Mobilization of hematopoietic stem cells

Mobilization of hematopoietic stem cells

Blood Reviews (2000) 14, 205–218 © 2000 Harcourt Publishers Ltd doi: 10.1054/ blre.2000.0138, available online at http://www.idealibrary.com on Haema...

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Blood Reviews (2000) 14, 205–218 © 2000 Harcourt Publishers Ltd doi: 10.1054/ blre.2000.0138, available online at http://www.idealibrary.com on

Haematological oncology

Mobilization of hematopoietic stem cells

S. Fu, J. Liesveld

Hematopoietic stem cell transplantation has been extensively exploited as a therapeutic and research modality and has revolutionized current patient care. At present, more and more medical centers use peripheral blood progenitor cells for transplantation by mobilizing hematopoietic stem cells from bone marrow to peripheral blood because of potential advantages of peripheral blood stem cell transplantation over bone-marrow transplantation. Different effective mobilization regimens have been developed recently with chemotherapeutic agents, hematopoietic growth factors or their combination. This article reviews current developments related to hematopoietic stem cell mobilization including the biology of hematopoietic stem cells, strategies for mobilization, management for mobilization failure, mechanisms of mobilization, and side effects during mobilization. Finally, the Initiation-Amplification-Emigration-Adaptation Model is proposed to help aid understanding of the mechanisms of hematopoietic stem cell mobilization and to stimulate development of novel and optimal mobilization strategies for patient care. © 2000 Harcourt Publishers Ltd aspirations from the posterior iliac crests and in some cases from the anterior crests, while the peripheral blood progenitor cells can be collected by leukapheresis after mobilization with chemotherapy and/or hematopoietic growth factors.2,3 Hematopoietic stemcell mobilization refers to a process of expanding circulating hematopoietic stem cell numbers. Potential advantages of peripheral blood progenitor cell transplantation include the avoidance of marrow harvest and anesthesia and the ability to expand the pool of eligible patients to include those with marrow involvement with tumor or prior pelvic radiation therapy, which frequently precludes marrow harvest.4 Another advantage of using peripheral blood progenitor cells compared to marrow has been enhanced engraftment of myeloid cells and platelets, resulting in significantly decreased medical support such as blood product use, antibiotic use and length of hospitalization.5,6 At present, more and more medical centers use peripheral blood progenitor cells for transplantation by mobilizing hematopoietic stem cells with chemotherapeutic agents and/or hematopoietic growth

INTRODUCTION The development of successful hematopoietic stem-cell transplantation as a therapeutic and research modality has revolutionized patient care in hematology and oncology, and has provided a curative method for some diseases including cancers and genetic diseases. Hematopoietic stem cells can be obtained from bone marrow, peripheral blood and umbilical cord blood, and from autologous, syngeneic and allogeneic donors.1 The marrow harvest is obtained under general or spinal anesthesia via multiple needle punctures and

Siqing Fu MD, PhD, Jane Liesveld MD, University of Rochester Medical Center, Department of Internal Medicine Hematology/Oncology, Blood and Marrow Transplant Program, 601 Elmwood Avenue, Box 610, Rochester, NY 14642, USA. Correspondence to: Jane Liesveld MD, University of Rochester Medical Center, Department of Internal Medicine, Hematology/ Oncology, Blood and Marrow Transplant Program, 601 Elmwood Avenue, Box 610, Rochester, NY 14642, USA. Tel.: +1 (716) 275 4099; Fax: +1 (716) 473 4314; E-mail: [email protected] rochester.edu

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factors.7 Success of mobilization is a prerequisite for success of transplantation. Thus, review of the known and unknown areas of mobilization has potential impact for understanding and maximizing the process.

THE BIOLOGY OF HEMATOPOIETIC STEM CELLS A hematopoietic stem cell must have two characteristics:8 the ability to differentiate to all hematopoietic lineages and the ability to maintain hematopoiesis over a life-span by a self-replication process.9 After hematopoietic stem cells were found to circulate routinely in the peripheral blood, initial studies using animal transplant models had positive results with these peripheral progenitor cells to establish sustained hematopoietic function.10 In the mid-1980s, investigators reported successful autologous peripheral blood stem cell autografts for patients with different diseases from several different medical centers working independently. Then, transplantation of allogeneic bloodderived cells was documented to result in sustained hematopoietic function of donor origin. The above clinical trials provided evidence for functional hematopoietic stem cells in the human circulation. There is the possibility that two general hematopoietic stem cell compartments exist in the bone marrow, one that frequently cycles and is the primary source of blood production over time, and another that rarely cycles but represents the major source of engraftment post-transplant.11 Only long-term engraftment of all blood lineages after bone marrow transplantation can truly test hematopoietic stem cell function. The invitro assays including CFU-C (a colony-forming unit culture assay), LTCIC (a long-term culture-initiating cell assay) and CAFC (a cobblestone area forming cell assay) provide supplemental tools to test hematopoietic stem cell function. Both LTCIC and CAFC assays are labor-intensive. The CFU-C assay is not reliably predicative of engraftment potential. Thus, phenotypic analysis of cell-surface antigens by flow cytometry has become a promising method to assess the quality of a potential transplant product if hematopoietic stem cells have specific antigen phenotype on the cellular surface.12 The human hematopoietic stem cell phenotype includes expression of CD34 and CD90 (Thy-1) antigens in the absence of a series of other antigens that mark differentiation lineage such as CD38.13,14 These CD34+CD90+CD38- hematopoietic cells which constitute 0.05% to 0.1% of human marrow and circulating hematopoietic cells are capable of giving rise to long-term repopulating hematopoietic cells of multiple lineages. CD34 antigen may function as an

adhesion molecule, based on homology to selectin and functional studies, and Thy-1 antigen may be involved in triggering signal-transduction cascades.15 In human postnatal hematopoietic tissues, 0.1 to 0.5% of CD34+ cells expressed VEGFR2 (vascular endothelial growth factor receptor 2 or KDR receptor).16 Primitive uncommitted hematopoietic stem cells are restricted to the CD34+KDR+ cell fraction. Another characteristic phenotype such as expression of MDR1 (gp170, multiple drug resistance phenotype) has been successfully used to define a primitive subset of CD34+ cells and even to predict the existence of hematopoietic stem cells that are CD34–, which may represent precursors of the CD34+ stem cells with better engraftment over long periods of time.17 New evidence from aging donors suggested even in the non-transplant setting of human hematopoiesis, the ability of the hematopoietic stem cell to self-replicate is not unlimited. The studies on the kinetics of cell division among hematopoietic stem cells suggested hematopoietic stem cells were quiescent with respect to the cell cycle and few of them were in cycle at any given time. These cycling hematopoietic stem cells failed to engraft the nucleated cell lineages in irradiated recipient animals when transplanted intravenously. Hematopoietic stem cells mobilized from the marrow by chemotherapy and/or hematopoietic growth factors were believed to be largely out of cell cycle when collected from the peripheral blood by leukapheresis.18 Thus, control of the cell cycle and expression of specific molecules are keys to understanding hematopoietic stem-cell biology and transplantation. As noted above, the CD34+ peripheral blood cells represent a pool of hematopoietic progenitor cells which include a small percentage of hematopoietic stem cells. To find a parameter to predict the quality of the collected CD34+ peripheral blood cells for engraftment, we examined the use of the ratio of CFU-GM vs CD34+ cells as a index to correlate with prompt reconstitution of hematopoietic lineages. Before transplantation, the infused number of CD34+ cells per kg and the infused number of CFU-GM per kg from all the studied patients at University of Rochester in 1999 were identified. The ratio of CFU-GM vs CD34+ cells was calculated. The recipient patients, most of whom received more than 2 ×106 CD34+ cells/kg (90%) or more than 5 ×106 CD34+ cells/kg (40%), were monitored with daily complete blood cell count until patients achieved 50 000 platelets/µl. Linear regression analyses were conducted to determine whether the ratio of CFU-GM vs CD34+ cells as a parameter for the quality of the collected CD34+ cells correlated with prompt reconstitution of platelets. Data as shown in Figure 1 supported significantly that the ratio of CFU-GM vs CD34+ cells could be applied to predict

Mobilization of hematopoietic stem cells

prompt reconstitution of platelets (P=0.031 and P=0.024 for days to reach platelet 20,000 per microliter and 50,000 per microliter, respectively). Further analyses showed that the average day to reach a platelet count of 20 000 per microliter was day 20 if the ratio was less than 1.4 and day 26 if the ratio was more than 1.4 with P-value <0.04, and the average days to reach platelet 50 000 per microliter was day 22 if the ratio was less than 1.4 and day 31 if the ratio was more than 1.4 with P-values <0.03. Therefore, successful engraftment is determined not only by the quantity, but also by the quality of mobilized peripheral blood progenitor cells.

STRATEGIES FOR HEMATOPOIETIC STEM CELL MOBILIZATION (TABLE 1) Autologous and allogeneic peripheral blood progenitor cell transplants have been increasingly performed for an effective curative treatment modality for a variety of disorders, including malignancy, aplasia, immunodeficiency and genetic disorders and are rapidly replacing conventional bone-marrow transplantation, not only due to decreased risks and greater convenience for the donor, but also because transplant with peripheral blood progenitor cells results in more rapid engraftment with less medical support.1,19–22 Collection of enough hematopoietic stem cells for transplantation from peripheral blood during steadystate marrow function usually requires leukapheresis from more than 40 L of blood. Thus, mobilization of hematopoietic stem cells from marrow to peripheral blood before leukapheresis is desirable. The number of circulating peripheral blood progenitor cells is greatly increased during the recovery phase following chemotherapy with myelosuppressive, but not myeloablative, agents such as cyclophosphamide.23 Hematopoietic growth factors were also found to significantly increase the number of peripheral blood progenitor cells. When chemotherapy and hematopoietic growth factors are combined, a synergistic effect resulting in increased peripheral blood progenitor cells occurs. Therefore, substantially larger numbers of peripheral blood progenitor cells can be collected following mobilization with hematopoietic growth factors, chemotherapy or their combination. By the use of the CD34 cell surface marker as a means of identifying the number of primitive hematopoietic stem cells contained in a peripheral blood progenitor cell collection, most reports suggested that a target number of 5 ×106 CD34+ cells/kg be infused for sustained neutrophil, platelet, and red blood cell recovery.24,25 Thus, the goal of peripheral blood progenitor cell mobilization is to collect at least 5 ×106 CD34+ cells/kg after leukapheresis, and after positive and

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Table 1 Strategies for stem-cell mobilization 1. Use of myelosuppressive but not myeloablative chemotherapy 2. Use of hematopoietic growth factors 3. Use of chemotherapy and hematopoietic growth factors in combination Single Growth Factors Combination chemotherapy chemotherapy Regimens agents (Utilized with G-CSF) Cyclophosphamide G-CSF ICE Paclitaxel GM-CSF TEC SCF CP Flt-3 ligand DICEP The above are examples of regimens that have been used for progenitor cell mobilization. See text for definitions of abbreviations and acronyms.

negative stem-cell selection, if such procedures are to be performed. Although 5 ×106 CD34+ cells/kg is an optimal goal for mobilization, successful transplantation with as few as 1 ×106 mobilized CD34+ cells/kg have been demonstrated in several clinical trials26 and most centers routinely perform transplantation with 2 ×106 CD34+ cells/kg with acceptable engraftment success. In contrast to the typical bone-marrow collection performed in an operating room during general anesthesia, peripheral blood progenitor cell collection is accomplished by using a leukapheresis device in an outpatient setting without anesthesia.27–29 First,placement of central venous catheters is needed for leukapheresis, administration of high-dose therapy and medical support of patients during recovery after transplant.30 Then mobilization is achieved with chemotherapeutic agents and/or hematopoietic growth factors. Finally, the leukapheresis device is programmed to collect cells with characteristics of low-density monocytes or lymphocytes. Instructions for collecting peripheral blood progenitor cells are available from manufacturers. Each leukapheresis procedure requires about 2.5 to 4 h to process 8 to 15 L of blood, which can be repeated daily as needed.31 Usually two or three standard-volume leukapheresis procedures are required to harvest a sufficient number of hematopoietic stem cells.32 These peripheral blood progenitor cells can be further manipulated for negative and positive selection to meet the patient’s need.33,34 The utilization of chemotherapeutic agents in mobilization Mobilization originated from an observation made in the 1970s that circulating hematopoietic stem cells increased following chemotherapy with myelosuppressive agents. Administration of myelosuppressive agents produces a mobilization of hematopoietic stem cells from marrow to peripheral blood about 2 weeks

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later as recovery from the nadir of peripheral cytopenia starts.35,36 Cyclophosphamide and paclitaxel were used frequently, among many chemotherapeutic agents.37–39 Mobilization by chemotherapeutic agents alone was used widely during the 1980s and early 1990s and has been replaced with hematopoietic growth factor-induced or hematopoietic growth factor plus chemotherapeutic agent-induced mobilization.40 The utilization of hematopoietic growth factors in mobilization The initial fear of using hematopoietic growth factors as mobilizing agents was that administration of hematopoietic growth factors to mobilize hematopoietic stem cells might stimulate circulating hematopoietic stem cells to differentiate from the uncommitted to the committed stage so that fewer primitive hematopoietic stem cells could be collected.41 The utilization of hematopoietic growth factors in mobilization has been used widely after first reports in 1988 of the mobilizing effects of G-CSF (granulocyte colony-stimulating factor) and GMCSF (granulocyte-macrophage colony-stimulating factor) and a later report of long-term hematopoietic reconstitution in patients transplanted with only hematopoietic growth factor mobilized peripheral blood progenitor cells.42 Several hematopoietic growth factors have been used as mobilizing agents to procure autologous as well as allogeneic peripheral blood progenitor cells.43 The kinetics of hematopoietic stem cell mobilization are similar with different hematopoietic growth factors, with peak levels of peripheral blood progenitor cells achieved after 5 to 10 days of hematopoietic growth factor treatment.44,45 A broad spectrum of hematopoietic cells are mobilized, including primitive hematopoietic stem cells, megakaryocytic, erythroid and myeloid progenitors as well as mature neutrophils. However, the efficacy of mobilization is not similar among different hematopoietic growth factors, ranging from a fivefold to a 500-fold increase of peripheral blood progenitor cells over baseline.46,47 G-CSF is the most commonly used hematopoietic growth factor for mobilization among others, including GM-CSF, SCF (stem cell factor, c-kit ligand), flt-3 ligand, IL1 (interleukin 1), IL3(interleukin 3), IL7 (interleukin 7), IL11 (interleukin 11), and IL12 (interleukin 12).48–55 In addition, G-CSF has been shown to act synergistically with other hematopoietic growth factors to induce hematopoietic stem cell mobilization. The maximum mobilization effect occurs on about the fifth day of G- CSF administration. Escalation of G-CSF doses up to 16 µg/kg/12 h is well tolerated and results in the collection of a higher number of peripheral blood progenitor

cells in healthy donors.56 Ninety-one percent of GCSF-stimulated normal donors produce more than 2 ×106 CD34+ cell/kg by a single leukapheresis. The combination of sequential and concurrent G-CSF with GM-CSF has been reported to be more effective than mobilization with G-CSF alone.57–59 The utilization of chemotherapy agents and hematopoietic growth factors in mobilization Although hematopoietic growth factors such as GCSF are generally used in more and more mobilization protocols, the optimal schedules and regimen for priming hematopoietic stem cells still remain to be established.60 Daily administration of hematopoietic growth factors following administration of mobilization doses of chemotherapy provides more effective mobilization of hematopoietic stem cells than either chemotherapy or hematopoietic growth factor treatment alone.61,62 This combination of chemotherapy and hematopoietic growth factors generates a maximum mobilization effect about 7 to 14 days later, which can last for several days during leukapheresis.63 Usually this correlates with rebound of the white blood cell count after nadir. In addition, their combination can be used for their anti-tumor effect, to avoid tumor cell contamination in the collections and to maximize hematopoietic stem-cell mobilization for autologous transplantation.64 An ideal regimen for autologous transplantation should be highly active against the target tumor cells as well as be capable of mobilizing hematopoietic stem cells for collection.65 Among many regimens, single chemotherapeutic agent such as cyclophosphamide66 or paclitaxel67,68 can be used to combine with G-CSF to mobilize hematopoietic stem cells. Multicyclic and dose-intensive chemotherapy in combination with hematopoietic growth factors may allow development of optimal regimens which are safe and well tolerated, highly active against metastatic tumor cells and capable of excellent hematopoietic stem-cell mobilization.69 Successful reports on mobilization induced by the combination strategy were published in chronic myelogenous leukemia with the mini-ICE regimen (idarabine, cytarabine and etoposide plus G-CSF),70 in lymphoma with the IVE regimen (ifosfamide, etoposide and epirubicin plus G-CSF),71,72 in metastatic breast cancer with the TEC regimen (paclitaxel, etoposide and cyclophosphamide plus G-CSF)73 or the CP regimen (cyclophosphamide and paclitaxel plus G-CSF),37 in lung cancer and lymphoma with the ICE regimen (ifosfamide, carboplatin and etoposide plus G- CSF),74 in some hematologic and solid malignancies with the DICEP regimen (dose-intensive cyclophosphamide, etoposide and cisplatin plus G-CSF),75,76 and many others.

Mobilization of hematopoietic stem cells

MANAGEMENT FOR MOBILIZATION FAILURE (TABLE 2) It is clear that previous chemotherapy and radiotherapy can impair the mobilization of sufficient hematopoietic stem cells to proceed with transplantation, especially with previous exposure to alkylating agents such as chlorambucil and purine analogues.77,78 Successful mobilization of hematopoietic stem cells is determined by successful engraftment.79,80 With infusion of adequate peripheral blood progenitor cells, neutrophil recovery will usually occur about 9–10 days post-transplant in almost all patients, and platelet recovery will occur about 10–11 days posttransplant, of whom 5–35% will experience delays in platelet engraftment.81 Therefore, prompt engraftment of platelets can be an index for successful mobilization, which is correlated with the CD34+ cells transplanted. Several retrospective studies document a minimum safe dose of 1 ×106 CD34+ cells/kg, especially according to the ideal body weight. Below this level, up to 80% of patients have delayed platelet engraftment and experience a significant risk of severe hemorrhage. It appears that the optimal CD34+ cell dose for prompt platelet engraftment is 5 ×106 CD34+ cells/kg, with more than 85% of patients having prompt platelet engraftment by day 14 post-transplant. Using the number of collected CD34+ cells/kg after mobilization and leukaphereses as a criterion, three groups of patients can be described as follows (See also Table 3): the non-mobilizable patient who does not accumulate the minimum cell dose of 1 ×106 CD34+ cells/kg after repeated leukaphereses, the poorly mobilizable patient who can reach the minimum cell dose but cannot accumulate the optimal cell dose of 5 ×106 CD34+ cells/kg after repeated leukaphereses, and the mobilizable patient who can easily accumulate the optimal cell dose of 5 ×106 CD34+ cells/kg in less than 5 leukaphereses.82 Several factors are associated with the inability to mobilize and collect 5 ×106 CD34+ cells/kg.83,84 Multivariate analyses have demonstrated that the number of chemotherapy cycles (>6 cycles), regardless of previous radiotherapy, and prior use of alkylating agents are associated with poor hematopoietic stem cell mobilization.85 Other factors include type of malignancy, age greater than 60 years, bone marrow metastases, prior radiotherapy to marrow-producing

Table 2 Donor classification based on mobilization potential 1. The non-mobilizable patient <1 × 106 CD34+ cells/kg* 2. The poorly mobilizable patient >1 ×106 CD34+ cells/kg 3. The mobilizable patient ≥5 ×106 CD34+ cells/kg *Amount able to be collected in ≥5 leukaphereses.

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Table 3 Factors associated with poor mobilization 1. 2. 3. 4. 5. 6.

Numbers of previous chemotherapy cycles Prior use of alkylating agents Type of malignancy Age > 60 years Prior radiotherapy to marrow-producing sites Inadequate mobilization regimen

sites, and the mobilization regimen.86 Certain chemotherapeutic agents such as melphalan, nitrosoureas, procarbazine, nitrogen mustard, fludarabine and platinum compounds appear to be associated with poor mobilization capacity.87,88 It is apparent that the alternative approach for patients with mobilization failure is to find an HLAmatched donor for allogeneic transplantation or to expand ex vivo the mobilized or marrow hematopoietic stem cells. Currently only few medical centers can handle this expensive strategy of ex vivo hematopoietic stem cell manipulation, which is beyond the scope of this review. If the matched donor is not available, several other strategies can be tried for this group of patients in spite of data indicating that currently available strategies do not ensure that patients with mobilization failure will achieve adequate CD34+ cell/kg for transplantation. An apparent strategy for hematopoietic stem-cell collection is to perform a bonemarrow harvest, which may be of some benefit in selected patients. Recent studies suggest that bone marrow harvesting is of limited value, especially in the non-mobilizable patient group.84 Thus, when fewer than 1 ×106 CD34+ cells/kg are mobilized, a different approach instead of bone marrow harvesting should be considered. The choices left are remobilization with the same regimen in escalated doses, mobilization with a different regimen such as multi-drug, muli-cyclic and dose-intensive chemotherapy and its combination with hematopoietic growth factors, or the combination of several hematopoietic growth factors (Table 4). Remobilization with the same regimen generally produces the same poor mobilization until the complete hematopoietic recovery from prior chemotherapy.89 Mobilization with a hematopoietic growth factor alone cannot be achieved for 4–5 weeks after the chemotherapy has been completed. Several remobilization strategies were evaluated recently.90,91 A higher dose of cyclophosphamide at 7 g/m2 instead of 4 g/m2 for the second mobilization produces a significant increase in peripheral blood progenitor cells. Higher doses of cyclophosphamide are not well tolerated, however, due to organ toxicity. Remobilization with escalated doses of G-CSF alone from 5 µg/kg/day to 32 µg/kg/day is effective in at least one-third of patients with mobilization failure. In one study, 96% of patients mobilized with a higher dose of G-CSF at

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Table 4

Alternative mobilization strategies

1. For selected patients, find a suitable allogeneic donor. 2. Marrow harvest 3. Escalated doses of the same mobilization regimen; e.g., higher doses of G-CSF 4. Mobilization with a different regimen Chemotherapy plus growth factor where growth factors alone had been utilized. Growth factor combinations (G-CSF + GM-CSF) (G-CSF + SCF) (G-CSF + Flt-3 ligand)

32 µ/kg/day achieved 2 ×106 CD34+ cells/kg in three collections, compared with 83% mobilized with GCSF at 10 µg/kg/day.92 Obviously, this benefit with escalating doses of chemotherapeutic agents and/or hematopoietic growth factors must be balanced against the associated morbidity and costs.73 Mobilization with a different regimen may produce better results.93,94 For patients initially mobilized with GM-CSF plus IL3, the switch to cyclophosphamide and GM-CSF mobilizes a significantly higher peripheral blood progenitor cells, 1.5 vs 4.1 ×106 CD34+ cells/kg. The most commonly utilized chemotherapeutic agent cyclophosphamide at 4–7 g/m2 produces a 5 to 30-fold increase in the number of CD34+ cells/kg in the peripheral blood. The addition of paclitaxel and/or etoposide produces better results than cyclophosphamide alone, as does the addition of GM-CSF, which results in a two-to five-fold greater increase in the number of CD34+ cells/kg after mobilization and leukapheresis. A randomized phase II trial done with poorly mobilizable patients revealed that the total CD34+ cell collection was significantly higher in the group mobilized with G-CSF and SCF or G-CSF and ftl-3 ligand than that with G-CSF alone.95–99 Forty-four percent of patients in the combination group reached the target dose of 5 ×106 CD34+ cells/kg and only 15% did not reach the minimum dose of 1 ×106 CD34+ cells/kg, compared with 17 and 26% in the G- CSF alone group respectively. Therefore, a combination of multiple chemotherapeutic agents68,100 such as paclitaxel, cyclophosphamide, etoposide, anthracyclines, ifosfamide, cisplatin, carboplatin and ara-C, with hematopoietic growth factors appears to be most effective in mobilizing hematopoietic stem cells.

MECHANISMS OF HEMATOPOIETIC STEM CELL MOBILIZATION (TABLE 5) The in vivo studies on the transition of hematopoietic stem cells during embryonic development from both embryonic and extraembryonic sites to fetal liver and finally to bone marrow, and the homing and trafficking

Table 5 Proposed mechanistic influences on mobilization 1. Alteration in adhesion receptor density or affinity 2. Changes related to chemokine or cytokine influences examples: IL-8, G-CSF 3. Release of metalloproteinases 4. Activation of intracellular signal transduction pathways 5. Alteration of proliferation and cell cycle status 6. Possible role of increased blood flow/crowding

of hematopoietic stem cells post transplantation provide us much information to help understand mechanisms of hematopoietic stem-cell mobilization. Although one or more common pathways are believed to be involved in mobilization and the individual parts remain unlinked, several mechanisms are obviously associated based on current limited in vivo and in vitro studies. Below is a summary of the previous studies in the area of mobilization and a description of the proposed initiation-amplification-emigration and adaptation model for mobilization.

The characteristics of circulating hematopoietic stem cells in steady or mobilized state The human bone marrow can be divided into two compartments: vascular and extravascular. Hematopoiesis occurs almost exclusively in the extravascular compartment. Exchange between these two compartments takes place at the level of sinusoids, which form the blood–bone-marrow barrier with a layer of endothelium.101 Thus, mobilization of hematopoietic stem cells takes place when these cells leave the stromal support system and migrate through the endothelial layer into the vascular compartment. Changes in the surface antigen profile, expression of adhesion molecules, proliferative state and responsiveness to hematopoietic growth factors and chemokines are associated with this process.102,103 By utilizing the CD34 antigen as a marker, most circulating hematopoietic progenitor cells in steady state and mobilized peripheral blood progenitor cells are found to be in either G0/G1 or in a prolonged G1 phase. They are ready for cycling soon after exposure to hematopoietic cytokines in vitro, in contrast to bone-marrow hematopoietic progenitor cells which have a longer latency and a higher percentage of S phase progenitors. Changes of surface antigen and adhesion molecule expression are different between these two groups of circulating hematopoietic stem cells.104 For example, an increased L-selectin level and a decreased VLA-4 (the β1 integrin CD29 and the α4 integrin CD49d, very late antigen 4) level are noted in circulating mobilized primitive CD34+ cells,105 Expression of C-kit (CD117, the stem cell factor

Mobilization of hematopoietic stem cells

receptor) and CXCR4 (the receptor for stromal cell derived factor-1) expression decrease in both circulating primitive CD34+ cells in steady state and circulaing mobilized primitive CD34+ cells.106 The role of hematopoietic growth factors and other chemokines Hematopoietic growth factors and chemokines are demonstrated in many clinical trials as effective and safe mobilizing agents for both autologous and allogeneic transplantation. The kinetics of mobilization range from a few minutes with some chemokines to several days with some hematopoietic growth factors. Cross-talk between hematopoietic stem cells, marrow microenvironment and other systems via hematopoietic growth factors and chemokines plays a major role in mobilization. The importance of hematopoietic growth factors and chemokines in mobilization is that they are functioning not only as a initiation trigger, but also as highly activated transmitters for this complicated cross-talking network, functioning directly as chemoattractants in emigration and supporting survival of the mobilized peripheral blood progenitor cells as well.107 Because of its potency and lack of serious toxicity, G-CSF is the most commonly used mobilizing agent.108 The role of G-CSF in mobilization has been intensively explored recently. Obviously, G-CSF can work as an initiation trigger, primarily targeting myeloid cells as well as endothelial cells. Administration of G-CSF significantly increases the percentage and absolute number of CD34+ cells in the peripheral blood,110 which have similar expression patterns of CD13, CD33, CD38, HLA-DR and CD90 antigens, but decrease expression of c-kit (CD117), VLA4 (CD49d-CD29), VLA5 (CD49e-CD29: α5β1 integrins, very late antigen 5), LFA1 (CD11a-CD18: the αLβ2 integrins, leukocyte function-associated antigen 1), LFA3 (CD58, leukocyte function-associated antigen 3) and L-selectin (CD62L) compared with their circulating counterparts in steady state; decreased responsiveness to SDF1 (stromal cell derived factor 1)111 induced chemotaxis through a transendothelial migration assay due to decreased expression of CXCR4; decreased adhesion capacity to preformed stroma; and decreased percentage of CD34+ cells in active cell cycle. The data from G-CSF receptor knock-out mice demonstrate that the presence of G-CSF receptors is required for hematopoietic stem-cell mobilization with G-CSF, cyclophosphamide, and IL8 but not with hematopoietic growth factors flt-3 and IL-12.112 The chemokine IL8 is the best characterized member of the ELR+ (Glu-Leu-Arg motif) and C-X-C

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(Cys-X-Cys motif) chemokines and has capacity for mobilizing hematopoietic stem cells up to 100-fold within 1 h.113 After a single administration of IL8, neutropenia is observed within 5 min and is followed by neutrophilia after several hours, associated with increases in several circulating hematopoietic growth factors. Multiple factors including GM-CSF can induce IL8 production. At least three mechanisms are involved in the IL8 induced mobilization. Firstly, it requires the intact G-CSF system shown as above. Secondly, it can be completely blocked by pretreatment with the anti-LFA1 antibody, suggesting the role of the β2 integrins in this pathway. Finally, IL8 can stimulate the rapid release of metalloproteinases such as gelatinase B113 which, in turn, cleave the marrow extracellular matrix and mobilize hematopoietic through the damaged blood–bone-marrow barrier.114 The role of the adhesion molecule system The hematopoietic stem cells are surrounded by stromal cells such as fibroblasts, adipocytes, macrophages and endothelial cells, and other hematopoietic progenitor cells. There are intensive cell–cell and cell–matrix interactions through the adhesion molecule system which includes several distinct superfamilies: integrins, immunoglobulins, selectins and proteoglycan molecules.114 Integrins are heterodimeric membrane glycoproteins composed of α and β subunits, which not only mediate cell–cell interactions, but also serve as signal transducers influencing many aspects of cell behavior such as cell migration, proliferation, differentiation and apoptosis. Integrins are greatly influenced in their actions by signals from coexpressed hematopoietic growth factor receptors or other adhesion molecules. This system is very complicated due to the same type of receptors or ligands expressed on both hematopoietic stem cells and stromal cells. Disturbance in adhesive interactions between hematopoietic stem cells and their marrow microenvironment by mobilizing agents results in release of hematopoietic stem cells into the peripheral blood. Integrins VLA4 and LFA1 as well as L-selectin are believed to be involved in the mobilization pathway.105 Decreased expression of VLA4 on mobilized hematopoietic stem cells by chemotherapy and/or hematopoietic growth factors plays a major role in mobiliztion.115 Treatment with anti-VLA4 monoclonal antibodies as well as antibodies to VLA4-binding receptor VCAM1 (CD106, vascular cell adhesion molecule 1) can induce mobilization, which is independent of GCSF, IL3 and IL7 receptors.116 A simple de-adhesion step in the VLA4 system is not sufficient to elicit

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mobilization of hematopoietic stem cells into peripheral blood, since an antibody of the same isotype that binds VLA4 but does not block its function, has no impact on mobilization. The intact functional cellular signal transduction pathway is necessary for this pathway to function. The role of extracellular matrix and metalloproteinases The emigration of hematopoietic stem cells through an endothelial layer of blood–bone-marrow barrier is thought to occur in sinusoids near interendothelial cell junctions and in regions where the endothelial cell basement membrane extracellular matrix is thinned or absent. The marrow extracellular matrix is composed of collagens, glycoproteins, glycosaminoglycans and matrix metalloproteinases,114,117 including gelatinases, collagenases and stromelysins. Damage of this blood–bone-marrow barrier and destruction of more mature hematopoietic progenitor cells by chemotherapeutic agents are supposed to be involved in the process of hematopoietic stem-cell mobilization. IL8 is chemotactic for neutrophils and induces the release of metalloproteinases and stimulates transendothelial emigration. Metalloproteinases as a group of enzymes, such as gelatinases, collagenases and stromelysins, degrade marrow extracellular matrix during the induction of mobilization. The role of intracellular signal transduction pathways Anti-VLA4 antibody without functional blocking activity does not mobilize hematopoietic stem cells. The roles of hematopoietic growth factors and adhesion molecules in mobilization require interaction with their functional receptors. The Janus kinasesignal transducer and activator of transcription (JakStat) pathway is widely used by multiple hematopoietic growth factor and chemokine families.118 Mouse knockouts, especially Stat 4 and Stat5ab knockouts, are expected to generate definitive information to further explore mechanisms of hematopoietic stem-cell mobilization. Negative regulation of the Jak-Stat pathway by tyrosine phosphatases, such as SHP1, is important as well. Deregulation of the Jak-Stat pathway in hematopoietic malignancies may provide indirect information on the involvement of this pathway in mobilization. As expected, anti-VLA4 antibody can induce mobilization in S1/S1d mice (lack expression of membrane-bound kit ligand in the marrow microenvironment with normal kit receptor on hematopoietic cells), but not in W/Wv mice (lack expression of kit receptor on hematopoietic cells with intact marrow

microenvironment). However, anti-VCAM1 antibody cannot induce mobilization in both S1/S1d and W/Wv mice, because a functional c-kit receptor is required for either anti-VLA4 or antiVCAM1 antibody induced mobilization, which does not occur in S1/S1d mice with an abnormal marrow microenvironment response and in W/Wv mice with unresponsive hematopoietic stem cells. Further experiments strengthen this idea that a functional kit receptor and its signal transduction pathway are important in the mobilization process. The tyrosine phosphatase SHP1 is a downstream negative effector of the c-kit receptor signal transduction pathway. Lack of SHP1 expression in me/mev mice results in augmented and prolonged activation of kit receptor phosphorylation, which, in turn, can result in remarkable hematopoietic disorders, including autoimmunity, massive expansion of myeloid cells and splenomegaly. The unexplained splenomegaly may be due to the increased migratory capacity of hematopoietic stem cells in these me/mev mice. It is not clear that downmodulation of the c-kit receptors on mobilized peripheral blood progenitor cells found with different mobilizing agents plays a role to induce mobilization or just reflects a negative feedback of the c-kit receptor signal transduction pathway activated by these mobilizing agents through receptor-mediated endocytosis. This is reflective of the dilemma presented by most proposed mechanisms of mobilization, that is whether the observed changes represent a causative influence on mobilization or a consequence of the mobilization treatment. The role of hematopoietic stem-cell proliferation, differentiation and apoptosis A wide spectrum of hematopoietic progenitor cells can be mobilized from uncommitted stem cells to committed hematopoietic progenitor cells. The more efficient the mobilization, the more primitive hematopoietic progenitor cells are mobilized. Ex vivo hematopoietic stem-cell-expansion strategies from either marrow or peripheral blood in non- and poorlymobilizable patients are often unsuccessful, indicating that hematopoietic stem cells with intrinsic defects in proliferation resulting from prior chemotherapy and/or radiotherapy cannot be mobilized. Hematopoietic stem-cell proliferation before emigration is believed to occur and plays an important role in this process since most of the mobilized CD34+ cells in peripheral blood are not in active cell cycle.119,120 Administration of G-CSF causes increased angiogenesis and marrow blood flow and stimulates a significant increase in the colonogenic capacity of all the progenitors evaluated.121 Profound proliferation within marrow can cause reorganization of existing

Mobilization of hematopoietic stem cells

The initiation-amplification-emigration-adaptation model of mobilization It is of great interest to establish the relationship among different mechanisms involved in hematopoietic stem cell mobilization and to define the role and sequence of different factors in a common pathway to induce mobilization. The role of cyclophosphamide locates upstream to the G-CSF system, while the cellular protein tyrosine kinase pathway locates downstream to the VLA4 system. The relationship between the VLA4 system and the G-CSF system in mobilization is not clear at present, but can be defined if the expression of VLA4 can be knocked out. Based on the previous observations of in-vivo and in-vitro studies, and synthesis of several postulates, the initiation-amplificationemigration-adaptation model as shown in Figure 1 may represent a common pathway involved in the process of hematopoietic stem cell mobilization. In the initiation phase, mobilizing agents act on their primary targets to initiate a network of reactions to induce mobilization. Thus, an indirect effect of mobilizing agents on hematopoietic stem cells is more important than its direct effect in this initiation phase. In the

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pools of hematopoietic progenitor cells and remodel marrow blood flow and anatomic structure. Increased proliferation and overcrowding, per se, cannot lead to emigration automatically. In flt3 ligand treated mice, proliferation within bone marrow peaks early at day 3, but peak emigration is present much later. In cyclophosphamide treated G-CSF receptor knockout mice, no mobilization response is noted, but an extensive proliferation within marrow is present. An increase of CD34+/CD90+CD13+ cells and a decrease of CD34+/CD90+/CD13- and CD34+/CD90-/CD13- cells is found during G-CSF administration, indicating a progressive differentiation toward committed cells from more primitive uncommitted cells. But the absolute number of mobilized primitive uncommitted cells is also significantly increased. Therefore, the optimal mobilizing regimens prevent further differentiation and maximize hematopoietic stem-cell collection. Although there are few studies on the anti-apoptosis of hematopoietic stem cells involved in mobilization, it is apparent that this mechanism plays a important role to keep the mobilized peripheral blood progenitor cells alive in a new environment.122,123 Compared with peripheral blood progenitor cells in steady state, G-CSF mobilized peripheral blood progenitor cells are significantly less apoptotic.124 Chemotherapeutic agents, hematopoietic growth factors, chemokines, adhesion molecules and extracellular matrix associated with mobilization are all involved in the process of apoptosis.

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amplification phase, cross-talk among hematopoietic stem cells, marrow microenvironment, and systemic status via hematopoietic growth factors, chemokines, intensive cell–cell and cell–matrix interactions, and their intercellular signal transduction pathways are involved. This may result in proliferation and phenotypic changes in hematopoietic stem cells for the next emigration step. In the emigration phase, the changed mobilizable hematopoietic stem cells lose their responses to stromal chemoattractants, demonstrate decreased adhesive affinity to the marrow microenvironment and, therefore, exit into blood through an endothelial layer. Obviously, damage to this layer may facilitate, but not necessarily result in shift of hematopoietic stem cells into peripheral blood if there are no coincident changes of hematopoietic stem cells and marrow microenvironment. In the adaptation phase, the modification of the mobilized cellular context and surface expression profile provide them a better chance to survive in the peripheral blood. Prevention of apoptosis by alternation of their proliferative status to G0/G1 phase, and inhibition of hematopoietic stem-cell homing by downregulating

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surface antigen expression, may be very important in this phase.125 Although this mobilization model lacks solid support from systemic experimental data, it may help us better understand mechanisms of mobilization at the early stage and develop more sophisticated studies to shed more light on the pathways responsible for hematopoietic stem-cell mobilization. SIDE EFFECTS DURING MOBILIZATION Complications of hematopoietic stem-cell mobilization include those due to the procedures and those due to administration of chemotherapeutic agents and/or hematopoietic growth factors.126,127 The side effects of procedures related to central venous catheter placement and leukapheresis include line-related infection and leukapheresis-induced thrombocytopenia and coagulation abnormality with anticoagulants during collection.128 Tumor cells or abnormal hematopoietic stem cells from bone marrow can be co-mobilized to contaminate mobilized peripheral blood progenitor cells, which may require further purging.129,131 The side effects caused by chemotherapeutic agent administration depend on the dosage and the type of drug used, which may result in a disturbed immune system, infection, secondary malignancy, pulmonary, cardiovascular, hematologic, GI, endocrine, fertility, ophthalmologic, CNS and psychosocial consequences.132 The side effects caused by hematopoietic growth factor administration

include bone pain, headache, fatigue, arthralgia, myalgia, low-grade fever, nausea, cough, dyspnea, local skin rash, reduced platelet aggregation, hypercoagulable status by increasing levels of FVIII:C and thrombin generation, and abnormal serum chemistry with elevated levels of C-reactive protein, LDH, alkaline phosphatase, sodium and uric acid, and slightly decreased levels of albumin, total bilirubin, BUN and potassium.133 In general, the side effects caused by the above mobilizing agents are well tolerated and rarely fatal. These side effects can sometimes be lessened by coadministration of NSAIDs, narcotics, antihistamines and/or steroids. A large number of neutrophils mobilized with G-CSF appear to be functional without significant toxicity for at least 48 h following infusion of reconstituted cryopreserved preserve products, which provides a possible means to fight against infection, reduce or eliminate post-transplant neutropenia and help rapid platelet recovery because G-CSF mobilized neutrophil products are highly contaminated with platelets. CONCLUSIONS Since the 1980s, the peripheral blood progenitor cell transplantation has almost supplanted conventional bone-marrow transplantation because of more rapid hematopoietic recovery, less morbidity, decreased medical support and lower total costs. If a dose of 5

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×106 CD34+ cells/kg is transplanted, prompt and sustained hematopoietic recovery including neutrophils and platelets will occur in most of the transplanted patients. Although the studies on mobilization mechanisms remain at the early stage, practical mobilization regimens with chemotherapeutic agents and/or hematopoietic growth factors have been developed for current patient care, which are generally safe, well tolerated and effective.1,134 One emphasis of clinical studies has been to identify optimal mobilization regimens that can maximize hematopoietic stem-cell collection with the least toxicity. A better understanding of the mechanisms that regulate hematopoietic stem-cell mobilization may lead to the design of novel and optimal mobilization strategies addressing simplicity, efficacy and cost.135

REFERENCES 1. Anderlini P and Korbling M. The use of mobilized peripheral blood stem cells from normal donors for allografting. Stem Cells 1997; 15(1): 9–17. 2. Demirer T, Bensinger WI, Buckner CD. Peripheral blood stem cell mobilization for high-dose chemotherapy. J Hematotherapy 1999; 8(2): 103–113. 3. DiPersio JF, Khoury H, Haug J et al. Innovations in allogeneic stem-cell transplantation. Semi Hematol 2000; 37 (1 suppl 2): 33–41. 4. Lefrere F, Hermine O, Audat F et al. The feasibility of peripheral blood stem cell collection for autograft following failure in bone marrow aspiration. Hematol Cell Therapy 1998; 40(3): 133–137. 5. Visani G, Lemoli R, Tosi P et al. Use of peripheral blood stem cells for autologous transplantation in acute myeloid leukemia patients allows faster engraftment and equivalent disease-free survival compared with bone marrow cells. Bone Marrow Transplant 1999; 24(5): 467–472. 6. Champlin RE, Schmitz N, Horowitz MM et al. Blood stem cells compared with bone marrow as a source of hematopoietic cells for allogeneic transplantation. Blood 2000; 95(12): 3702–3709. 7. Pratt G, Johnson RJ, Rawstron AC et al. Autologous stem cell transplantation in chronic myeloid leukaemia using Philadelphia chromosome negative blood progenitors mobilised with hydroxyurea and G-CSF. Bone Marrow Transplant 1998; 21(5): 455–460. 8. Cooper DD and Spangrude GJ. (R)evolutionary considerations in hematopoietic development. Ann New York Acad Sci 1999; 872: 83–93. 9. Spangrude GJ, Cooper DD. Paradigm shifts in stem cell biology. Sem Hematol 2000; 37(1 suppl) 2: 3–10. 10. Atwater S, Corash L. Advances in leukocyte differential and peripheral blood stem cell enumeration. Curr Opin Hematol 1996; 3: 71–76. 11. Bradford GB, Williams B, Rossi R et al. Quiescence, cycling and turnover in the primitive hematopoietic stem cell compartment. Exp Hematol 1997; 25: 445–453. 12. Vantelon JM, Koscielny S, Brault P et al. Scoring system for the prediction of successful peripheral blood stem cell (PBSC) collection in non-Hodgkin’s lymphoma (NHL): application in clinical practice. Bone Marrow Transplant 2000; 25(5): 495–499. 13. Imbert AM, Bagnis C, Galindo R et al. A neutralizing antiTGF-betal antibody promotes proliferation of CD34+Thy-1+ peripheral blood progenitors and increases the number of transduced progenitors. Exp Hematol 1998; 26(5): 374–381.

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14. Millar BC, Millar JL, Shepherd V et al. The importance of CD34+/CD33- cells in platelet engraftment after intensive therapy for cancer patients given peripheral blood stem cell rescue. Bone Marrow Transplant 1998; 22(5): 469–475. 15. Humeau L, Bardin F, Maroc C et al. Phenotypic molecular, and functional characterization of human peripheral blood CD+/Thy1+ cells. Blood 1996; 87: 949–955. 16. Ziegler BL, Valtieri M, Porada GA et al. KDR receptor: a key marker defining hematopoietic stem cells. Science 1999; 285: 1553–1558. 17. Goodell MA, Rosenzweig M, Kim H et al. Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species. Nat Med 1997; 3: 1337–1345. 18. Lemoli RM, Tafuri A, Fortuna A et al. Cycling status of CD34+ cells mobilized into peripheral blood of healthy donors by recombinant human granulocyte colony-stimulating factor. Blood 1997; 89(4): 1189–1196. 19. Bertolini F, de Vincentiis A, Lanata L et al. Allogeneic hematopoietic stem cells from sources other than bone marrow: biological and technical aspects. Haematologica 1997; 82(2): 220–238. 20. Russell NH, McQuaker G, Stainer C et al. Stem cell mobilisation in lymphoproliferative diseases. Bone Marrow Transplant 1998; 22(10): 935–940. 21. Anderlini P, Przepiorka D, Korbling M et al. Blood stem cell procurement: donor safety issues. Bone Marrow Transplant 1998; 21(Suppl 3): S35–S39. 22. Schulman KA, Birch R, Zhen B et al. Effect of CD34(+) cell dose on resource utilization in patients after high-dose chemotherapy with peripheral-blood stem-cell support. J Clin Onc 1999; 17(4): 1227. 23. Krieger MS, Schiller G, Berenson JR et al. Collection of peripheral blood progenitor cells (PBPC) based on a rising WBC and platelet count significantly increases the number of CD34+ cells. Bone Marrow Transplant 1999; 24(1): 25–28. 24. Perez-Simon JA, Martin A, Caballero D et al. Clinical significance of CD34+ cell dose in long-term engraftment following autologous peripheral blood stem cell transplantation. Bone Marrow Transplant 1999; 24: 1279–1283. 25. Scheid C, Draube A, Reiser M et al. Using at least 5×10(6)/kg CD34+ cells for autologous stem cell transplantation significantly reduces febrile complications and use of antibiotics after transplantation. Bone Marrow Transplant 1999; 23(11): 1177–1181. 26. Gandhi MK, Jestice K, Scott MA et al. RE The minimum CD34 threshold depends on prior chemotherapy in autologous peripheral blood stem cell recipients. Bone Marrow Transplant 1999; 23(1): 9–13. 27. Gowans ID, Hepburn MD, Clark DM et al. The role of the Sysmex SE9000 immature myeloid index and Sysmex R2000 reticulocyte parameters in optimizing the timing of peripheral blood stem cell harvesting in patients with lymphoma and myeloma. Clin Lab Haematol 1999; 21(5): 331–336. 28. Hiruma K, Nakayama S and Okuyama Y. A comparative study of a new, fully automated procedure and the standard mononuclear cell program using the Cobe Spectra for peripheral blood stem cell collection. Therapeutic Apheresis 1998; 2(4): 273–276. 29. Rowley SD, Loken M, Radich J et al. Isolation of CD34+ cells from blood stem cell components using the Baxter Isolex system. Bone Marrow Transplant 1998 21(12): 1253–1262. 30. Tichelli A, Passweg J, Hoffmann T et al. Repeated peripheral stem cell mobilization in healthy donors: time- dependent changes in mobilization efficiency. Bri J Haematol 1999; 106(1): 152–158. 31. Kiel K, Cremer FW, Ehrbrecht E et al. First and second apheresis in patients with multiple myeloma: no differences in tumor load and hematopoietic stem cell yield. Bone Marrow Transplant 1998; 21(11): 1109–1115. 32. Smolowicz AG, Villman K, Berlin G et al. Kinetics of peripheral blood stem cell harvests during a single apheresis. Transfusion 1999; 39(4): 403–409.

216

Blood Reviews

33. Glaspy JA. Economic considerations in the use of peripheral blood progenitor cells to support high-dose chemotherapy. Bone Marrow Transplant 1999; 23(suppl 2): S21–S27. 34. Lemoli RM, Fortuna A, Motta MR et al. Concomitant mobilization of plasma cells and hematopoietic progenitors into progenitors into peripheral blood of multiple myeloma patients: positive selection and transplantation of enriched CD34+ cells to remove circulating tumor cells. Blood 1996; 87: 1625–1634. 35. Benet I, Prosper BF, Marugan I et al. Mobilization of peripheral blood progenitor cells (PBPC) in patients undergoing chemotherapy followed by autologous peripheral blood stem cell transplant (SCT) for high risk breast cancer (HRBC). Bone Marrow Transplant 1999; 23(11): 1101–1107. 36. Ojeifo JO, Wu AG, Miao Y et al. Docetaxel-induced mobilization of hematopoietic stem cells in a murine model: kinetics, dose titration, and toxicity. Exp Hematol 2000; 28(4): 451–459. 37. Klein JL, Rey PM, Dansey R et al: Cyclophosphamide and paclitaxel as initial or salvage regimen for the mobilization of peripheral blood progenitor cells. Bone Marrow Transplant 1999; 24: 959–963. 38. Shea TC: Mobilization of peripheral blood progenitor cells with paclitaxel-based chemotherapy. Semi Onc 1997; 24(1 suppl 2): S2–105–7. 39. Verma UN, van den Blink B, Pillai R et al. Paclitaxel vs cyclophosphamide in peripheral blood stem cell mobilization: comparative studies in a murine model. Exp Hematol 1999; 27(3): 553–560. 40. Lefrere F, Makke J, Fermand J et al. Blood stem cell collection using chemotherapy with or without systematic G-CSF: experience in 52 patients with multiple myeloma. Bone Marrow Transplant 1999; 24(5): 463–466. 41. Shpall EJ. The utilization of cytokines in stem cell mobilization strategies. Bone Marrow Transplant 1999; 23(suppl 2): S13–S19. 42. Mielcarek M, Torok-Storb B. Phenotype and engraftment potential of cytokine-mobilized peripheral blood mononuclear cells. Curr Opin Hematol 1997; 4(3): 176–182. 43. Tabbara IA, Ghazal CD, Ghazal HH. The role of granulocyte colony-stimulating factor in hematopoietic stem cell transplantation. Cancer Investigation 1997; 15(4): 353–357. 44. Kawano Y, Watanabe T, Takaue Y. Mobilization/harvest and transplantation with blood stem cells, manipulated or unmanipulated. Ped Transplant 1999; 3(suppl 1): 65–71. 45. Li K, Wong A, Li CK et al. Granulocyte colony- stimulating factor-mobilized peripheral blood stem cells in betathalassemia patients: kinetics of mobilization and composition of apheresis product. Exp Hematol 1999; 27(3): 526–532. 46. Kroger N, Zeller W, Hassan HT et al. Stem cell mobilization with G-CSF alone in breast cancer patients: higher progenitor cell yield by delivering divided doses (2 ×5 microg/kg) compared to a single dose (1 ×10 microg/kg). Bone Marrow Transplant 1999; 23(2): 125–129. 47. Lefrere F, Bernard M, Audat F et al. Comparison of lenograstim vs filgrastim administration following chemotherapy for peripheral blood stem cell (PBSC) collection: a retrospective study of 126 patients. Leuk Lymph 1999; 35(5–6): 501–550. 48. Carlo-Stella C, Cesana C, Regazzi E et al. Peripheral blood progenitor cell mobilization in healthy donors receiving recombinant human granulocyte colony-stimulating factor. Exp Hematol 2000; 28(2): 216–224. 48. Gomez-Espuch J, Moraledz JM, Ortuno F et al. Mobilization of hematopoietic progenitor cells with paclitaxel (taxol) as a single chemotherapeutic agent, associated with rhG-CSF. Bone Marrow Transplant 2000, 25: 231–235. 49. Chang Q, Harvey K, Akard L et al. Comparison of the distribution of progenitor cells in G-CSF-mobilized peripheral blood and steady-state bone marrow after counterflow centrifugal elutriation. Biol Blood Marrow Transplant 1999; 5(5): 328–335.

50. De la Rubia J, Martinez C, Solano C et al. Administration of recombinant human granulocyte colony- stimulating factor to normal donors: results of the Spanish National Donor Registry. Bone Marrow Transplant 1999; 24: 723–728. 51. Facon T, Harousseau JL, Maloisel F et al. Stem cell factor in combination with filgrastim after chemotherapy improves peripheral blood progenitor cell yield and reduces apheresis requirements in multiple myeloma patients: a randomized, controlled trial. Blood 1999; 94(4): 1218–1225. 52. Basser RL, To LB, Begley CG et al. Rapid hematopoietic recovery after multicycle high-dose chemotherapy: enhancement of filgrastim-induced progenitor-cell mobilization by recombinant human stem-cell factor. J Clin Onc 1998; 16(5): 1899–1908. 53. Bearman SI. Use of stem cell factor to mobilize hematopoietic progenitors. Curr Opin Hematol 1997; 4(3): 157–162. 54. Weaver A, Testa NG. Stem cell factor leads to reduced blood processing during apheresis or the use of whole blood aliquots to support dose-intensive chemotherapy. Bone Marrow Transplant 1998; 22(1): 33–38. 55. Kolbe K, Peschel C, Rupilius B et al. HG Peripheral blood stem cell (PBSC) mobilization with chemotherapy followed by sequential IL-3 and G-CSF administration in extensively pretreated patients. Bone Marrow Transplant 1997; 20(12): 1027–1032. 56. Kroger N, Zeller W, Fehse N et al. Mobilizing peripheral blood stem cells with high-dose G-CSF alone is as effective as with Dexa-BEAM plus G-CSF in lymphoma patients. Brit J Haematol 1998; 102(4): 1101–1106. 57. Avigan D, Wu Z, Gong J et al. Selective in vivo mobilization with granulocyte macrophage colony-stimulating factor (GMCSF)/granulocyte-CSF as compared to G-CSF alone of dendritic cell progenitors from peripheral blood progenitor cells in patients with advanced breast cancer undergoing autologous transplantation. Clin Cancer Res 1999; 5(10): 2735–2741. 58. Fischmeister G, Kurz M, Haas OA et al. G-CSF versus GMCSF for stimulation of peripheral blood progenitor cells (PBPC) and leukocytes in healthy volunteers: comparison of efficacy and tolerability. Ann Hematol 1999; 78(3): 117–123. 59. Spitzer G, Adkins D, Mathews M et al. Randomized comparison of G-CSF + GM-CSF vs G-CSF alone for mobilization of peripehral blood stem cells: effects on hematopoietic recovery after high-dose chemotherapy. Bone Marrow Transplant 1997; 20(11): 921–930. 60. Weaver A, Chang J, Wrigley E et al. Randomized comparison of progenitor-cell mobilization using chemotherapy, stem-cell factor, and filgrastim or chemotherapy plus filgrastim alone in patients with ovarian cancer. J Clin Onc 1998; 16(8): 2601–2612. 61. Demirer T, Buckner CD, Storer B et al. Effect of different chemotherapy regimens on peripheral-blood stem-cell collections in patients with breast cancer receiving granulocyte colony-stimulating factor. J Clin Onc 1997; 15(2): 684–690. 62. Demirer T, Buckner CD and Bensinger WI: Optimization of peripheral blood stem cell mobilization. Stem Cells 1996; 14: 106–116. 63. Watanabe T, Kawano Y, Kanamaru S et al. Endogenous interleukin-8 (IL-8) surge in granulocyte colony-stimulating factor-induced peripheral blood stem cell mobilization. Blood 1999; 93(4): 1157–1163. 64. Tricot G, Gazitt Y, Leemhuis T et al. Collection, tumor contamination, and engraftment kinetics of highly purified hematopoietic progenitor cells to support high dose therapy in multiple myeloma. Blood 1998; 91: 4489–4495. 65. van der Loo JC, Hanenberg H, Cooper RJ et al. Nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse as a model system to study the engraftment and mobilization of human peripheral blood stem cells. Blood 1998; 92(7): 2556–2570. 66. Breban M, Dougados M, Picard F et al. Intensified-dose (4 gm/m2) cyclophosphamide and granulocyte colonystimulating factor administration for hematopoietic stem cell mobilization in refractory rheumatoid arthritis. Arthritis & Rheumatism 1999; 42(11): 2275–2280.

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67. Burtness BA, Psyrri A, Rose M et al. A phase I study of paclitaxel for mobilization of peripheral blood progenitor cells. Bone Marrow Transplant 1999; 23(4): 311–315. 69. Takahashi M, Yoshizawa H, Tanaka H et al. A phase I dose escalation study of multicyclic, dose-intensive chemotherapy with peripheral blood stem cell support for small cell lung cancer. Bone Marrow Transplant 2000; 25: 5–11. 70. Sureda A, Petit J, Brunet et al. Mini-ICE regimen as mobilization therapy for chronic myelogenous leukemia patients at diagnosis. Bone Marrow Transplant 1999; 24: 1285–1290. 71. McQuaker I, Haynes A, Stainer C et al. Mobilisation of peripheral blood stem cells with IVE and G-CSF improves CD34+ cell yields and engraftment in patients with non-Hodgkin’s lymphomas and Hodgkin’s disease. Bone Marrow Transplant 1999; 24(7): 715–722. 72. McQuaker IG, Haynes AP, Stainer C et al. Stem cell mobilization in resistant or relapsed lymphoma: superior yield of progenitor cells following a salvage regimen comprising ifosphamide, etoposide and epirubicin compared to intermediate-dose cyclophosphamide. Bri J Haemat 1997; 98(1): 228–233. 73. Bilgrami S, Feingold JM, Bona RD et al. Dose-intense paclitaxel, etoposide and cyclophosphamide: a safe and active regimen for tumor cytoreduction and stem cell mobilization in metastatic breast cancer. Bone Marrow Transplant 2000; 25(2): 123–130. 74. Moskowitz CH, Bertino JR, Glassman JR et al. Ifosfamide, carboplatin, and etoposide: a highly effective cytoreduction and peripheral-blood progenitor-cell mobilization regimen for transplant-eligible patients with non-Hodgkin’s lymphoma. J Clin Onc 1999; 17(12): 3776–3785. 75. Rauck AM, Ruymann FB, Klopfenstein K et al. Mobilization of peripheral blood stem cells in children using G-CSF after carboplatin containing myelosuppressive chemotherapy. J Clin Apheresis 1998; 13(4): 146–154. 76. Stewart DA, Guo D, Morris D et al. Superior autologous blood stem cell mobilization from dose-intensive cyclophosphamide, etoposide, cisplatin plus G-CSF than from less intensive chemotherapy regimens. Bone Marrow Transplant 1999; 23(2): 111–117. 77. Lowenthal RM, Faberes C, Marit G et al. Factors influencing haemopoietic recovery following chemotherapy-mobilised autologous peripheral blood progenitor cell transplantation for haematological malignancies: a retrospective analysis of a 10-year single institution experience. Bone Marrow Transplant 1998; 22(8): 763–707. 78. Laszlo D, Galieni P, Raspadori D et al. Fludarabine containing-regimens may adversely affect peripheral blood stem cell collection in low-grade non Hodgkin lymphoma patients. Leuk Lymph 2000; 37(1–2): 157–161. 79. Buckstein R, Imrie K, Spaner D et al. Stem cell function and engraftment is not affected by “in vivo purging” with rituximab for autologous stem cell treatment for patients with low-grade non-Hodgkin’s lymphoma. Semi Onc 1999; 26(5 suppl 14): 115–122. 80. Burns LJ, Weisdorf DJ, DeFor TE et al. Enhancement of the anti-tumor activity of a peripheral blood progenitor cell graft by mobilization with interleukin 2 plus granulocyte colonystimulating factor in patients with advanced breast cancer. Exp Hematol 2000; 28(1): 96–103. 81. Desikan KR, Barlogie B, Jagannath S et al. Comparable engraftment kinetics following peripheral-blood stem-cell infusion mobilized with granulocyte colony-stimulating factor with or without cyclophosphamide in multiple myeloma. J Clin Onc 1998; 16(4): 1547–1553. 82. Stiff PJ. Management strategies for the hard-to-mobilize patient. Bone Marrow Transplant 1999; 23 (suppl 2): S29–S33. 83. Brown RA, Adkins D, Goodnough LT et al. Factors that influence the collection and engraftment of allogeneic peripheral-blood stem cells in patients with hematologic malignancies. J Clin Onc 1997; 15(9): 3067–3074.

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84. Watts MJ, Sullivan AM, Leverett D et al. Back-up bone marrow is frequently ineffective in patients with poor peripheral-blood stem-cell mobilization. J Clin Onc 1998; 16(4): 1554–1560. 85. Moskowitz CH, Glassman JR, Wuest D et al. Factors affecting mobilization of peripheral blood progenitor cells in patients with lymphoma. Clin Cancer Res 1998; 4(2): 311–316. 86. Watts MJ, Sullivan AM, Jamieson E et al. Progenitor-cell mobilization after low-dose cyclophosphamide and granulocyte colony-stimulating factor: an analysis of progenitor-cell quantity and quality and factors predicting for these parameters in 101 pretreated patients with malignant lymphoma. J Clin Onc 1997; 15(2): 535–546. 87. Fischer T, Neubauer A, Mohm J et al. Outcome of peripheral blood stem cell mobilization in advanced phases of CML is dependent on the type of chemotherapy applied. Annals of Hematology 1998 77(1–2): 21–26. 88. Knudsen LM, Rasmussen T, Jensen L et al. Reduced bone marrow stem cell pool and progenitor mobilisation in multiple myeloma after melphalan treatment. Medical Onc 1999; 16(4): 245–254. 89. Perry AR, Watts MJ, Peniket AJ et al. Progenitor cell yields are frequently poor in patients with histologically indolent lymphomas espeeially when mobilized within 6 months of previous chemotherapy. Bone Marrow Transplant 1998; 21(12): 1201–1205. 90. Marshall E, Woolford LB, Lord BI. Continuous infusion of macrophage inflammatory protein MIP-1alpha enhances leucocyte recovery and haemopoietic progenitor cell mobilization after cyclophosphamide. Bri J Cancer 1997; 75(12): 1715–1720. 91. Martinez C, Sureda A, Martino R et al. Efficient peripheral blood stem cell mobilization with low-dose G-CSF (50 microg/m2) after salvage chemotherapy for lymphoma. Bone Marrow Transplant 1997; 20(10): 855–851. 92. Lie AK, Hui CH, Rawling T et al. Granulocyte colonystimulating factor (G-CSF) dose-dependent efficacy in peripheral blood stem cell mobilization in patients who had failed initial mobilization with chemotherapy and G-CSF. Bone Marrow Transplant 1998; 22(9): 853–857. 93. Emmons RV, Andre M et al. The administration of 10 microg/kg granulocyte colony-stimulating factor (G-CSF) alone results in a successful peripheral blood stem cell collection when previous mobilization with chemotherapy and hematopoietic growth factor failed. Leuk Lymph 1999; 34(1–2): 105–109. 94. Reiser M, Josting A, Draube A et al. A Successful peripheral blood stem cell mobilization with etoposide (VP-16) in patients with relapsed or resistant lymphoma who failed cyclophosphamide mobilization. Bone Marrow Transplant 1999; 23(12): 1223–1228. 95. Glaspy JA, Shpall EJ, LeMaistre CF et al. Peripheral blood progenitor cell mobilization using stem cell factor in combination with filgrastim in breast cancer patients. Blood 1997; 90(8): 2939–2951. 96. Moskowitz CH, Stiff P, Gordon MS et al. Recombinant methionyl human stem cell factor and filgrastim for peripheral blood progenitor cell mobilization and transplantation in nonHodgkin’s lymphoma patients—results of a phase I/II trial. Blood 1997; 89(9): 3136–4750. 97. EJ, Wheeler CA, Turner SA et al. A randomized phase 3 study of peripheral blood progenitor cell mobilization with stem cell factor and filgrastim in high-risk breast cancer patients. Blood 1999; 93(8): 2491–2501. 98. Molineux G, McCrea C, Yan XQ et al. Flt-3 ligand synergizes with granulocyte colony-stimulating factor to increase neutrophil numbers and to mobilize peripheral blood stem cells with long-term repopulating potential. Blood 1997; 89(11): 3998–4004. 99. Pless M, Wodnar-Filipowicz A, John L et al. Synergy of growth factors during mobilization of peripheral blood precursor cells with recombinant human Flt3-ligand and granulocyte colonystimulating factor in rabbits. Exp Hematol 1999; 27(1): 155–161.

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100. Honkoop AH, van der Wall E, Feller N et al. Multiple cycles of high-dose doxorubicin and cyclophosphamide with GCSF mobilized peripheral blood progenitor cell support in patients with metastatic breast cancer. Ann Onc 1997; 8(10): 957–962. 101. To LB, Haylock DN, Simmons PJ et al. The biology and clinical uses of blood stem cells. Blood 1997; 89: 2233–2258. 102. Kroger N, Zeller W, Hassan HT et al. Difference between expression of adhesion molecules on CD34+ cells from bone marrow and G-CSF-stimulated peripheral blood. Stem Cells 1998; 16(1): 49–53. 103. Perey L, Peters R, Pampallona S et al. Extensive phenotypic analysis of CD34 subsets in successive collections of mobilized peripheral blood progenitors. Bri J Haematol 1998; 103(3): 618–629. 104. Pecora AL. Impact of stem cell on hematopoietic recovery in autologous blood stem cell recipients. Bone Marrow Transplant 1999; 23 (suppl 2): S7–S12. 105. Bellucci R, De Propris MS, Buccisano F et al. Modulation of VLA4 and L-selectin expression on normal CD34+ cells during mobilization with G-CSF. Bone Marrow Transplant 1999; 23: 1–8. 106. Roberts MM, Swart BW, Simmons PJ et al. Prolonged release and c-kit expression of haemopoietic precursor cells mobilized by stem cell factor and granulocyte colony stimulating factor. Bri J Haematol 1999; 104(4): 778–784. 107. Watanabe T, Dave B, Heimann DG et al. GM-CSFmobilized peripheral blood CD34+ cells differ from steadystate bone marrow CD34+ cells in adhesion molecule expression. Bone Marrow Transplant 1997; 19(12): 1175–1181. 108. Watts MJ, Ings SJ, Leverett D et al. ESHAP and G-CSF is a superior blood stem cell mobilizing regimen compared to cyclophosphamide 1.5 g m(-2) and G-CSF for pre-treated lymphoma patients: a matched pairs analysis of 78 patients. Bri J Cancer 2000; 82(2): 278–282. 109. Link DC. Mechanisms of granulocyte colony stimulating factor-induced hematopoietic progenitor cell mobilization. Semi Hematol 2000; 37(1 suppl 2): 25–32. 110. Rumi C, Rutella S, Teofili L et al. RhG-CSF-mobilized CD34+ peripheral blood progenitors are myeloperoxidase negative and noncycling irrespective of CD33 or CD13 coexpression. Exp Hematol 1997; 25(3): 246–251. 111. Lataillade JJ, Clay D, Dupuy C et al. Chemokine SDF-1 enhances circulating CD34(+) cell proliferation in synergy with cytokines: possible role in progenitor survival. Blood 2000; 95(3): 756–768. 112. Liu F, Poursine-Laurent J, Link DC. The granulocyte colonystimulating factor receptor is required for the mobilization of murine hematopoietic progenitors into peripheral blood by cyclophosphamide or interleukin 8 but not flt-3 ligand. Blood 1997; 90: 2522–2528. 113. Pruijt JF, Fibbe WE, Laterveer L et al. Prevention of interleukin 8-induced mobilization of hematopoietic progenitor cells in rhesus monkeys by antibodies to the metalloproteinase gelatinase B. Proc Natl Acad Sci 1999; 96: 10863–10868. 114. Fibbe WE, Pruijt JF, van Kooyk Y et al. The role of metalloproteinases and adhesion molecules in interleukin 8-induced stem cell mobilization. Semi Hematol 2000; 37(1 suppl 2): 19–24. 115. Papayannopoulou T. Mechanisms of stem/progenitor cell mobilization: the anti-VLA4 paradigm. Semi Hematol 2000; 37(1 suppl 2): 11–18. 116. Papayannopoulou T, Priestley GV, Nakamoto B. AntiVLA4/VCAMI induced mobilization requires cooperative signaling through the kit receptor/mkit ligand. Blood 1998; 91: 2231–2239. 117. Janowska-Wieczorek A, Marquez LA, Nabholtz JM et al. Growth factors and cytokines upregulate gelatinase expression in bone marrow CD34(+) cells and their transmigration through reconstituted basement membrane. Blood 1999; 93(10): 3379–3390.

118. Ward AC, Touw I, Yoshimura A. The Jak-Stat pathway in normal and perturbed hematopoiesis. Blood 2000; 95: 19–29. 119. Morrison SJ, Wright DE, Weissman IL. Cytophosphamide/granulocyte colony stimulating factor induces hematopoietic stem cells to proliferate prior to mobilization. Proc Natl Acad Sci 1997; 94: 1908–1913. 120. Uchida N, He D, Friera AM et al. The unexpected G0/G1 cell cycle status of mobilized hematopoietic stem cells from peripheral blood. Blood 1997; 89: 465–472. 121. Perez-Oteyza J, Ramos P, Testa NG et al. High-dose granulocyte-colony stimulating factor (G-CSF) in vitro induces the growth of high proliferative potential colony forming cells (HPP-CFC) in patients undergoing blood stem cell mobilization. Exp Hematol 1997; 25(6): 516–520. 122. Anthony RS, McKelvie ND, Cunningham AJ et al. Flow cytometry using annexin V can detect early apoptosis in peripheral blood stem cell harvests from patients with leukaemia and lymphoma. Bone Marrow Transplant 1998; 21(5): 441–446. 123. Staknke K, Hecker S, Kohne E et al. CD95 (APO1/FAS)mediated apoptosis in cytokine-activated hematopoietic cells. Exp Hematol 1998; 26: 844–850. 124. Philpott NJ, Prue RL, Marsh JC et al. G-CSF-mobilized CD34 peripheral blood stem cells are significantly less apoptotic than unstimulated peripheral blood CD34 cells: role of G-CSF as survival factor. Bri J Haematol 1997; 97(1): 146–152. 125. Whetton AD, Graham GJ. Homing and mobilization in the stem cell niche. Trend Cell Biol 1999; 9(6): 233–238. 126. Dicke KA, Hood DL, Arneson M et al. Effects of short-term in vivo administration of G-CSF on bone marrow prior to harvesting. Exp Hematol 1997; 25: 34–38. 127. Falzetti F, Aversa F, Minelli O et al. Spontaneous rupture of spleen during peripheral blood stem-cell mobilisation in a healthy donor. Lancet 1999; 353(9152): 555. 128. Murata M, Harada M, Kato S et al. Peripheral blood stem cell mobilization and apheresis: analysis of adverse events in 94 normal donors. Bone Marrow Transplant 1999; 24: 1065–1071. 129. Franklin WA, Glaspy J, Pflaumer SM et al. Incidence of tumor-cell contamination in leukapheresis products of breast cancer patients mobilized with stem cell factor and granulocyte colony-stimulating factor (G-CSF) or with GCSF alone. Blood 1999; 94(1): 340–347. 130. Irving JA, Lennard A, Storey N et al. Analysis of CD34 populations in mobilised peripheral blood stem cell harvests and in bone marrow by fluorescent in situ hybridisation for the bcr/abl gene fusion in patients with chronic granulocytic leukaemia. Leukemia 1999; 13(6): 944–949. 131. Johnson RJ, Rawstron AC, Richards S et al. Circulating primitive stem cells in paroxysmal nocturnal hemoglobinuria (PNH) are predominantly normal in phenotype but granulocyte colony-stimulating factor treatment mobilizes mainly PNH stem cells. Bood 1998; 91: 4504–4508. 132. Krishnan A, Bhatia S, Slovak ML et al. Predictors of therapy-related leukemia and myelodysplasia following autologous transplantation for lymphoma: an assessment of risk factors. Blood 2000; 95(5): 1588–1593. 133. Keung YK, Suwanvecho, S Cobos E. Anaphylactoid reaction to granulocyte colony-stimulating factor used in mobilization of peripheral blood stem cell. Bone Marrow Transplant 1999; 23(2): 200–201. 134. Pan L, Teshima T, Hill GR et al. Granulocyte colonystimulating factor-mobilized allogeneic stem cell transplantation maintains graft-versus-leukemia effects through a perforin-dependent pathway while preventing graft-versus-host disease. Blood 1999; 93(12): 4071–4078. 135. Akard LP, Thompson JM, Dugan MJ et al. Matched-pair analysis of hematopoietic progenitor cell mobilization using G-CSF vs. cyclophosphamide, etoposide, and G-CSF: enhanced CD34+ cell collections are not necessarily cost-effective. Biol Blood Marrow Transplant 1999; 5(6): 379–385.