Current Pharmaceutical Design - Volume 8, Issue 5, 2002

Cytokines are centrally involved in the regulation of normal hematopoiesis, the production of mature blood cells by bone marrow stem cells. Cytokines influence stem survival, proliferation, and differentiation commitment, as well as controlling the orderly maturation of progenitor cells into functional leucocytes, erythrocytes, and platelets. Acute leukemias result from malignant transformation of bone marrow stem cells. Although cytokines do not appear to be centrally involved in the pathogenesis of acute leukemias, leukemic cells express receptors for many of the cytokines regulating normal hematopoiesis, particularly GCSF, GM-CSF, IL-3, and stem cell factor. These molecules have demonstrable effects on acute leukemia cells in vitro, inducing proliferation and enhancing survival, but their biological activity when administered as recombinant proteins in pharmaceutical doses to patients with active leukemia are less well understood. Because of the stimulatory effects of cytokines such as G-CSF and GM-CSF on normal hematopoiesis in vitro and in normal individuals, these two molecules have been extensively studied in randomised clinical trials of chemotherapy for cancer, including acute leukemia. Concerns about the potential for G-CSF and GM-CSF to accelerate the growth of acute myeloid leukemia, which expresses receptors for both molecules, have not been realised. Conversely, the concept of using either of these two cytokines to induce acute myeloid leukemia cells into active DNA synthesis, thus potentially sensitising them to the effects of S-phase-specific drugs, has not been shown to be clinically beneficial. Both G-CSF and GM-CSF have been demonstrated to accelerate the recovery of normal granulopoiesis after intensive initial cytotoxic chemotherapy for acute leukemia, significantly shortening the duration of severe treatment-induced neutropenia, and resulting in a number of tangible benefits including reduction in infection, use of intravenous antibiotics, and duration of hospital stay. However, the final role for these agents in the treatment of acute leukemia remains controversial and still to be fully defined.

The aetiology of the myeloproliferative disorders and, in particular, of the myeloid leukaemias is unknown. The transformation of cells is primarily due to molecular aberrations leading to excessive cellular signalling and proliferation. In addition cytokines and their receptors may play a role in leukaemogenesis by increasing the proliferative capacity of leukaemic cells and extending their life span. Chemotherapeutic agents are regularly used to treat patients with leukaemia but they are nondiscriminatory treatments that kill both healthy and cancer cells. Consequently patients receiving chemotherapy suffer unwanted toxicities in both the haematological and other systems. Therapies that specifically target malignant cells sparing normal cells are being investigated in a number of contexts. Cytokine antagonists can target growth factor-dependent cells by obstructing the interaction between cytokine and receptor. In this review we will discuss the myeloproliferative disorders in particular the leukaemias, the cytokines involved in leukaemogenesis, and the therapeutic potential of new agents that block specific cytokines.

Prevention of hemorrhage secondary to thrombocytopenia has generally been managed by the transfusion of platelets. The need for such transfusion is related to the depth and duration of ”critical“thrombocytopenia, a level that until recently was hotly debated. However a number of clinical trials have established that transfusion at a platelet count greater than 10 x 10 9 / L was safe in the absence of factors associated with increased tendency to bleed. Trying to predict which patients are at risk of bleeding due to thrombocytopenia has proven difficult outside of those scenarios that inevitably cause severe thrombocytopenia, such as treatment of leukemia and myeloablative chemotherapy with stem cell support. Models have been proposed but have yet to be validated. Of greater importance is the need for proof that intensive treatment of solid tumours with growth factor support leads to improved outcomes.The discovery of platelet growth factors raised expectations that an effective method for abrogating thrombocytopenia would be soon available in the clinic. The cytokines initially described were pleiotropic in nature, and stimulation of platelet production was generally modest. However, one of these agents, interleukin-11, was successfully shown to reduce the incidence of severe thrombocytopenia in patients receiving dose-intensive chemotherapy, and has now received approval from the FDA for this purpose. Initial clinical trials of thrombopoietin (TPO), the central regulator of megakaryocytopoiesis and thrombopoiesis, and its analogues showed these agents to be the most potent stimulators of thrombopoiesis and to be associated with few adverse effects. They have also been shown to enhance platelet recovery after chemotherapy, but early results from trials investigating their ability to prevent severe thrombocytopenia associated with the treatment of leukemia and bone marrow transplantation have been disappointing. In addition, subcutaneous administration of one of these agents, megakaryocyte growth and development factor, has been shown to induce the formation of antibodies that neutralize native TPO and cause thrombocytopenia. TPO remains a promising therapeutic agent, however its potential application is more limited than initially anticipated, and there are a number of obstacles to overcome before it finds an importance use in the clinic.

Haemopoietic progenitors mobilised into peripheral blood are now almost universally used in autologous haemopoietic stem cell transplantation in the treatment of a range of malignant and some nonmalignant disease. Although chemotherapy alone was initially used, all modern protocols now involve the use of cytokines, with or without chemotherapy. Important developments have included an in understanding of the importance of prior cancer therapy on progenitor yield, knowledge of the kinetics of mobilisation and development of necessary skills to collect and cryopreserve progenitors. More accurate measurement of haemopoietic progenitors and definitions of target cell yields for optimal haemopoietic recovery after highdose therapy have also contributed to more predictable outcomes and provide a reference point for newer mobilisation approaches. Although G-CSF based regimens are usually successful, some patients either fail to mobilise sufficient progenitors or require an excessive number of collections. Clinical studies with the early acting cytokine, stem cell factor, in combination with G-CSF have demonstrated increased progenitor yields in a range of patients which may translate to clinical benefit in selected situations. In animal models and to a lesser extent in humans, other cytokines such as thrombopoietin and Flt-3 ligand or a number of engineered small molecules with single or dual agonist activity for cytokine receptors (IL-3, Flt-3L, TPO, G-CSF), have also been found to be promising mobilising agents. Further research into the relative importance of cell proliferation, cellular adhesion and the role of accessory cells and other signalling events is leading to an improved understanding of the underlying mechanisms of haemopoietic progenitor mobilisation.Administration of appropriate high-dose chemotherapy followed by re-infusion of haemopoietic progenitor cells capable of long-term reconstitution has long had a place in the treatment of a number of malignant (largely haematological) and non-malignant diseases. For many years these progenitor cells were obtained by direct aspiration of bone marrow under general anaesthetic, hence the term bone marrow transplantation. However, it has also been recognized that haemopoietic stem cells may be recovered from peripheral blood, albeit in low numbers, and also from umbilical cord blood. Further empirical observations showed that the number of haemopoietic progenitors circulating in the blood could be transiently augmented after chemotherapy and / or administration of one or more of a number of cytokines. Refinements to the clinical practice of progenitor mobilisation, collection and enumeration have proved very successful such that in many cases peripheral blood stem cells (PBSC) have largely replaced bone marrow as the preferred source.

Keratinocyte Growth factor (KGF) is an epithelial cell growth factor of the fibroblast growth factor family and is produced by fibroblasts and microvascular endothelium in response to proinflammatory cytokines and steroid hormones. KGF is a heparin binding growth factor that exerts effects on epithelial cells in a paracrine fashion through interaction with KGF receptors. Preclinical data has demonstrated that KGF can prevent lung and gastrointestinal toxicity following chemotherapy and radiation and preliminary clinical data in the later setting supports these findings. In the experimental allogeneic bone marrow transplant scenario KGF has shown significant ability to prevent graft-versus-host disease by maintaining gastrointestinal tract integrity and acting as a ”cytokine shield“ to prevent subsequent proinflammatory cytokine generation. Within this setting KGF has also shown an ability to prevent experimental idiopathic pneumonia syndrome by stimulating production of surfactant protein A, promoting alveolar epithelialization and attenuating immune-mediated injury. Perhaps most unexpectantly, KGF appears able to maintain thymic function during allogeneic stem cell transplantation and so promote T cell engraftment and reconstitution. These data suggest that KGF will find a therapeutic role in the prevention of epithelial toxicity following intensive chemotherapy and radiotherapy protocols and in allogeneic stem cell transplantation.

Dendritic cells (DC) initiate tumor specific immune responses in animal studies and initial human trials suggest that certain tumor-antigen loaded DC preparations generate clinical responses.DC may be obtained from blood or generated in vitro from precursor cells. In vitro generation of DC from precursor cells, under the influence of cytokines, has been favoured to date as a source because of the greater numbers of DC produced. However, the different cytokine combinations and serum or plasma component(s) used, differentiate precursor cells into DC with different physiological properties and ultimate immunogenicity. Thus, the quality of in vitro cytokine derived DC may have a profound influence on clinical outcomes. The administration of certain growth factors, which increase the number of circulating blood DC, may provide an alternative source of DC for use in clinical trials.Although clinical trials in prostate cancer, melanoma and metastatic renal carcinoma patients are encouraging, some data suggest certain DC preparations and administration protocols are sub optimal, even potentially tumor enhancing. As basic scientific studies establish how to provide DC with stable phenotype, resistance to tumour inhibitory factors and high migratory capacity, the technology for producing cytokine derived DC in vitro using Good Manufacturing Practise (GMP) conditions needs to be developed. Future DC vaccination protocols will require careful control of the DC used for tumor-antigen loading and repetitive long term DC vaccination may be necessary to maintain effective anti-tumor immune responses.

Myeloid leukaemia cells are sensitive to attack by elements of the immune system as evidenced by the effects of T cell depletion, graft versus leukaemia and donor lymphocyte infusion on leukaemic recurrence. An implication is that the immune system can be manipulated to enhance anti-leukaemic effects by exogenous stimulation including the use of immunostimulatory cytokines. These could potentially be used in a controlled manner that avoids the clinical problems associated with graft-versus-host disease. The cytokine used most extensively to date is interleukin-2 (IL2), a molecule that induces T lymphocyte proliferation and the generation of MHC unrestricted cytotoxicity. Despite over 10 years of clinical experience, the data on efficacy in acute myeloid leukaemia remains unclear due to lack of adequate randomised trials. IL2 appears to be effective in patients with low level marrow infiltration by acute myeloid leukaemia (AML) blast cells. It is less effective when patients present or relapse with packed bone marrows. The logical assumption that IL2 treatment given during states of minimal residual disease will reduce the incidence or speed of disease recurrence remains to be adequately tested. IL2 administration is associated with characteristic clinical adverse effects and with specific immuno-haematological changes. The use of other cytokines for immune manipulation in patients with AML is so far essentially limited to the research laboratory. Potential uses include cytokine induced blast differentiation to dendritic cells and the use of irradiated cytokine gene transduced leukaemic cells as vaccines.