobox genes appear to be important for normal cellular development from progenitor cells in the adult organism [4]. Upon ligand stimulation of cytokine receptors, activities of a series of downstream stimulatory and inhibitory molecules are influenced which, in combination, determine the nature of the response. Apart from the potential use in cancer therapy, there is a number of nonneoplastic disorders associated with defects in hematopoiesis and immunity in which the use of cytokines may prove to be beneficial. Clinical application of cytokines has to take into account the physiological mode of action of these agents, i.e. endocrine, paracrine or autocrine. While in vivo augmentation of hematopoiesis is easily achieved by systemic administration, i.e. by endocrine mechanisms, stimulation of specific immunity appears to require paracrine secretion in most instances. Augmentation of specific immunity against cancer in man has remained elusive with cytokines were administered systemically. Remarkable progress, however, has been achieved in the enhancement of myelopoiesis by cytokines in clinical syndromes of myelosuppression.

Hematopoietic Cytokines

The hematopoietic growth factors are a family of glycoprotein hormones which regulate survival, self-renewal, proliferation, and differentiation of hematopoietic progenitor cells as well as the functional activities of mature cells. Although direct actions of these factors upregulate cell function, they may also induce the production of molecules which exert inhibitory effects on hematopoiesis. As an example CSF for granulocytes and macrophages (GM-CSF), which promotes growth and differentiation of myeloid cells, has been shown to induce production of tumor necrosis factor-alpha (TN F -alpha) by monocytes and granulocytes, which is known for its inhibitory action on several hematopoietic lineages [4,5,6]. Moreover, transforming growth factor-beta (TGF-beta), leukemia inhibiting factor (LIF), and possibly IL-l 0 may playa role in downmodulation of hematopoiesis [7,8,9]. On the other hand TNF-alpha which has been primarily recognized as a potent inhibitor [6], is also known to induce the production of growth promoting factors by hematopoietic cells [10,11]. Whereas accessory cells, present in the bone marrow micro-environment, including endothelial cells and fibroblasts, produce a broad spectrum of factors with hematopoietic activity under steady state conditions [12], the potential of mature hematopoietic cells including monocytes, granulocytes, T -lymphocytes and B-Iymphocytes to synthesize such factors is linked to cellular activation. Lectine stimulation results in growth factor expression by monocytes, granulocytes and T -lymphocytes. This unspecific induction can be replaced by more physiological stimuli. I L-I for example triggers granulocytes, monocytes and preactivated T -cells to synthesize and release hematopoietins. However, with a variety of stimulating agents that we used to induce growth factor production we found a restricted production pattern of colony stimulating factors by hematopoietic cells: i.e. monocytes and granulocytes share the capacity to produce CSF for granulocytes (G-CSF and M-CCSF) but fail to produce GM-CSF and IL-3, whereas T-cells exhibit the opposite behaviour [5, 13]. Hematopoietic growth factors and cytokines interact with their target cells via specific receptor structures. In general the numbers of growth factor receptors do not exceed a few hundred per cell. M-CSF receptors are exclusively present on cells derived from the monocyte macrophage lineage and possibly in very low number on neutrophils [14]. G-CSF receptors are present on cells derived from the neutrophil granulocyte lineage and with a much lower number on monocytes/macrophages [15]. The distribution of receptors for GM-CSF and IL-3 appears to be broader, including neutrophil and eosinophil granulocytes as well as monocytes/macrophages [16]. IL-l, IL-6 and TNF-alpha are molecules with pleiotropic activity with receptors on hematopoietic, non-hematopoietic and accessory cells. Mechanisms involved for signal transduction of CSFs after ligand binding to the receptor include tyrosine kinase activity of the receptor molecule as well as a link to GTP-binding proteins which results in an increase of intracellular calcium via activation of the phosphatidylinositol-specific phospholipase C [17-20]. Moreover, various CSFs have been shown to modulate the function of other enzyme systems, thereby regulating the intracellular A TP levels, glucose transport and Na + K +-dependent A TPase-systems [21-23]. The factors currently under active clinical investigation include multipotential colony-stimulating factor (multi-CSF or interleukin-3), granulocyte-macrophage CSF (GM-CSF), granulocyte CSF (G-CSF) and erythro poietin (EPO) which will be the focus of this discussion. More recently, also macrophage CSF (M-CSF), Interleukin-l, Interleukin-6 and others are being investigated clinically. Erythropoietin (EPO) is a glycoprotein hormone produced predominantly in the kidney and to a small extent in the liver. It regulates the proliferation and differentiation of erythroid progenitor cells to mature erythrocytes [24]. The mechanism by which a hypoxic stimulus triggers the production and release of EPO is still unknown. Evidence has been presented that the oxygen sensor is a heme protein [25]. EPO is a heavily glycosylated, 166 amino acid protein with a molecular mass of 34-39 kD. While glycosylation is a prerequisite for in vivo activity, in vitro activity is also provided by the non-glycosylated form. The EPO gene has been localized to chromosome 7q 11-q22 [26]. Like those of its natural counterpart, the major target cells of recombinant EPO have been identified as colonyforming progenitor cells committed to the erythroid lineage (CFU-E, colony-forming unit-erythroid) and to a minor extent as more immature erythroid progenitor cells, the BFU-E (burst-forming unit-erythroid). Recent in vitro data also indicate that EPO induces expansion of very early progenitor cells [27]. Granulocyte Colony-Stimulating factor (G-CSF) was molecular defined in 1985 [28] and cloned in 1986 [29,30]. Due to alternative splicing, two cDNAs representing a 177 amino acid protein form and a 174 amino acid protein form of human G-CSF have been isolated [2830]. The shorter version of the molecule seems to be more active in vivo. The gene which encodes G-CSF is located on chromosome 17 q11-q22. G-CSF is a rather lineage-specific hematopoietic growth factor, in that it acts on cells capable of forming one differentiated cell type: the neutrophil granulocyte. In combination with other hematopoietic growth factors it acts synergistically to stimulate a broader spectrum of colony-forming cells [28]. As an example for the extremely rapid clinical development of these new agents, G-CSF entered clinical trials in 1987 and was approved world-wide in 1991-1992. The human gene encoding Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) is located on chromosome 5q21-5q32 [31]. Due to variable glycosylation, the molecular mass of the mature protein which comprises 127 amino acids, ranges from 14 to 35 kD [32]. A variety of cells producing GM-CSF have been identified, among them monocytes, fibroblasts, endothelial cells, epithelial cells, and T -lymphocytes [33]. GM-CSF stimulates granulocyte/macrophage and eosinophil colony formation in vitro and acts in combination with erythropoietin as erythroid burst-promoting activity [34]. In addition to its effect on progenitor cell differentiation, GM-CSF also induces a variety of functional changes in mature cells. It increases neutrophil phagocytic activity, inhibits the random migration of neutrophil granulocytes [35], and induces the production of other cytokines (e.g. tumor necrosis factor, interleukin-l) by these same cells [36, 37]. It also induces macrophage tumor cytotoxicity [38], activates macrophages to synthesize MHC class II molecules, to augment antigen presentation [39], and to release oxygen radicals [40]. As for GM- and G-CSF, the gene for interleukin-3 (IL-3) has been located on chromosome 5 q23-q31 [41]. The protein is produced by activated T -lymphocytes and has a molecular mass of 14-28 kD. 1L-3 is a multilineage hematopoietin which promotes the growth and differentiation of various myeloid and lymphoid progenitor cells including early multipotent progenitors such as blast colony-forming units and mixed colonies. The colonies produced in response to IL-3 contain eosinophils, basophils, neutrophils, mast cells, megakaryocytes, macrophages, and erythroid cells.

Hematopoietic (HPGF) in the Pathogenesis of Acute Myeloid Leukemia (AML)

In recent years, our laboratory has investigated a series of questions addressing the possible pathogenetic role of dysregulation of cytokine expression in AML. The first question was, whether AML progenitor cells require HPGF for proliferation ex vivo and, subsequently, in vivo (see below). In 38 of 54 AML bone marrow samples tested, there was a requirement for exogenous HPGF for growth in culture while the remaining 16 cases demonstrated growth in culture in the absence of exogenous HPGF ("autonomous growth"). After having demonstrated HPGF dependency for the majority of cases, the second question addressed the possible cellular sources of HPGF in AML. While unstillulated normal blood and bone marrow cells under our experimental conditions fail to release any HPGF or accumulate respective mRNAs, the majority of AML samples were found to release HPGF and to accumulate HPGF mRNA constitutively. Selected experiments were performed to study if the addition of neutralizing anti-GM-CSF monoclonal antibody was able to inhibit spontaneous colony growth of AML populations which produced GM-CSF. Exposure resulted in a 20-70% reduction of colony growth, whereas proliferation of AML cells which did not express GM-CSF were not affected by anti-GM-CSF monoclonal antibodies. This result suggests that GMCSF can be involved in an autocrine mechanism which promotes proliferation of leukemic cells. Overall, there appeared to be a correlation between the constitutive release of HPG F and spontaneous ("autonomous") growth in culture [42]. Our series of 54 AML populations have also been analysed for the expression of IL-2 and IL4 mRNA. None of the samples has been found to produce these cytokines. Direct effects of CSFs and cytokines, which are produced by AML populations have been investigated focussing on growth regulation of leukemic cells. Whereas GM-CSF, G-CSF and IL-3 added to the agar assays at concentrations of 50 ng/ml, induced growth of leukemic colony forming units (L-CFU) in approximately 30-50% of the AML samples tested, M-CSF added at a concentration of 1000/ml induced growth in only one AML cell population of 19 samples tested [42]. IL-l-beta and TNF-alpha failed to promote growth of leukemic cells via direct action [43]. Since autocrine stimulation could explain autonomous proliferation in vivo only in some patients we investigated next, whether paracrine induction of HPGF could contribute to AML progenitor cell proliferation. Whereas TNF-alpha has been shown to exert inhibitory action on normal and malignant hematopoiesis when added to cultures at high concentrations [6], we were interested in investigating if AML supernatants containing TNF-alpha may induce CSF release by accessory cells present in the bone marrow environment. Therefore, we added culture supernatants containing TNFalpha constitutively secreted by AML cells to human umbilical cord vein endothelial cells and performed Northern blot analyses of exposed endothelial cells. TNF-alpha stimulated endothelial cells were found to produce GM- and G-CSF mRNA and released these proteins; this induction could be inhibited by pretreatment of AML supernatants with anti- TNF monoclonal antibody prior to exposure of endothelial cells. Other investigators demonstrated similar data when exposing endothelial cells to AML supernatants containing IL- 1beta [44]. IL-6 failed to induce endothelial cell production of colony-stillulating factors [43]. We then studied IL-I-beta and GM-CSF, which are known to be potent function-inducing molecules in myeloid cells [5,45], for their potential to induce CSF production in CSF-negative AML populations. None of the AML samples tested (n = 6) responded to these mediators with CSF production. Concentrations for GM-CSF were chosen up to lOO ng/ml and for IL-I-beta up to 10 /ml. However , it has recently been reported that G M -CSF production by AML blasts may be inducible with TNFalpha at a concentration of 50 ng/ml [46]. We were further interested to investigate the CSF gene structure of AML populations constitutively producing the respective CSF species. For each, G- and GM-CSF we analysed 10 AML samples by Southern blot analysis using multiple restriction enzyme digestions. None of the study samples showed a change of the G- or GM-CSF gene structure as compared to germ line configuration. Under our experimental conditions, many samples of AML blasts show an activated. GM-CSF expression which is not seen in normal cells of the myeloid lineage under the conditions tested. In order to exclude lineage promiscuity of GM-CSF-producing AML samples, we studied the presence of immunoglobulin gene and T -cell receptor gene rearrangements. No AML sample, which produced GM-CSF, provided evidence for rearrangements of illmunoglobuline genes (IGh) nor of T -cell receptor genes. In another study with 69 patients with AML we found that IGh-rearrangements appeared in 10 cases with an additional kappa chain gene rearrangement in I case. Two patients within this group showed rearrangements of the T -cell receptor beta chain gene [47]. In an effort to investigate the molecular basis of constitutive cytokine expression in AML cells we have begun to study the activated expression of point mutated RAS genes in CSF- and cytokine-expressing AML populations. Fifty patients with AML have been tested for the presence of activating point mutations focussing on first and second nucleotides of codon 61 of N-RAS. Mutations of the 7 positions that have been investigated have been shown to be heterozygous, which was demonstrated by hybridization of the normal allele with wild type probes. Most often we found an association between IL6 expression in AML cells with N-RAS activation, whereby the occurring mutations always involved exon I. There was no clear correlation of N-RAS mutations with the expression of the other growth factors investigated. The reported co-incidence of alterations of myconcogenes with N-RAS oncogene was also investigated. The 12.5 kb ECOR I band, which comprises the total coding region of myc was unchanged and not amplified in all patients investigated [48]. Synthesis of factors with hematopoietic activity was also studied in patients with acute lymphoblastic leukemia (ALL). Northern blot analyses from 13 ALL did not reveal mRNA accumulation for G-CSF, GM-CSF, M-CSF and IL-6. Only two T -ALL populations showed an accumulation of IL-3 transcripts after prolonged autoradiography (unpublished). In another model, we investigated the possible contribution of suppressor cytokines to the suppression of normal hematopoiesis in patients with leukemia. As a model, we chose Hairy Cell Leukemia (HCL), a disease characterized by profound pancytopenia. Our studies demonstrated constitutive mRNA accumulation and protein release of TNF-alpha by HCL cells and not by other lymphoproliferative disorders [49]. The addition of neutralizing anti TNF monoclonal antibodies could in part restore normal progenitor cell proliferation in culture. in vivo correspond well to predictions based on in vitro studies in that the ontogenetically latest acting growth factor, G-CSF, induces the fastest rise, while IL-3, compatible with the role of an early stem cell factor, requires approximately 10 days for white blood cells to rise in the peripheral blood. In view of their pleiotropic and in vitro- synergistic properties, HPGF can be expected to also synergize in vivo, corresponding clinical trials are ongoing. In addition to stimulating production of various lymphohematopoietic cells, hematopoietic cytokines also lead to mobilization of progenitor cells from bone marrow into peripheral blood facilitating harvest by leukapheresis for subsequent stem cell transplantation [55,56]. Clinical studies have documented the effectiveness of recombinant EPO in correction of anemia in patients with end-stage renal disease [57] as well as in pre-dialysis patients [58]. In these patients only low levels of EPO can be demonstrated in serum. Although, pathogenetically, anemia in tumor patients is not characterized by EPO deficiency, we investigated whether exogenously administered EPO could stimulate erythropoiesis in these patients and thus contribute to correcting transfusiondependent anemia. The therapeutic effect of EPO in correcting chemotherapy-induced anemia in patients with normal renal function was demonstrated [59]. EPO response was accompanied by a decrease in serum ferritin. The requirement for red blood cell transfusion was reduced or eliminated by EPO therapy. EPO was also shown to be beneficial in the treatment of malignancy due to neoplastic bone marrow infiltration [60]. No side effects were noted during EPO therapy. This is in contrast to results in patients with endstate renal disease, in whom EPO therapy was associated with increases in blood pressure and blood viscosity leading to significant morbidity in some patients. This difference may possibly be explained by a predisposition of patients with renal disease to vascular complications and to their lack of functioning kidneys to regulate fluid balance and thus blood viscosity due to increased red cell mass. Like GM-CSF, G-CSF has been utilized in the prevention of chemotherapy-induced neutropenia [61-65] and in the setting of autologous bone marrow transplantation [66,67]. A dose-dependent increase in absolute neutrophil counts and shortening of the neutropenic period was observed. At higher doses up to a ten-fold increase in monocytes was also seen [63]. In most recent studies the incidence of severe infections was reduced following these cycles of chemotherapy combined with G-CSF. Neutropenia caused by marrow infiltration with low grade lymphoma (hairy cell leukemia) also improved after treatment with G-CSF [68] as with GM-CSF [69]. Toxicities in G-CSF trials in general have been limited consisting mainly of bone pain, presumably secondary to

Hematopoietic Cytokines as Therapeutic Agents

In a series of phase I-IV clinical trials we have investigated the in vivo biology of HPGF in man. G-CSF exclusively induces production of neutrophils and bands [50]. At doses up to 250 ug/m2 neutrophils rise until day 10, while thereafter a decrease is observed in spite of continued G-CSF administration, suggesting endogenous negative feed-back mechanisms. GM-CSF induces proliferation of several white blood cells: neutrophils and bands, predominantly, but also of eosinophils and moncytes [51]. Biological responses are not only dependent on dose but also on routes of administration, since s.c. administration almost exclusively induces neutrophils and bands, while continuous i. v. administration also leads to expansion of eosinophil and monocyte poopulations by GM-CSF. In contrast to G- and GM-CSF, IL-3 not only promotes expansion of all white blood cell subpopulation but also leads to increased reticulocyte and platelet counts (52-54). The kinetics of white blood cell increases bone marrow expansion. This adverse effect was seen in up to 25% of patients treated with an intravenous bolus > 30 uuuuuug/kg body weight; with subcutaneous administration and lower doses it was less frequently encountered. In some patients reversible elevation of serum alkaline phosphatase and lactic dehydrogenase have been noted as a result of increased neutrophil turnover, and rarely evidence of overshooting neutrophil activation such as the acute neutrophilic dermatosis (Sweet's syndrome) has been observed [68]. G-CSF is currently being tested by a number of investigators for its usefulness in the treatment of neutropenic disorders due to malignancies, cancer chemotherapy as well as in congenital (Kostmann's syndrome), cyclic, or idiopathic neutropenia [69- 71 ]. From the promising preliminary data it can be anticipated that in the near future recombinant G-CSF will have a beneficial impact on the natural history of these disorders. Although in vitro findings suggested a possible role for inducing indirect and direct antitumor effects, no such effects of GM-CSF have been observed as yet in any of the clinical studies [72- 78]. Apart from its possible role as antitumor agent, which requires further evaluation, GMCSF shows activity in reducing chemotherapy associated neutropenia. At higher doses (several-fold higher than required for amelioration of chemotherapy induced neutropenia), the dose limiting toxicities of GM-CSF reported were fever and, during continuous infusion into small veins, thrombophlebitis [74,78]. Effects of GM-CSF were also studied in the clinical setting of autologous bone marrow transplantation [74,79]. It has been shown that GM-CSF accelerates the rate of neutrophil recovery and increases the circulating pool of peripheral blood hematopoietic progenitors. No difference was seen between GM-CSF treated patients and control patients with respect to the first appearance of neutrophils in the circulation [80]. More recently, the benefits of GM-CSF therapy was also demonstrated after allogeneic bone marrow transplantation in situations of graft failure [81 ]. Administration of GM-CSF to patients with myelodysplastic syndrome has been described to normalize red cell, white cell, and platelet counts in some patients [76]. More recent studies, however, have not been able to confirm this optimistic report, demonstrating rises in neutrophil counts only. At higher GM-CSF doses an increase in leukemic blast cells in blood and bone marrow has been seen, indicating that GM-CSF can stimulate the proliferation of human leukemic blast cells as well as of normal hematopoietic cells in vivo [76,77,82]. Even a possible progression to frank leukemia has been observed [82]. Some ongoing studies are exploring the use of GM-CSF in acute myeloblastic leukemia, by augmenting the proportion of malignant cells recruited into the S-phase of the cell cycle and thus obtaining enhanced cytotoxic effects with drugs such as Ara-C, that kill cycle-activated cells [83,84]. The value of GM-CSF in the treatment of aplastic anemia appears to be limited to those patients with residual hematopoiesis as reported by Champlin and Nissen and their colleagues [85,86]. Combinations of hematopoietic growth factors acting on early progenitors with later-acting factors might have synergistic effects in accelerating repopulation of the bone marrow and warrant further investigation in this disease, possibly in combination with immunosuppression agents. This approach is currently being tested in a clinical trial (unpublished). Ultimately, GM-CSF may find a place in the treat ment of other nonmalignant conditions characterized by leukopenia (e.g. acquired immune deficiency syndrome) [87] or in the improvement of host defense in infectious disease. In preclinical murine and primate models, IL-3 has been shown to significantly elevate numbers of circulating leukocytes. A phase 1/11 trial in patients with bone marrow failure syndromes has revealed the following dose related hematological responses: increases in platelet counts, absolute leukocyte counts including neutrophils, monocytes, eosinophils, lymphocytes, and, to a lesser extent, basophils, reticulocyte counts, and bone marrow cellularity [52]. Side effects included fever, flushing, headache, and local irritation at the site of the subcutaneous injection at higher doses ( > 250 ug/m²). More efficacy is to be expected from the use of combinations of IL-3 with other late-acting hematopoietins [55,56].

Hematopoietic Cytokines in the Treatment of Leukemias

The available data on the therapeutic use of HPGF in various leukemias and other diseases infiltrating the bone marrow would suggest the following conclusions. EPO will stimulate erythrocyte production in settings of bone marrow infiltration by chronic lymphocytic leukemia or multiple myeloma, after chemotherapy, but only occasionally, if erythropoiesis is part of the neoplastic clone such as in MDS. A similar interpretation is suggested by the data obtained with GM-CSF in hairy cell leukemia (HCL): Since granulopoiesis is not part of the leukemic clone, it should and does respond to exogenous stimulation with CSF [69]. Similarly, it would be expected that neutrophil regeneration is accelerated by G- or GM-CSF after successful induction chemotherapy in AML, when the leukemic cells are eliminated and normal progenitor cells (PGC) available to respond [88,89]. As long as normal progenitor cells are available to respond, the balance can be shifted from leukemic to normal PGC, possibly independent of the suppressing mechanism (e.g. chemotherapy, TNF-alpha in HCL, unknown in CLL). As expected, a more complex pattern of response and non-response is seen in conditions where the cells targeted by the respective HPGF are intrinsically abnormal. Treatment results in myelodysplasia are variable, and even in patients that do respond in one or more lineage as judged by peripheral blood counts, a clinical benefit remains to be demonstrated. More recently, efforts to induce leukemic PGC more sensitive to chemotherapy are under investigation in several clinical trials [e.g. 91]. A third approach would be suggested by the observations made in patients with MDS whose blast cells expand during therapy. In these patients blasts tend to return to baseline and occasionally less than baseline upon discontinuation of cy to kine therapy. This observation would parallel in vitro studies where leukemic cell lines can be adapted to cytokine containing media, leading to cell death upon cytokine deprivation.

Cytokines in the Treatment of Chemotherapy Induced Cytopenia With or Without Autologous or Allogeneic Stem Cell Rescue

Increased neutrophil recovery after intensive chemotherapy and autologous or allogeneic stem cell rescue has been demonstrated in a series of clinical studies (for review see [92]). Since hematopoietic PGC are shifted from bone marrow to blood by chemotherapy, and even more pronounced when followed by cytokine therapy, peripheral blood PGC harvested under these conditions appear useful in further accelerating white blood cell and especially platelet recovery after intensified chemotherapy [55,56,93]. The question of dose intensification and its relevance to improved therapeutic efficacy can now be studied in an unprecedented way. While increased therapeutic efficacy remains to be our overall goal, reduced morbidity and, in some cases, mortality in cancer patients receiving cytotoxic therapy is a definitive and important step in this direction. The future will see the evaluation of novel cytokines alone as well as combinations of cytokines also in non-neoplastic conditions, e.g. infectious diseases. Molecular efforts correcting the pathophysiological defect or directly inducing antitumor effects by gene transfer into tumor or host cells [94] appear to be another promising step for cytokinebased treatments, especially involving cytokine requiring paracrine secretion for optimum activity and specificity.

Acknowledgements

I would like to thank my colleagues Drs. F. Herrman, A. Lindemann, L. Kanz, W. Erugger, W. Oster, M. Lübbert, F. Rosenthal and many others for working with me on the concepts discussed in this paper. I am grateful to Ms. H. Vogel for excellent secretarial assistance.

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