It's moving day: factors affecting peripheral blood stem mobilization and strategies for improvement

Mobilized peripheral blood stem cells (PBSC) have become the main source for autologous or allogeneic transplantation following myeloablative therapy in patients with haematolymphoid malignancies or solid tumours. Classical strategies for PBSC mobilization include administration of granulocyte colony-stimulating factor (G-CSF) alone or in combination with other cytokines or myelosuppressive chemotherapy. PBSC mobilization and collection have been optimized in numerous clinical trials, but a significant proportion of patients fails mobilization. In recent years, some of the underlying physiology has been elucidated, leading to the development of new mobilization strategies. Expression of the G-CSF receptor on stem cells is not required for mobilization. G-CSF or myelosuppressive therapy act via secondary pathways, including the chemokine stromal-derived factor-1 and its receptor CXCR4. New chemokine receptor agonists lead to a rapid and substantial PBSC mobilization. The most advanced data are available for AMD-3100, which acts synergistically with G-CSF in normal donors. This CXCR4 ligand appears to mobilize more primitive PBSC with increased repopulation capacity when compared with G-CSF. Wider recognition of these new developments may enable sufficient PBSC to be obtained for transplantation in poorly mobilizing patients, thus enabling them to receive potentially curative treatment. In the mid 1980s, PBSC became a source for transplantation and the 'modern era of stem cell transplantation' began (Juttner et al, 1985; Kessinger et al, 1986; Körbling et al, 1986, Reiffers et al, 1986). In the early 1990s, PBSC replaced bone marrow as the preferred source because of the relative ease of collection, the faster haematological recovery when compared with bone marrow transplantation (BMT), leading to fewer complications, and lower costs of the procedure (To et al, 1997). Furthermore, several studies have shown that the overall survival and disease-free survival was superior in patients that received PBSC compared with those that underwent BMT (Heldal et al, 2000; Bensinger et al, 2001), although other groups did not find a difference in survival (Vigorito et al, 1998; Blaise et al, 2000). By 1998, 95% of all autologous transplants and 25% of all allogeneic transplants used PBSC (Goldman & Horowitz, 2002). The number of allogeneic PBSC transplants has further increased in recent years. Despite the success of PBSC transplantation, the exact mechanisms involved in PBSC mobilization and homing are not yet clear. In adults, small amounts of PBSC are present in the peripheral blood (PB) during steady state haematopoiesis, as has been known for 40 years (Goodman & Hodgson, 1962; Dorie et al, 1979; Fruehauf et al, 1995), suggesting a continuous migration and exchange of haematopoietic stem cells between the bone marrow (BM) and other organs, such as the liver and spleen (Starzl et al, 1996; Nierhoff et al, 2000). This model is strongly supported by experiments in which parabiotic mice were surgically conjoined and shared a common circulation (Wright et al, 2001). They rapidly established stable, functional cross-engraftment of partner-derived haematopoietic stem cells (HSC) and maintained partner-derived haematopoiesis after surgical separation (Wright et al, 2001; Abkowitz et al, 2003). This recently published study confirmed that few HSC exit the marrow, transit the blood and re-enter the marrow during 3–12 weeks of parabiosis, and thus suggest that, under normal homeostatic conditions, HSC exit the marrow as a one-way process, potentially a death pathway. In contrast, however, cytokine exposure significantly increased the return of HSC to the marrow, as well as their mobilization to circulating blood (Abkowitz et al, 2003). The proliferation of HSC is increased during mobilization, followed by subsequent migration and egress from the BM in the PB (Abkowitz et al, 2003). Mobilization of haematopoietic stem cells can be achieved by a diversity of agents (Table I) including haematopoietic growth factors, such as stem cell factor (SCF), ftl-3 ligand (Brasel et al, 1996), interleukin 3 (IL-3) (Orazi et al, 1992), granulocyte–macrophage colony stimulating factor (GM-CSF) (Socinski et al, 1988) or granulocyte colony-stimulating factor (G-CSF) (Dührsen et al, 1988), chemotherapeutic agents, combinations of cytokines and chemotherapeutic agents or certain chemokines. The use of haematopoietic growth factors like GM-CSF led to a 45-fold increase of circulating progenitor cells in mice (Brasel et al, 1997), whereas for G-CSF an increase of up to 100-fold in mice (Molineux et al, 1990; de Haan et al, 1995) was shown. Numerous other studies have not detected significant differences between the numbers of GM-CSF- and G-CSF-mobilized PBSC, as has been reviewed elsewhere (Gazitt, 2002). The combination of a mobilization chemotherapy with G-CSF leads to an additional sevenfold increase in mobilization yield compared with chemotherapy alone, whereas an additional fivefold increase in the PBSC yield was detected with the combination of chemotherapy and GM-CSF. The objective of this review is to give an overview of the variables that influence PBSC yields. Emphasis is placed on new strategies to optimize the blood stem cell mobilization in the clinical setting, which is particularly relevant for heavily pretreated patients. There are different definitions for 'stem cells' and 'progenitor cells' as well as for 'CD34+ cells' used in the actual literature. This causes confusion. We would support the use of the definition of Weissman (2000): stem cells are regarded as clonogenic cells capable of self-renewal and multilineage differentiation. Progenitor cells are oligo-lineage cells that are already more restricted in their differentiation potential and are not able to self-renew. The expression of the antigen CD34 and lineage negativity are often used in clinical practice as a surrogate markers for haematopoietic stem and progenitor cells. The percentage of CD34+ cells in peripheral blood under steady-state conditions is about 0·06% and about 1·1% in bone marrow (Fruehauf et al, 1995; Körbling & Anderlini, 2001). They are thought to play an important role both in short-term engraftment, for rapid haematopoietic reconstitution in patients receiving BM ablative doses of irradiation and chemotherapy, as well as long-term, stable engraftment. Nevertheless, their characterization remains difficult, as has recently been reviewed elsewhere (Wagers et al, 2002). At least two primitive cell subsets can be defined by their immunophenotype: CD34+/CDw90(Thy1)+ (Craig et al, 1993) and CD34+/CD38– cells (Terstappen et al, 1991). Besides the well-characterized CD34+ HSC, there is evidence that CD34-negative HSC with engraftment potential and distinct HSC characteristics exist in mice as well as humans (Goodell et al, 1997). Interestingly, corresponding to their functional differences, CD34+ cells from the BM and the PB show a differential expression of genes: BM CD34+ cells cycle more rapidly, whereas mobilized PBSC are more deeply arrested in G0 and are composed of a greater number of quiescent stem cells (Uchida et al, 1997; Fruehauf et al, 1998). Steidl et al (2002) showed that nine cell-cycle-associated genes (e.g. CDC25A, B-MYB, PLK, CDC20, UBCH10) and 11 genes required for DNA synthesis (e.g. ligase 1) were expressed at distinctly higher levels in BM compared with circulating CD34+ cells, indicating a higher cycling activity of the former. The expression of apoptosis genes, including caspases 3, 4 and 8, was higher in circulating CD34+ cells, whereas the expression of anti-apoptotic cytoplasmic antiproteinase 2 was decreased. This might serve as a physiological mechanism to prevent uncontrolled proliferation once a haematopoietic stem cell has left the BM. The BM provides a suitable microenvironment for stem cells. It is a reservoir for haematopoietic stem and progenitor cells (Reya et al, 2001), endothelial progenitors (Lyden et al, 2001), and possibly neuronal, muscle, liver and pancreas progenitor cells (Krause et al, 2001). It is not known whether these primitive regenerative cells are one or many cell populations. Under steady-state conditions, most of the stem cells are maintained in G0 phase of the cell cycle by interaction with the stromal cells in the BM (Cheng et al, 2000), while there is only a small proportion of stem cells in S or G2/M phase of the cell cycle. Apparently, adhesive interactions between the CD34+ haematopoietic stem cells with cellular and matrix components of the BM environment are involved in mobilization (Papayannopoulou et al, 2001). Primitive haematopoietic stem cells express a wide range of cell adhesion molecules (CAM), including members of the integrin, selectin, immunglobulin superfamily and CD44 families of adhesion molecules. BM stromal cells express the ligands of many of these CAM. The role and relative contributions of the many individual CAM-ligand pairs in the homing, lodgement and retention of primitive haematopoietic stem cells within the BM is complex and is not fully elucidated. Mobilization of haematopoietic stem cells is believed to result from cytokine-induced functional changes in the adhesion profile expressed by the haematopoietic stem cells, facilitating egress from the BM. Mobilized CD34+ cells show significant reduction of c-kit, CD18+/CD11a+, CD49d, CD62L and CXCR4 compared with steady-state BM and PB (Kollet et al, 2001; Lapidot & Petit, 2002; Petit et al, 2002). The most extensively characterized adhesion molecules are: CD49d [very late antigen (VLA)-4] and its ligand, fibronectin (Papayannopoulou et al, 2001); CD49e (VLA-5) and its receptor, vascular adhesion molecule-1 (VCAM-1); and CD62L (L-Selectin), which mediates the initial contact of leucocytes with the endothelium and is also highly expressed on circulating CD34+ cells, suggesting its role for the homing of stem cells following transplantation (Dercksen et al, 1995; Fruehauf et al, 2003). In the mouse model, it was shown that VLA-4 and VCAM-1 were involved in the retention of stem cells in the marrow, as in vivo treatment of the mice with antibodies to VLA-4 or VCAM-1 induced CD34+ mobilization (Kikuta et al, 2000; Papayannopoulou et al, 2001). The reduced c-kit (CD117) expression before the egress of HSC in the circulation is inversely correlated with stem cell yield. Recently, it was demonstrated that the activation of the metalloproteinase (MMP)-9, leading to the release of c-kit-L, is a decisive checkpoint for the mobilization of haematopoietic stem cells as it promotes the recruitment of stem cells into peripheral blood (Heissig et al, 2002). The importance of metalloproteinases in the mobilization process has also been shown by studies on IL-8-induced mobilization. This HSC mobilization is preceded by a rise in MMP-9, leading to degradation of critical extracellular matrix components in the rhesus monkey model, and neutralizing antibodies directed against MMP-9 partially blocked IL-8-induced HSC mobilization (Pruijt et al, 1999). A higher expression level of CXCR4 is found on BM CD34+ cells compared with PB CD34+ cells (Deichmann et al, 1997). The upregulation of CXCR4 and the reduction of its ligand, stromal cell-derived factor-1 (SDF-1), is regarded as a prerequisite for mobilization (Petit et al, 2002). SDF-1 is expressed by BM stromal cells. The differential expression of this chemokine receptor supports the model that the interaction of SDF-1 and CXCR4 is crucial for the adhesion and differentiation of haematopoietic progenitors in BM (Peled et al, 1999; Fig 1). Consistent with these observations, 'poor mobilisers' showed significantly higher SDF-1, CXCR4 and VLA-4 expression than 'good mobilisers' (Gazitt & Liu, 2001; Gazitt et al, 2001) irrespective of the growth factor used for mobilization. Blockage of the G-protein-coupled CXCR4 receptor signalling by pertussis toxin led to PBSC mobilization in an experimental setting (Papayannopoulou et al, 2002). Proposed role of the CXCR4/SDF-1 chemokine receptor/ligand pair for mobilization of haematopoietic stem cells (HSC). G-CSF induces downregulation of SDF-1 in bone marrow stroma and upregulates matrix metalloproteinase-9 (MMP-9), neutrophil elastase (NE) and cathepsin G (CG). In the presence of proteases, an N-terminal cleavage of the CXCR4 receptor occurs. The CXCR2 agonist GROβ, or its derivative SB-251353, act via induction of MMP-9. The SDF-1 agonist CTCE0021 competes with stroma-bound SDF-1 for CXCR4 binding. The small molecule AMD-3100, a bicyclam, is also a partial CXCR4 agonist. The cyclic peptide TC14012 is an inverse CXCR4 agonist that does not signal and reduces PBSC mobilization. Chemokine receptor signalling occurs through the G 1 alpha (G1α) protein (Vlahakis et al, 2002). N,N-terminal CXCR4 epitope. G-CSF is the most established agent for the mobilization of stem cells in current clinical practice (Table I). Interestingly, it appeared that G-CSF receptor is required for G-CSF-induced proliferation of HSC in the BM but not for G-CSF-induced mobilization, as has been shown in G-CSF-receptor-deficient mice (Liu et al, 2000): G-CSF-dependent signals seem to act indirectly to mobilize stem cells from the BM. In this model, the first step toward HSC mobilization is the activation of a subset of mature haematopoietic cells by the mobilization stimulus such as G-CSF; the second step is the generation of secondary signals (protease release, SDF-1 modulation) by these activated cells, which in turn leads to mobilization. Changes in the BM microenvironment might be more important for a successful stem cell mobilization than changes within the HSC. Mobilization of HSC by G-CSF coincides in vivo with the cleavage of the N-terminus of CXCR4 on HSC resident in the BM (Fig 1). The N-terminus of CXCR4 on PB-derived CD34+ HSC, isolated from G-CSF-mobilized donors, was similarly cleaved (Levesque et al, 2002). More systematic exploration of this network resulted in the assumptions that: (1) neutrophil elastase (NE), cathepsin G and MMP-9 are not required for HSC mobilization by G-CSF in mice; (2) VCAM-1 cleavage may contribute, but is not required for HSC mobilization by G-CSF; (3) G-CSF treatment induces a decrease in BM SDF-1 protein that correlates well with HSC mobilization, but this can occur in the absence of neutrophil elastase and cathepsin G (Liu et al, 2000). Overnight culture of G-CSF-primed PBSC in a protease-free environment restored the expression of the N-terminal CXCR4 epitope. Therefore, it is likely that recovery of full-length CXCR4 expression occurs on HSC in the PB, facilitating their homing into the BM of the recipient (Levesque et al, 2002). These results suggest that the CXCR4/SDF-1 pathway plays a key role in PBSC mobilization (Fig 1). The aim of PBSC mobilization is to obtain at least 2·0 × 106 CD34+ cells per kg body weight (bw) as there is a clear inverse correlation between the amount of CD34+ cells infused below this threshold and the achievement of a rapid and sustained engraftment following myeloablative treatment (Siena et al, 1991, 2000). There are numerous studies analysing predictive factors for mobilization and the characteristics of the 'poor mobilisers'. Sautois et al (1999) showed that a leucocyte count of > 5 × 109/l, more than 5% myeloid progenitors in the PB and more than 0·02 × 109 CD34+ cells/l PB were associated with higher PBSC yields in patients mobilized with chemotherapy and G-CSF. The good correlation between the CD34+ cell count in peripheral blood and the yield in the PBSC collection has been shown by many groups (Haas et al, 1994; Ho et al, 1996). In a study of 41 consecutive patients, Marques et al (2000) reported that an early total white blood cell recovery was predictive of a low number of apheresis and high CD34+ cell yield. Healthy donors have been reported to show a high interindividual variation in PBSC mobilization using G-CSF (Grigg et al, 1995; Anderlini et al, 1997; Holm, 1998; de la Rubia et al, 2002). The roles of donor age and the G-CSF administration schedule are controversial. A significant negative influence of these two variables on the CD34+ cell mobilization has been shown when the aim was to collect ≥ 4·0 × 106 CD34+/kg bw (de la Rubia et al, 2002). This could not be confirmed by others or the correlation seen was rather weak (Miflin et al, 1996; Tabilio et al, 1997). Consequently, Anderlini et al (1997) concluded that it is not possible to exclude a healthy individual as PBSC donor because of clinical characteristics associated with a low PBSC yield. In terms of tolerance and mobilization capacity of CD34+ cells, G-CSF was proven to be superior to GM-CSF in healthy donors (Fischmeister et al, 1999). The acute G-CSF-associated side affects are generally mild: bone and muscle pain, and slight spleen enlargement (Platzbecker et al, 2001). Severe allergic reactions are rare. Several reports have demonstrated that G-CSF administration induces a transient mild hypercoagulable state, therefore, caution should be applied when considering donors with a history of peripheral vascular disease, myocardial infarction or stroke (Gutierrez-Delgado & Bensinger, 2001). In conclusion, the long-term side-effects of G-CSF administration to normal donors need to be further studied (Beelen et al, 2002), and a longer follow-up using large registry databases is required (Gutierrez-Delgado & Bensinger 2001; de la Rubia et al, 2002). The interindividual differences in PBSC mobilization may be partially due to genetic background. Mouse studies have shown that mobilization efficacy is the result of multigene traits controlled by loci on chromosomes 2 and 11 (Hasegawa et al, 2000). In general, poorly mobilizing mice strains also have low stem cell concentrations in the BM. As SDF-1 is constitutively expressed by BM stromal cells and other tissues, and has also an important role for stem cell homing (Shirozu et al, 1995), Benboubker et al (2001) investigated the relationship between SDF-1 gene polymorphism and mobilization capacity in 63 patients with malignancies. They demonstrated that the presence of the SDF-1 3′-A allele was a predictive factor for high levels of circulating CD34+ cells. As the SDF-1 genotype was not absolutely predictive for moderate or poor mobilization, interference with others factors must be considered. Little is known about the association of certain human leucocyte antigen (HLA) genotypes and mobilization potential. We and others have observed that PB CD34+ cells and colony forming cells during steady-state haematopoiesis are measures of a patient's mobilisable pool after G-CSF and chemotherapy (Fruehauf et al, 1995, 1999; Haug et al, 1997), or after G-CSF alone (Brown et al, 1996; Husson et al, 1996; Ashihara et al, 2002). BM CD34+ cells were not predictive of the mobilization yield. Prospective studies are necessary to confirm that the expression level of molecular markers, such as CXCR4 or VLA-4 etc., really can predict poorly mobilizing patients. In stem cell disorders, the diagnosis the PBSC mobilization of patients with acute myeloid show lower CD34+ cell in the peripheral blood compared with other malignancies. patients may have marrow of CD34+ cells with a release of these cells into the peripheral blood during the apheresis procedure than observed in et al, 1999). the other et al (1997) found that of patients with were able to collect sufficient PBSC for transplantation > × 106 CD34+ after induction with and first with and The mobilization capacity of patients with haematological malignancies in general, lower than in patients with solid tumours. patients with and patients are compared with or patients, or patients with other solid et al, 1998; et al, 1998; Gazitt et al, 1999; Sautois et al, 1999). This is most likely to be due to the more of patients with haematolymphoid malignancies. mobilization in patients with has also been associated with an increased of the development of or secondary after transplantation et al, 2002). The results the influence of age on the PBSC yield are as already for the healthy donors. In there was correlation between PBSC yield and the patient's age et al, 1998; et al, 2001). patients with an age of than years was associated with PBSC mobilization et al, 2000). The for the influence of reported a significantly higher of mobilization in than in et al, 2001). studies could not confirm this but the lower amount of total CD34+ cells in the to the lower body weight of compared with correlation between CD34+ cell yield and was observed in et al, 2001). BM does not seem to be an factor with significant influence on PBSC mobilization, although reduction of within the marrow environment may the of successful mobilization et al, 1999). In the results are some that BM significantly affects et al, 1997; et al, while others have to show et al, 1997; et al, 1998). These may be to the of the bone marrow with cells and the high of studies that and chemotherapy with agents mobilization et al, 1997; & 1998; et al, 1998; et al, 1998; et al, 1998; et al, 2001). is particularly and a low before mobilization results in reduced CD34+ cell mobilization et al, et al, 1996; et al, 1997; et al, 1997; et al, 2002). was proven in several studies to be associated with poor PBSC mobilization (e.g. et al, as well as other agents such as or more than chemotherapy et al, 1998). of and were and steady-state G-CSF PBSC mobilization and et al, 2002). to predict poor mobilization have been for certain et al (2001) found that in patients a between diagnosis and mobilization therapy of more than was an as well as a high BM with cells and exposure to agents for a mobilization of × 106 CD34+/kg et al (2000) to × 109 PB, and found low white blood cell and exposure to and an longer than from the of treatment to be In an of patients, et al (2001) found PBSC mobilization in those received at least two of received a high number of different chemotherapy with a chemotherapy > and received more than of results were seen by et al, in consecutive and patients underwent autologous transplantation between and predictive for poor were a of significant increase in white blood cell count on the first of apheresis and the number of Interestingly, found that poor mobilization a after for patients. This was not confirmed by et al did not find difference survival and overall survival in good and poorly mobilizing patients. and did not a correlation between the of chemotherapy and several have shown that mobilization is by a treatment when the is (Haas et al, 1994; et al, 1996; et al, 2002). et al (1999) for patients, that a of more than was sufficient to marrow and PBSC mobilization a is not in with a high of such as or could be a possible therapy to during this et al, 1994; et al, 2000). We observed a that patients received more than of chemotherapy their CD34+ cell in the PB et al, and showed a reduced number of CD34+ cells in the PB (e.g. 5% of all patients with 4 the CD34 than of all patients with > This is in with studies that PBSC collection should be early in the of to stem cell et al, 1997; et al, 1998; et al, 2002). is a used mobilization et al (2001) showed that at 4 haematological and with an CD34+ collection compared with while others found a of on the mobilization et al, 1997). and G-CSF mobilized sufficient PBSC side-effects in patients, leading to a high proportion of after transplantation with et al, 2001). A combination and followed by G-CSF administration mobilized sufficient PBSC in of heavily pretreated patients et al, 2001). with significant activity against and with G-CSF was used to mobilize PBSC with in these patients et al, 2000). with has been shown to be for PBSC mobilization in patients with compared with other et al, 2000). PBSC mobilization with and G-CSF with or in 40 cell patients led to higher numbers of CD34+ cells in the circulation in the of the overall collection et al, 2001). There are only a few studies that with the of apheresis and mobilization to the mobilization et al in a of patients, the PBSC collection at a of in patients receiving and and a of in patients were mobilized with and G-CSF. Interestingly, the were with a at the first apheresis and rapid of CD34+ cell count in the and a steady mobilization in the The concluded that the mobilization should be into before PBSC G-CSF and GM-CSF are the most used cytokines for PBSC mobilization in patients. difference in their mobilization was found for these two cytokines (Grigg et al, 1995; et al, 1998). The to these cytokines is therefore, a higher G-CSF is in poorly mobilizing patients (Grigg et al, 1995; et al, 1996; 1999). This is only the cytokine is applied after chemotherapy, the mobilization yield is not on the G-CSF et al, 1998). Several studies have shown that PBSC mobilization is more using chemotherapy and cytokines like compared with cytokines alone et al, 1995; et al, 1997; et al, 1998). It therefore, to use a chemotherapy with G-CSF or other in patients with A subset of patients, often heavily not PBSC mobilization. et al (1999) that poor or heavily pretreated patients could be with a high G-CSF to obtain sufficient CD34+ cells. the other after chemotherapy the mobilization yield is not on the G-CSF that there is for of G-CSF in this et al (2000) found that a combination therapy with and G-CSF is a sufficient