Stem Cell Mobilization
Michele H. Cottler-Fox, Tsvee Lapidot, Isabelle Petit, Orit Kollet, John F. DiPersio, Successful blood and marrow transplant (BMT),
stem cell interactions. On the basis of this under-
both autologous and allogeneic, requires the
standing, new approaches to mobilization have
infusion of a sufficient number of hematopoietic
been designed and are now starting to undergo
progenitor/stem cells (HPCs) capable of homing to
clinical testing.
the marrow cavity and regenerating a full array of
In Section II, Dr. Michele Cottler-Fox describes
hematopoietic cell lineages in a timely fashion. At
factors predicting the ability to mobilize the older
present, the most commonly used surrogate
patient with myeloma. In addition, clinical ap-
marker for HPCs is the cell surface marker CD34,
proaches to improving collection by individualiz-
identified in the clinical laboratory by flow
ing the timing of apheresis and adjusting the
cytometry. Clinical studies have shown that
volume of blood processed to achieve a desired
infusion of at least 2 × 106 CD34+ cells/kg recipient
product are discussed. Key to this process is the
body weight results in reliable engraftment as
daily enumeration of blood CD34+ cells. Newer
measured by recovery of adequate neutrophil and
methods of enumerating and mobilizing autolo-
platelet counts approximately 14 days after
gous blood HPCs are discussed.
transplant. Recruitment of HPCs from the marrow
In Section III, Dr. John DiPersio and colleagues
into the blood is termed mobilization, or, more
provide data on clinical results of mobilizing
commonly, stem cell mobilization.
allogeneic donors with G-CSF, GM-CSF and the
In Section I, Dr. Tsvee Lapidot and colleagues
combination of both as relates to the number and
review the wide range of factors influencing stem
type of cells collected by apheresis. Newer meth-
cell mobilization. Our current understanding
ods of stem cell mobilization as well as the
focuses on chemokines, proteolytic enzymes,
relationship of graft composition on immune
adhesion molecules, cytokines and stromal cell-
reconstitution and GVHD are discussed.
during injury and inflammation. Currently, mobilized INFLUENCING STEM CELL MOBILIZATION
cells are the preferable and major source of stem andprogenitor cells harvested for autologous and alloge- Tsvee Lapidot, PhD,* Isabelle Petit, PhD, neic transplantations because of the higher yield of these cells, leading to faster engraftment and decreased pro-cedural risks compared with harvested BM cells. The Recruitment of hematopoietic stem and progenitor cells emerging picture of stem cell mobilization involves in- to the blood following treatment with chemotherapy, terfering with the physiological interplay between mes- or cytokines, is a clinical process termed mobilization. enchymal stromal and hematopoietic cells regulating This process mimics enhancement of the physiological both bone and BM remodeling processes, which also release of stem cells and progenitors from the bone mediate stem/progenitor cell proliferation and migra- marrow (BM) reservoir in response to stress signals tion. The mobilization process is initiated by stress-in-duced activation of neutrophils and osteoclasts by che-motherapy and repeated stimulation with cytokines such * The Weizmann Institute of Science, Rehovot 76100, Israel as granulocyte colony-stimulating factor (G-CSF), re-sulting in shedding and release of membrane-bound Acknowledgments: Special thanks go to Drs. Michele Fox, stem cell factor (SCF), proliferation of progenitor cells, Luen Bik To, and Josef Vormoor for fruitful discussions andfor critical reviews of this manuscript.
as well as activation and/or degradation of adhesion molecules such as VLA-4 and P/E selectins. The dy- Definitive, Repopulating Stem Cells
namic secretion and inactivation of the chemokines stro- Are Migrating Cells
mal cell derived factor-1 (SDF-1)/CXCL12 and During late embryonic development, both human and interleukin-8 (IL-8)/CXCL8, and multiple cycles of murine stem cells migrate via the blood circulation from inactivation and degradation of BM components by pro- the fetal liver to the BM and repopulate this tissue with teolytic enzymes, such as elastase, cathepsin G, pro- high levels of immature and maturing cells. These in teinase 3, CD26, and various matrix metalloproteinases turn are released into the circulation, while a small pool (MMPs), are implicated as major players in progenitor of undifferentiated stem and progenitor cells within the mobilization. These mechanisms will be reviewed in BM is maintained throughout life. Prior to their local- ization in the murine fetal liver, hematopoietic stem cells The road to stem cell mobilization began in the can be isolated from the aorta-gonad-mesonephros 1960s, with publication of a few reports documenting (AGM) region and the yolk sac even before comple- the presence of hematopoietic stem cells in the periph- tion of blood circulation development.5 However, some eral blood of mice, dogs, and monkeys, followed by of these stem cells are not definitive, since they cannot reports revealing low levels of progenitors in the hu- rescue and repopulate lethally irradiated recipients be- man circulation during steady-state homeostasis. How- cause of their inability to migrate in the host circula- ever, steady-state progenitors in the circulation of mice tion and home to and repopulate the BM. These were shown to be inferior to BM as a source of repopu- predefinitive stem cells isolated from the murine yolk lating stem cells. More important, although sporadic sac can still engraft the liver of newborn mice precon- clinical transplantations documented successful he- ditioned as fetuses with chemotherapy if the pre- matopoietic recovery, in most cases graft failure was definitive stem cells are directly injected into this or- detected in autologous, syngeneic, and fully matched gan.6 Furthermore, an additional maturation step pro- related allogeneic transplantations, using steady-state vided to predefinitive progenitors isolated from the peripheral blood cells. Moreover, the first successful AGM region, in the form of in vitro incubation on stro- human blood leukocyte transplantations were not bet- mal cells and stimulation with cytokines, shifts these ter than BM as a source of repopulating stem cells (re- cells into definitive stem cells with functional migra- viewed in Korbling and Fliender1 and To et al2). While tion, homing, and repopulation potential.5 Murine em- the levels of human progenitors in the circulation dur- bryos that lack the chemokine SDF-1 or its receptor ing steady-state homeostasis are low, they are signifi- CXCR4 have multiple lethal defects, including impaired cantly elevated in patients with myeloproliferative dis- seeding of the fetal BM (reviewed in Lapidot and Petit7).
orders.3 Similarly, a transient increase in circulating We have shown the essential roles of SDF-1/CXCR4 progenitors was documented in dogs treated with dex- interactions in a functional, preclinical model for hu- tran sulfate and humans treated with endotoxin or other man stem cells using non-obese diabetes severe com- stress-inducing mobilization agents. These encourag- bined immune deficient (NOD/SCID) mice as recipi- ing results were followed by preliminary reports in the ents. Homing and repopulation in both primary and late 1970s documenting increased levels of progeni- serially transplanted mice with human CD34 stem cells tors in the circulation of patients after chemotherapy was dependent on CXCR4 signaling,8,9 which is dy- treatment with cyclophosphamide and other drugs.
namically expressed by human progenitors.10 Moreover, Technical improvements including in vitro colony as- SDF-1-mediated migration of human CD34 cells in vitro says and the availability of large-scale harvesting of correlated with their in vivo repopulation potential in human progenitors by continuous-flow leukopheresis transplanted patients.11 In conclusion, stem cell motil- enabled closer examination of chemotherapy-/drug- ity (migration, homing, and release) is essential for BM induced mobilization in treated patients.1,2 In the early repopulation and for the development of the hemato- 1980s, To and colleagues reported high levels of pro- genitors in the circulation of leukemia patients in earlyremission.2 Clinical studies by this group were the first Hematopoietic Stem Cells in the Circulation
to document the beneficial faster repopulation in pa- Can Home Back to the BM
tients transplanted with autologous mobilized periph- The role of circulating stem cells in steady-state ho- eral blood (PBL). Initially, the mobilization protocols meostasis is currently unknown. While some progeni- were based on chemotherapy alone. However, after the tors are needed for seeding of the thymus, which re- discovery of human G-CSF by Welte et al,4 mobiliza- quires migration of lymphocyte precursors from the tion protocols began to include G-CSF, which today is BM, this process is markedly reduced in adult life.12 Stem cells migrate also to nonhematopoietic organs such as the liver, especially during liver injury/inflammation, Bone and BM Remodeling Processes
creating an alarm situation and transmitting stress sig- Are Mutually Regulated
nals that mobilize and recruit stem cells as part of or- Proliferation and release of hematopoietic stem and gan repair.13 Ramshaw et al suggest that circulating stem progenitor cells require dynamic cycles of BM destruc- cells can reengraft the BM, documenting successful tion/restructuring, which seem to be linked to bone re- engraftment of unconditioned murine recipients trans- modeling by osteoclast/osteoblast interactions. More- planted with high doses of BM cells.14 In support of over, both processes are likely to be orchestrated by this approach, Wright et al elegantly demonstrated rapid the same factors. For example, continuous G-CSF treat- clearance of intravenous transplanted mouse stem cells ment to prevent neutropenia has also been shown to from the murine circulation within a few minutes. By induce osteoporosis in some juvenile patients with se- using genetically marked parabiotic mice with a shared vere congenital neutropenia18 and in transgenic mice circulation, they showed that the low levels of circulat- that overexpress this cytokine.19 More important, there ing stem cells can durably reengraft the BM, suggest- is a dramatic increase in the number of murine osteo- ing continuous release and reengraftment of the BM as clasts in response to repetitive G-CSF stimulation, lead- sequential events taking part in physiological pro- ing to osteoclast-mediated bone resorption during stem cesses.15 In parallel, Abkowitz et al, using the same para- cell mobilization and calcium release, which can in- biotic model, revealed only low levels of reengrafted duce detachment of hematopoietic progenitors from stem cells with secondary recipient repopulation po- fibronectin in vitro.20 Unexpectedly, treatment of mice tential (1%–2.5%) in the partner BM as opposed to high with pamidronate, an inhibitor of osteoclast-mediated levels of progenitors in the spleen. These investigators bone resorption, together with G-CSF increased the suggest the release of stem cells into the circulation levels of mobilized progenitors in the murine circula- functions also as an apoptotic pathway for the BM to tion.20 Osteoclasts secrete high levels of IL-8, a mobili- regulate excess amounts of progenitors because of lim- zation-inducing chemokine.21,22 Another chemokine, ited numbers of stem cell niches.16 Of interest, these SDF-1, which also participates in G-CSF–mediated investigators also applied G-CSF- and SCF-induced mobilization, induces osteoclasts to secrete the mobilization to the parabiotic mice pairs, documenting metalloproteinase MMP-9.23 This enzyme is involved a dramatic 4- to 10-fold increase (from 1%–2.5% to in stem cell mobilization by shedding the membrane- 10.1%) in the levels of reengrafted stem cells with sec- bound cytokine SCF within the BM and releasing this ondary repopulation potential in the partner BM. These cytokine into the circulation.24 MMP-9 also induces results demonstrate that inducing the release of stem migration of osteoclasts to the cortical bone, adjacent cells in response to stress signals also increases their to the SDF-1 and stem cell–rich endosteum region, to homing back and reengraftment of the stimulated/ form resorption pits.23,25 These results suggest that the stressed BM, confirming the repopulation potential of role of osteoclasts in mobilization could be related to mobilized progenitors harvested for clinical protocols.
the rapid increase in their numbers and to the secretion These results also support the hypothesis that tissue of chemokines and proteolytic enzymes. Activation and repair of damaged BM can be supported by intensified secretion of proteolytic enzymes lead to cleavage and homing and reengraftment of circulating hematopoi- degradation of the BM extracellular matrix, adhesion etic stem cells, in response to stress signals, which is molecules, cytokines, and chemokines and therefore similar to migration of mature leukocytes to sites of facilitate transendothelial migration and stem cell re- inflammation as part of the immune system host de- lease from the BM.7 These processes mediate both pe- fense. The concept of BM niches occupied by station- ripheral bone and medullar BM remodeling as well as ary, quiescent stem cells may be an oversimplification stem cell proliferation and release by inactivating the of a more dynamic situation. Higher proliferative and BM microenvironment. Subsequently, stem cells mi- migratory turnover of definitive, repopulating stem cells grate via the circulation, home back to the BM, and involves their release into the circulation, migration, repopulate the damaged/destructured sites in this or- homing, and reengraftment of the BM as part of their gan as part of tissue repair and the continuous replen- physiological development. Bradford et al revealed that ishment of the blood with new immature and maturing even the most primitive murine stem cells in the BM hematopoietic cells of all lineages, while maintaining must cycle at least once every 30 days.17 a small pool of undifferentiated stem and progenitorcells within their renewed BM niches. In support ofthis hypothesis, by applying complementary DNA ar-ray technology, 2 recent studies revealed high expres-sion of the proteolytic enzyme proteinase 3 and low expression of a proteinase 3 inhibitor in isolated hu- agent, is usually administered daily at a dose of 5–10 man BM CD34 cells compared with enriched mobi- µg/kg for 5–10 days, alone or after chemotherapy.2 lized CD34 cells isolated from G-CSF-treated healthy However, a substantial number of patients, especially donors.26,27 These results suggest that proteinase 3, with patients having extensive chemotherapy that leads to its ability to cleave connective tissue in the BM, is BM aplasia, older patients, and a minority of healthy needed for maintaining a niche for hematopoietic stem individuals, are poor mobilizers.7 For example, increas- and progenitor cells. Recent findings document that ing age and longer intensive chemotherapy treatment proteinase 3 also cleaves the cell cycle checkpoint p21, in old multiple myeloma patients inversely correlate inducing cells to enter the cell cycle.28 This could ex- with successful mobilization.30 These patients are usu- plain the higher levels of cycling BM CD34 cells com- ally treated with higher doses of G-CSF, GM-CSF fol- pared with the more quiescent mobilized PBL CD34 lowed by G-CSF, or a combination of G-CSF and SCF.
cells.26,27 Immature mesenchymal bone-forming osteo- Recently, it was reported that some chronic myeloid blasts lining the stem cell–rich endosteum region se- leukemia (CML) patients treated with high doses of crete high levels of SDF-1.25 In response to stimulation imatinib (Glivec, STI 571) suffer hematological toxic- with this ligand, hematopoietic stem cell–derived os- ity and can develop drug-induced neutropenia.31 In ad- teoclasts secrete MMP-9. 23,24 Thus, peripheral periosteal dition, some treated patients failed to mobilize well, bone and medullar BM destruction and remodeling are requiring discontinuation of the drug during the mobi- sequential events mediated by the same factors, i.e.
lization process and the addition of SCF to G-CSF, to MMP-9, G-CSF, SCF, IL-8, and SDF-1, which regu- successfully overcome the poor mobilization.32 How- late both new bone formation and stem cell self-renewal, ever, while SCF stimulation increases the levels of mo- bilized CD34 progenitors, this treatment also has sideeffects such as allergic reaction due to activation of mast Stress-Induced Mobilization of Stem
cells. These studies stress the need to identify and char- and Progenitor Cells
acterize the mechanisms of stem cell mobilization in Applying single or multiple stress signals interferes with order to develop better strategies for poor mobilizers.
steady-state homeostasis, creating an alarm situation Chemotherapy followed by repetitive G-CSF–induced leading to increased release of immature and maturing mobilization is a multistep process. Anchorage of stem hematopoietic cells from the BM into the blood circu- and progenitor cells to the BM microenvironment by lation, which occurs naturally during inflammation and activation of adhesion interactions, which are facilitated injury. For example, increased levels of G-CSF and by membrane-bound SCF or SDF-1, needs to be dis- other cytokines secreted by long-distance runners dur- rupted.33 Papayannopoulou elegantly demonstrated that ing marathons cause blood leukocytosis, in particular the integrin VLA-4 plays a critical role in stem cell higher levels of CD34/CD38-positive committed pro- mobilization since anti-VLA-4 antibodies prevent ad- genitor cells, but not more primitive CD38-negative hesion molecules expressed on the surface of hemato- stem cells compared with control individuals, reflect- poietic progenitors from binding their ligand VCAM-1 ing adaptation of BM activity to stress induced by ex- expressed by stromal cells. This induces rapid mobili- tensive exercise.29 Increased cell release from the BM zation of primate and murine stem cells, which also reservoir is part of the immune system host defense involves signaling via the c-kit receptor.33 Moreover, during inflammation as a result of infection- or injury- as a result of inactivation during the mobilization pro- mediated release of stress signals. This release is in- cess, mobilized CD34 cells have lower levels of VLA- duced clinically or in animal models by a wide range 4 and c-kit expression compared with BM progeni- of molecules and/or treatments: DNA damaging, single tors.26,27 In addition, sulfated polysaccharides such as chemotherapeutic drugs such as cyclophosphamide Fucoidan lead to rapid mobilization of progenitors34 (Cy), or combined chemotherapy regimens such as since they compete for adhesion interactions with iphosphamide, carboplatin and etoposide (ICE) and selectins and for SDF-1 binding to the BM endothe- etoposide, methylprednisolone, ara-c and cisplatin lium via its nonsignaling C-terminus, inducing inacti- (ESHAP); cytokines such as G-CSF, granulocyte-mac- vation of selectin interactions as well as release of SDF- rophage colony-stimulating factor (GM-CSF), SCF, and 1 into the circulation. Levesque et al revealed increased flt-3 ligand; and chemokines such as IL-8, Mip-1α, release of elastase and cathepsin G within murine BM Groβ, and SDF-1.7 These molecules differ in their mode during Cy- and G-CSF–induced mobilization; peak lev- of administration, the time frame needed to achieve els were reached during stem cell egress.35 These en- mobilization, the type of cells mobilized, and the effi- zymes cleaved VCAM-1 expressed by stromal cells, ciency. G-CSF, the most commonly used mobilization preventing adhesion of progenitors via VLA-4 as part tors.8,39 In addition, other factors such as complement In addition to their distinctive adhesive properties, C3a improve SDF-1–mediated directional migration of mobilized CD34 progenitors differ from their BM coun- human progenitors and the in vivo homing of murine terparts in several ways. Besides reduced VLA-4, c- progenitors. However, some of these factors can be kit, and CXCR4 expression, a significantly higher per- species-specific or absent in immune-deficient mice.40 centage of mobilized progenitors are noncycling qui- Of interest, priming of mobilized human progenitors escent cells.26,27 Prior to mobilization, hematopoietic by several inflammatory molecules improves their progenitors proliferate within the BM, and this feature chemotactic responses to SDF-1.41 We have demon- could involve proteinase 3–mediated cleavage of p21 strated increased levels of SDF-1 production in the in response to neutrophil stimulation.28 Mobilized pro- murine BM in response to DNA-damaging chemo- genitors have higher levels of the proapoptotic genes therapy drugs, including Cy and 5-fluorouracil (5-FU), caspase 3, 4, and 8 and reduction in inhibitors of most probably to prevent cell death since this chemokine apoptosis such as antiproteinase 2, compared with hu- is also a survival factor for stem cells.25,42,43 Rafii and man BM CD34 cells,26 supporting the hypothesis of Hattori et al demonstrated that the increased levels of Abkowitz et al that release into the circulation may also SDF-1 in response to stress-mediated 5-FU mobiliza- serve as an apoptotic pathway for stem cells.16 Enhanced tion in mice induce release of MMP-9, which cleaves repopulation documented with mobilized progenitors and releases membrane-bound SCF, leading quiescent is due to significantly higher cell doses, in particular stem cells to proliferate in the BM prior to their mobi- committed progenitors, which lead to faster neutrophil lization into the circulation.24 SDF-1 mediates secre- and platelet recovery.7 However, better leukemia-free tion of MMP-2 and MMP-9 from human CD34 cells,44 survival and overall survival were recently documented and these proteolytic enzymes inactivate SDF-1 by in acute myeloid leukemia (AML) patients transplanted cleaving a few amino acids in the N terminus.45 We with increased cell doses of matched BM cells com- demonstrated that each injection of G-CSF stimulates pared with lower doses of BM cells and more impor- mesenchymal cells such as immature osteoblasts to se- tant, also compared with high cell doses of matched crete SDF-1, leading to a transient increase in SDF-1 mobilized PBL, suggesting BM as a superior source of levels within the BM. This oscillating increase is fol- stem cells for HLA-identical allogeneic transplanta- lowed by a profound decrease due to inactivation by tions.36 New protocols aimed at increasing the levels of proteolytic enzymes, mostly by neutrophil elastase, with long-term repopulating stem cells as the major source the lowest levels of this ligand released during cell mo- while maintaining short-term repopulating cells need bilization.46 Levesque et al reported that in addition to inactivation of BM SDF-1 by elastase and cathepsin G,these proteolytic enzymes can also cleave part of the SDF-1/CXCR4—Key Regulators of Stem Cell
CXCR4 receptor N terminus in the BM,47 partially in- Homing and Mobilization
activating SDF-1 signaling and directional migration.
Mobilized human CD34 progenitors express reduced Of interest, neutrophils secrete the proteolytic enzymes levels of the SDF-1 receptor CXCR4, which correlates elastase, cathepsin G, and proteinase 3 in response to with improved mobilization,11,37 suggesting involvement G-CSF, and neutrophil elastase also inactivates G-CSF,48 of SDF-1/CXCR4 interactions in the mobilization pro- which is essential for regulating release of neutrophils cess. Overexpression of SDF-1 in the murine circula- from the BM. However, expression of G-CSF recep- tion leads to stem cell mobilization.38 In functional pre- tors by neutrophils is not required because of an indi- clinical animal models for human stem cells such as rect mechanism that involves proteolytic reduction of preimmune sheep and immune-deficient NOD/SCID SDF-1 within the murine BM.49 Of interest, the central mice, mobilized CD34 cells are inferior in their repopu- role of elastase in regulating release of BM leukocytes lating potential compared with similar cell doses of is suggested by inherited genetic mutations in the CD34 cells obtained from human BM. This is most elastase gene, leading to 2 forms of neutropenia in ju- probably due to their reduced levels of surface CXCR4, venile patients (severe congenital neutropenia, or which is needed for SDF-1–mediated directional hom- Kostmann disease, and cyclic neutropenia) that require ing and repopulation in transplanted mice and increased continuous treatment with G-CSF.7 CD26, another pro- levels of short-term repopulating cells.7 These results teolytic enzyme that inactivates SDF-1, is expressed should be interpreted with caution, since short-term by human CD34 progenitors and is involved in G-CSF– stimulation with human SCF upregulated surface CXCR4 expression on human CD34 cells and murine A role for CXCR4 signaling in cell egress from the SCF is much less potent in stimulating human progeni- BM to the circulation emerges from several studies. We found that in parallel to gradual reduction of BM SDF- apologize to those whose excellent work could not be 1 during G-SCF administration, CXCR4 expression is elevated within the human and murine BM, reachingpeak levels at the time of mobilization.46 While some II. MOBILIZING THE OLDER PATIENT WITH MYELOMA
CXCR4 inhibitors also lead to release of human pro-genitors,51 and treatment with pertussis toxin, which blocks Gαi-mediated signaling in 7 transmembrane Gcoupled receptors such as CXCR4, induced mobiliza- Individually Optimized Collection of HPCs
tion in mice,52 SDF-1/CXCR4 interactions are alsoneeded for cell egress. Treatment of mice with G-CSF Enumeration of HPCs
and neutralizing anti-CXCR4, anti-SDF-1 Ab, or in- Variables having an impact on the ability to collect he- hibitors of CD26 reduced the mobilization levels.46,50 matopoietic progenitor cells (HPCs) may be donor re- Finally, warts, hypogammaglobulinemia, immunodefi- lated or procedure related. Donor-related variables in- ciency and myelokathexis (WHIM) syndrome, a rare clude age, previous chemotherapy, mobilization regi- inherited immunodeficiency disease associated with men, and platelet count at the time of mobilization. Pro- mutations in CXCR4, is characterized by neutropenia cedure-related variables include central access devices and B-cell lymphopenia. In one case, an affected indi- as well as variables inherent to the different cell sepa- vidual was born with cardiac malformation, resembling ration devices used. Nonetheless, at present, the basis the murine model in which knocking out CXCR4 or for optimizing HPC collection is the ability to deter- SDF-1 results in a lethal phenotype associated with lack mine when to start collection, based on the ability to of BM seeding, B-cell development, and cardiac sep- enumerate CD34+ cells in the peripheral blood of a do- tum formation.53 Thus, SDF-1/CXCR4 interactions may nor/patient on a daily basis. It has been demonstrated have a role in the regulation of the routine and active repeatedly that the best currently available predictor of egress of progenitor and maturing cells from the BM an adequate collection is the number of CD34+ cell/µL in the blood on the morning of collection, both for good Taken together, these results decipher key mecha- mobilizers and for poor mobilizers.1-5 A number of au- nistic insights into stress-induced mobilization that thors have recommended starting collection when a mimic and amplify naturally occurring recruitment of particular number of CD34+ cells/µL is present (usu- progenitors during alarm situations. We suggest the BM ally a number between 8 and 20) in order to increase as a reservoir for immature and maturing hematopoi- the likelihood of collecting at least 2–4 × 106 CD34+ etic cells to be released into the circulation upon stress cells/kg in a single apheresis, i.e., an acceptable num- signals, to migrate to injured sites, and to contribute to ber of HPCs for either one or two autologous trans- host defense and tissue repair. The steady-state balance plants or a single allogeneic transplant.6,7 in the BM is disrupted, leading to transient increased In the past it was important that each center deter- production of SDF-1, and proliferation and activation mine a number of its own as the starting point for aph- of neutrophils and osteoclasts. Release of proteolytic eresis, given the historic difficulty in comparing flow enzymes is followed by shedding of membrane-bound cytometry results from center to center.8 At present, SCF, proliferation of hematopoietic progenitors, in- however, there are two commercially available single creasing surface CXCR4 expression and inactivation platform tests available for measuring CD34+ cells in of SDF-1, G-CSF, the BM adhesion machinery, and blood (ProCount from Becton-Dickinson, Mt View, CA, extra cellular matrix (ECM). These events are intensified and StemKit from Beckman-Coulter, Fullerton, CA), in each cycle of stimulation by G-CSF, eventually lead- so that any center using the same technology should ing to release of progenitors into the circulation (Figure
have a reasonable chance of success using the same 1 contains a model of the process; see Appendix, page
numbers. More importantly, it is possible to use the 602). A better understanding of the process by which pro- number of CD34+ cells/µL obtained using single plat- genitors egress from the BM will eventually lead to the form technology in a predictive formula which allows development of improved mobilization protocols, in par- the clinician to know when to start apheresis in order to ticular for patients who are poor mobilizers.
* Department of Pathology and Myeloma Institute for Because of the complexity of stem cell mobilization Research and Therapy, University of Arkansas for Medical and the wide range of agents inducing this process, we Sciences, 4301 W Markham Street, MS 517, Little Rock AR could not discuss many interesting studies, and we optimize collection and how many liters of blood need Timing of apheresis
to be processed in order to collect a given number of There is a fall in circulating leukocytes immediately CD34+ cells9 using the formula in Figure 3. While re-
after granulocyte colony-stimulating factor (G-CSF) has sults of collection using this formula have only been been given, with the peak of CD34+ cell mobilization reported for the COBE Spectra cell separators, it seems approximately 3–6 hours after each dose is given sub- likely that it would work with any continuous flow aph- cutaneously, paralleling its half-life.15-17 To optimize col- eresis device if the appropriate value for machine col- lection, it seems reasonable therefore, to wait at least 1 hour after giving the injection, trying to time the dura- Sadly, despite the consensus that daily CD34+ cell tion of the collection for the period which includes the numbers in the blood are the best currently available rise in leukocytes and as much of the peak as possible.
predictor for achieving an acceptable HPC collection,8 For example, at least one center routinely waits for 2 the practice has not been universally adopted because hours after the injection, since their average collection it is both time consuming and relatively expensive. Al- lasts about 2–3 hours.5 It is possible that the apparent ternatives that have been reported to be useful include ability of large volume leukapheresis lasting 4–5 hours measuring the number of CD34+ cells in the blood the to “recruit” CD34+ cells into the blood, reported by sev- day before starting collection, with or without the total eral groups but not others,18,19 in fact simply reflects white cell count, or change in white cell count from the the timing of the apheresis collection relative to the dose day before apheresis to the day of apheresis,4 and the of G-CSF. With the arrival of pegylated filgrastim rapidity of rise in white cell count and platelet count.
(Neulasta, Amgen, Thousand Oaks, CA) it is possible More recently, the Food and Drug Administration that the relationship of collection yields relative to the (FDA) has approved the use of an HPC window on an time of dosage will cease to be a factor in collection.
automated cell counter (Sysmex, Kobe, Japan) and thisnumber may be used to predict when to start apheresis.10 Predicting Who Will Mobilize Poorly
The HPC number measured by the Sysmex does not The effect of age has been shown to be a continuous, correlate well with CD34+ cell number in the blood (nor incremental variable in the myeloma population, with does CD133, an antigen expressed by the more primi- no threshold past which there is an accelerated decline tive CD34+ cells11 [Cottler-Fox et al, in preparation]), in mobilization of CD34+ cells.20 That is, the older the but since it is now recognized that not all HPCs express patient, the fewer stem cells are likely to be collected, measurable CD3412 it has been presumed that the HPCs but there is no fixed age past which it is impossible to measured by the Sysmex include some that are CD34–.
collect HPCs. Over and above age, however, two other Thus, the HPCs measured by Sysmex are available rap- variables are statistically significant: number of months idly and less expensively, and may serve as a guide for of previous chemotherapy and platelet count at the time starting apheresis, but they are difficult to use in the of mobilization.20 In 85% of myeloma patients over age 70 who had < 12 months of therapy and a platelet count HPCs expressing high levels of the enzyme aldehyde > 200 × 109/L, it was possible to obtain ≥ 4 × 106 CD34+ dehydrogenase (ALDH) are believed to be pluripotent cells/kg in a single apheresis using individually opti- and generally express CD34 on their surface.13 A com- mized collection conditions after combined chemo- mercial assay has now been developed for this intracellu- therapy and growth factors. Those patient over 70 years lar enzyme (Aldecount, Stemco Biomedical, Durham, of age with > 12 months of prior therapy and platelets NC). As this assay detects both CD34+ and CD34– cells, < 200 × 109/L, however, were poor mobilizers.
and appears to differentiate viable from non-viable cells,14its use may eventually lead to a major change in how Choosing a Mobilization Regimen
HPCs are enumerated in transplant grafts.
Mobilization with chemotherapy and growth factors hasbeen shown in a number of settings to be significantlymore effective than growth factors alone. Nonetheless,it is often the case that the toxicity of chemotherapy Figure 3. Predictive formula that allows the clinician to know when to start apheresis in order to optimize collection and how
many liters of blood need to be processed in order to collect a given number of CD34+ cells.

CD34+ cell/µL blood × machine collection efficiency # of L blood to process = # CD34+ cells desired ÷ makes this combination less attractive in the fragile dence of allergic reactions to this agent and the need older patient. It is therefore important to know that in for observation after it is given have made it difficult to the subgroup of myeloma patients over the age of 70 move into standard clinical practice. It is not currently who were predicted to be poor mobilizers (> 12 months prior therapy and platelets < 200 × 109/L), as many stemcells were collected with growth factors alone as with Newer agents
chemotherapy and growth factors together. Thus, for Longer lasting variants of G-CSF (pegfilgrastim, this group of patients it seems worthwhile trying to Amgen) and erythropoietin (darbopoietin, Amgen) are mobilize with growth factors alone in order to avoid now available and are in clinical trials as mobilizing toxicity, assuming the disease itself does not require agents. They have the benefit of very long half-lives chemotherapy at the time of attempted mobilization.
and so add an important measure of patient convenienceand the probability that timing of collection may be Current standard agents
more flexible without sacrificing optimal collections.
G-CSF (filgrastim, Amgen) has become the standard A new factor (AMD3100, AnorMed, Vancouver, against which all other mobilization agents are mea- Canada), which is a reversible inhibitor of the binding of sured. This is because it has been shown to both mobi- stromal derived factor (SDF-1a) to its cognate receptor lize more CD34+ cells and have less toxicity than any CXCR4, is currently in clinical trials as a mobilizing agent.
other single agent against which it has been tested to It is the first agent to be tried for mobilization based on a date. It is not completely without toxicity, however, rational understanding of its mechanism of action rela- given that there have been deaths attributed to throm- tive to HPC-stromal cell interactions (see Section I). While bosis (acute myocardial infarction and stroke) in sib- it mobilizes CD34+ cells adequately on its own, it signifi- ling donors,21 possibly related to receptors on platelets cantly improves the mobilization capacity of G-CSF when for G-CSF.22 Also, a recent study of serial ultrasounds used in combination with G-CSF in mice. Clinical trials in donors receiving G-CSF prompted by 4 reports of in humans with various diseases are in progress, includ- splenic rupture related to G-CSF demonstrated univer- sal enlargement of the spleen during mobilization, withregression of size after discontinuing the growth fac- Remobilization
tor.23,24 Other effects of G-CSF which are shared with For the patient who fails to mobilize the necessary num- granulocyte-macrophage colony-stimulating factor ber of cells for transplant on the first attempt, but for (GM-CSF) include pain, nausea, vomiting, diarrhea, whom it is clear that transplant is the best option, two insomnia, chills, fevers, and nightsweats.25,26 decisions need to be made simultaneously: when to re- GM-CSF (sargramostim, Berlex, Richmond, CA) mobilize, and with what? When to remobilize is still a as a single agent is used less often today for mobiliza- subject of debate, and depends to some extent on tion than G-CSF, because it mobilizes somewhat less whether the failed mobilization was with chemotherapy well than G-CSF27 and because of a relatively higher plus growth factor or growth factor alone. Although at incidence of both mild and severe side effects.28 How- least one set of authors recommend immediate ever, the fact that it can be more cost effective, and that remobilization with growth factors for a patient who there are reports of improved immune reconstitution has not mobilized adequately after chemotherapy plus with GM-CSF relative to G-CSF,29-32 has caused some growth factor,39 others feel that at least 2-3 weeks off clinicians to reconsider its use. Further, for the patient, growth factor prior to remobilization offers the best or normal donor who has failed to mobilize adequately chance of success.40-42 If adequate cells are given for an on G-CSF alone, the combination of GM-CSF with or autologous transplant, it is even possible to collect followed by G-CSF has been shown to be efficacious.33-36 enough for a second transplant during the period of Erythropoietin, now commonly used among can- white blood cell recovery following the first transplant.43 cer patients undergoing chemotherapy to maintain he- It is also possible to collect HPCs at least one year after moglobin in the near normal range, also has some abil- a prior transplant: of 38 myeloma patients who at- ity to mobilize CD34+ cells.37 In the hard to mobilize tempted such a collection, 36 achieved an adequate patient, its use may therefore be doubly beneficial.
number of cells for transplant (Cottler-Fox et al, inpreparation).
Alternative agents
What to use for a repeat mobilization attempt for a Stem cell factor (SCF) has been shown to be an excel- specific patient and disease may be a complex deci- lent mobilizing agent, particularly when used in com- sion. However, some general guidelines may be found bination with G-CSF.38 Unfortunately, the high inci- in the literature. First, mobilization with chemotherapy plus growth factor will generally yield more CD34+ cells 6. If patient achieves a CR with transplant but does than growth factor alone (for the single exception see not mobilize adequately to collect during the leu- #2 in the Algorithm for Mobilizing Myeloma Patients kocyte recovery phase post-transplant, consider at- below).44,45 Second, chemotherapy plus G-CSF and SCF tempting collection with combination growth fac- is more effective than chemotherapy plus G-CSF tors at least 1 year after transplant.
alone.38 Further, chemotherapy with either sequentialor concurrent GM-CSF plus G-CSF may be more ef- III. MOBILIZATION OF ALLOGENEIC STEM CELLS
fective than G-CSF alone,45 as erythropoietin may im-prove the response to G-CSF.46 Finally, retrospective John F. DiPersio, MD, PhD,* Dan Link, MD, analysis of data in myeloma patients from a single in- stitution has shown that for patients who have under-gone as many as 4 attempts at mobilization, only 1 of General Principles
the 4 may yield an adequate collection (Cottler-Fox et Although early attempts to use unmobilized peripheral al, in preparation). It is possible that AMD3100 will blood stem cells (PBSCs) for autologous stem cell trans- find a niche in this hard to mobilize population: in an plantation were problematic, the use of cytokines such ongoing trial at the University of Arkansas for Medical as G-CSF to enhance the peripheralization of CD34+ Sciences in myeloma patients who have previously cells and the collection of these stem cells using leuka- failed to collect at least 5 × 106 CD34+ cells/kg, it pro- pheresis procedures has become the standard for au- vides adequate mobilization in a significant number of tologous stem cell transplantation around the world.1 patients (Tricot et al, in preparation).
IBMTR and EBMT data suggest that over 80-90% ofall autologous stem cell transplants in the world are An Algorithm for Mobilizing Myeloma Patients
performed using cytokine or chemotherapy/cytokine Although many variables may affect the decision of how mobilized PBSCs as a source of stem cells.2 In addition and when to mobilize a myeloma patient, the following to reducing patient morbidity, the use of mobilized PBSCs has resulted in higher CD34 content of grafts, 1. Attempt first collection relatively early, i.e., with shorter hospital stays, and reduced engraftment times for both neutrophils and platelets as well as improved 2. Attempt collection first with chemotherapy plus lymphocyte recovery resulting in enhanced immuno- growth factor (except for patients over 70 with logic reconstitution when compared to patients receiv- > 12 months prior therapy and platelets < 200 × ing autologous BM.3-6 These beneficial effects of mo- 109/L, for whom growth factors alone may be tried bilized PBSCs as a source of stem cells for autologous first). The choice of growth factor (G-CSF versus stem cell transplantation have been confirmed in a num- GM-CSF) may depend on data under development regarding the importance of early immune recon- Based on the sustained success of using mobilized stitution on time to progression and long-term dis- PBSCs for autologous stem cell transplantation, inves- ease-free survival. Pegylated filgrastim may replace tigators began to pilot the use of PBSCs for allogeneic standard G-CSF if studies show it to be equivalent stem cell transplantation. Initial concerns focused on the possibility of increased risk of acute and chronic GVHD due to the presence of 10- to 50-fold increased a. patient is in CR or near-CR: wait at least 3 weeks, numbers of T cells present in mobilized PBSC prod- then remobilize with combination growth factors.
ucts. It was not clear if the function of mobilized allo- In the future, AMD3100 may be a possibility.
geneic T cells might be qualitatively altered resulting b. patient is not in CR or near-CR: give planned che- in even greater risk of GVHD or relapse. In addition, motherapy and combine it with sequential GM-CSF the risk of infusing increased numbers of cytomega- and G-CSF. Consider adding erythropoietin. In the lovirus (CMV)-positive granulocytes, dendritic cells, future, AMD3100 may be a possibility.
and monocytes into both CMV– and CMV+ recipients 4. If patient is not progressing, 3a and/or 3b may be remained unknown and potentially posed an increased repeated until adequate cells are collected.
risk to the recipient. On the other hand, mobilized 5. If patient is progressing, and adequate cells are PBSCs contain 3- to 4-fold more CD34+ cells, which available for autologous transplant (≥ 3 × 106 CD34+cells/kg), monitor CD34+ cells in the blood at thetime of leukocyte recovery, and consider collect- * Washington University School of Medicine, 660 S Euclid, ing HPCs if there is adequate mobilization.
might result in faster engraftment and more efficient ment and showed that CD34+ cells peaked in the blood transformation to complete donor chimersim.
between days +4 and +5 for G-CSF and days +5 and Early Phase 2 studies demonstrated that G-CSF had +6 after GM-CSF treatment. Data from Seattle sug- a generalized effect on the peripheralization of many gested that larger doses of G-CSF (16 µg/kg/day) may different types of allogeneic peripheral blood cells, not result in even higher CD34 yields at the time of pheresis just CD34+ cells. These data are consistent with the on day 5.24 We have assessed the effect of 5 days of of notion that G-CSF has a generalized effect on remod- G-CSF (10 µg/kg) on the numbers of leukocyte subsets eling the BM microenvironment, which results in the in the peripheral blood of 100 consecutive normal allo- egress of many types of cells including T cells and geneic PBSC donors (Figure 3; see Appendix, page
monocytes. Activation of neutrophils by cytokines such 602). It is clear from these data that G-CSF has a pleio- as G-CSF results in the release of proteases that facili- tropic effect of increasing the numbers of circulating tate the egress of HSCs from the BM microenviron- neutrophils and monocytes (WBC) as well as T cells ment. Interruption of the G-CSF signal through geneti- (both CD4 and CD8), NK cells and B cells. Korbling cally “knocking out” the G-CSF receptor results in not and Anderlini compared the allograft content after G- only the expected elimination of G-CSF-induced mo- CSF mobilization to cellular contents of BM harvests.
bilization of HSCs but also IL-8- and chemotherapy- These data suggest a 3- to 4-fold enhancement of CD34+ induced HSC mobilization in these G-CSF receptor cells and a 10- to 20-fold increase in the number of CD3+ knock-out mice.12,13 Although it is not completely clear T cells in PBSC products compared to BM harvests.25 which is the most important tether binding HSCs to themicroenvironment, LFA-1, VLA-4, CXCR4, and c-kit Factors Determining Mobilization and Outcomes
have all been implicated as critical stem cell adhesion It is well known that certain factors may help predict molecules.14-17 Likewise, a number of neutrophil-spe- those autologous stem cell recipients who might be cific enzymes have been implicated in mediating criti- expected to be “poor mobilizers.” These include extent cal cleavages that result in stem cell egress from the of previous treatment, treatment with certain drugs such microenvironment. These include neutrophil elastase, as nitrosoureas and certain diseases such as Hodgkin’s cathepsin G, proteinase 3, gelatinase B (MMP-9), and disease, non-Hodgkin’s lymphomas and preleukemic other metalloproteinases.18-21 Recent evidence has syndromes. No such data exist for allogeneic donors.
strongly implicated CD26, a CD34-associated protease, We have examined stem cell mobilization from over as the prime protease that may cleave SDF-1 off the 400 HLA-matched sibling donors since 1995. Using marrow microenvironment resulting in the release of G-CSF as the sole mobilizing agent, only 2.0% of nor- CD34+ HSCs into the periphery.22 Of interest, a col- mal donors mobilized with G-CSF (10 µg/kg/day; 20 L laborative effort of the Link, Simmons and Levesque exchange on day 5) did not achieve > 2 × 106 CD34/kg laboratories have shown that mice deficient in MMPq, and 25% did not achieve > 5 × 106 CD34/kg after a neturophil elastase and cathepsin-G and mice deficient single collection. These data have been recapitulated in dipeptidyl peptidase I (CD26) all mobilized by other groups. We have studied the few normal do- hematopietic precursors in response to G-CSF normally.
nors who did not achieve > 1 × 106 CD34/kg after 3 These data question the role of neutrophil specific pro- collections (1.0% of all normal donors in our data set).
teins in stem cell egress (Daniel Link, personal com- All of these donors underwent BM harvests as well but these all yielded < 1.0 × 106 CD34 cells consistent withthe notion that these poor allogeneic PBSC mobilizers Effects of G-CSF Mobilization on Allograft Content
were not defective in cytokine induced mobization per Although a number of cytokines and cytokine combi- se but had low levels of BM stem cell reserves. Brown nations have been used to mobilize autologous HSCs, et al26 correlated premobilization PB CD34/mL with only G-CSF and GM-CSF have been approved by the G-CSF-induced mobilization. None of the normal al- Food and Durg Administration (FDA) for use as au- logeneic donors who had < 2000 CD34/mL prior to tologous stem cell mobilizing agents. Thus, these have mobilization yielded > 5 × 106 CD34+ cells/kg while been the only cytokines used to mobilize allogeneic 95% of those normal donors with > 4000 CD34/mL PB PBSCs. The majority of the initial Phase 2 studies us- yielded > 5 × 106 CD34/kg after G-CSF mobilization.
ing mobilized PBSCs in an allogeneic setting utilized Although provocative, this has not been widely accepted G-CSF (10–16 µg/kg/day for 5 days). Leukapheresis as a method of identifying poor autologous or alloge- was performed on day 4 or day 5 after G-CSF treat- ment. Fischmeister et al23 followed CD34+ in the pe- In the mid- and late-1990s, a large number of small ripheral blood after either G-CSF or GM-CSF treat- Phase 2 studies were performed using mobilized PBSCs as a source of HSCs for allogeneic stem cell transplan- ies, 20% of normal donors required placement of a cen- tation. All of these studies yielded similar results. Al- tral line for apheresis. Eleven percent required more though neutrophil and platelet recovery was enhanced than 2 leukapheresis procedures and “serious compli- using cytokine mobilized allogeneic PBSCs, rates of cations” occurred in 1.1% of allogeneic PBSC collec- acute GVHD were similar or less than that documented tion versus 0.5% after BM harvest. Rowley et al31 uti- for BM as a source of allogeneic HSCs. The majority, lized an 11-point scale (0 = minimum and 10 = maxi- but not all of these early studies, demonstrated increased mum) for 23 different symptoms occurring during the actuarial rates of limited and extensive26-28 chronic first 14 days after either BM or PBSC collection. There GVHD (cGVHD). Cost and hospitalization appeared were no statistical differences between the PBSC and to be reduced compared to patients transplanted using BM groups for any of the symptom complexes or for allogeneic BM in these small Phase 2 studies.
Similar to many autologous PBSC studies, the num- ber of allogeneic CD34 cells infused correlated well Randomized Studies
with both neutrophil and platelet engraftment. Brown Many of the Phase 2 studies assessing the role of mobi- demonstrated that those allogeneic PBSC recipients who lized allogeneic PBSCs on GVHD, relapse and overall had > 5 × 106 CD34/kg infused had a 95% chance of survival are limited by design (Phase 2), paucity of pa- both neutrophil and platelet engraftment by day +15.26 tients, short-term follow-up, and heterogeneity of dis- In this study, no correlation could be found between eases for which allogeneic PBSC transplantation was GVHD or survival and the number of CD3+ cells in- performed. Several trials have provided insight into the fused. In one retrospective study by the MD Anderson relative effect of PBSC versus BM on GVHD, relapse, group, infusion of > 8 × 106/kg CD34 resulted in de- and survival. These include randomized studies, case creased survival presumably due to increased rates of control retrospective studies, and meta-analyses. Table
cGVHD associated complications.29 These data have 1 summarizes the results of all randomized trials com-
not been corroborated by other groups. Therefore, the paring BM and PB as a source of stem cells for alloge- infusion of high numbers (> 8 × 106 CD34/kg) of alloge- neic stem cell transplantation.32-38 Half of these trials neic stem cells remains a controversial negative predictor suffer from low numbers of patients. The 3 largest stud- for outcomes after allogeneic PBSC transplantation.
ies36-38 demonstrate no significant difference in overallsurvival when peripheral blood is compared to BM as a Effects on Donors
source of allogeneic stem cells. These studies, similar A major question is whether allogeneic stem cell har- to the smaller randomized trials, did demonstrate a sig- vesting results in less morbidity than BM harvesting nificant enhancement in both neutrophil and platelet for allogeneic stem cell donors. Anderlini et al30 re- recovery consistent with the significantly increased viewed 1448 mobilized allogeneic PBSC collections numbers of CD34 cells that are harvested in mobilized from the IBMTR and EBMT registries. G-CSF was used allogeneic PBSC products compared with BM.
in > 99% of donors. Similar to multiple Phase 2 stud- In one of the largest and best-designed studies, Table 1. Randomized trials comparing allogeneic peripheral blood to bone marrow.
PB, % BM, %
PB, % BM, %
PB, % BM, %
PB, % BM, %
a Engraftment (days) ANC > 500/mm3b Engraftment (days) Plt > 25000/mm3 Abbreviations: ANC, absolute neutrophil count; PLT, platelets; TRM, treatment-related mortality; aGVHD, acute graft-versus-host disease;cGVHD, chronic GVHD; PB, peripheral blood; BM, bone marrow.
Bensinger et al,38 using identical conditioning regimens, for patients with acute leukemia in first remission. In GVHD prophylaxis (cyclosporine and methotrexate), contrast, acute leukemia patients in second remission and post-transplant growth factor support (no G-CSF), and patients with CML in accelerated phase experienced found a slight advantage in both disease-free and over- lower TRM, improved DFS and overall survival when all survival in those patients receiving mobilized allo- allogeneic PBSCs were used as a source of stem cells.
geneic stem cells versus BM (P = .03 and P = .06, re- There was no apparent difference in the risk of relapse spectively). Although there was no difference in prob- after allogeneic PBSC versus BM transplantation. There ability of 2-year overall survival in the subgroup of was a trend toward lower relapse rates in patients with patients with less advanced disease (75% for PB and high-risk leukemia (acute leukemia in second remis- 72% for BM), those patients with more advanced dis- sion and CML in accelerated phase). The relative risk ease demonstrated a significantly enhanced overall sur- of both limited and extensive cGVHD was increased in vival when mobilized allogeneic PBSCs were used as recipients of allogeneic PBSCs (relative risk 1.3).
a source of stem cells (57% for PB and 33% for BM; P Mohty et al41 have performed the only long-term = .04). With a median follow-up for all surviving pa- follow-up of allogeneic PBSC and BM recipients fo- tients of 26 months (9-47 months), the cumulative inci- cusing specifically on the rates of cGVHD. At a me- dence of grade III-IV acute GVHD at 100 days was dian follow-up of 45 months (range 31–57 months), 64% in the PB group and 57% in the BM group (P = the 3-year cumulative incidence of cGVHD was 65% .35). The cumulative incidence of grade II-IV acute in the PBSC group (n = 53) and 36% in the BM group GVHD was 15% in the PB group and 12% in the BM (n = 48) (P = .004). Extensive chronic GVHD was also group (P = NS). Although the follow-up was relatively more frequent in the PBSC group (44% versus 17%; P short, the cumulative incidence of extensive cGVHD was 46% in the PB group and 35% in the BM group (P = .54).
These data suggest that although hematopoietic re- These results were inconsistent with many of the other covery is increased in recipients of allogeneic PBSC, smaller Phase 2 and Phase 3 studies, which all showed no there appears to be no increased risk of acute GVHD difference in rates of acute GVHD and increased rates of and a modest increase risk of cGVHD including exten- cGVHD in recipients of allogeneic PBSC.
sive cGVHD. Overall survival in recipients of alloge- A recent meta-analysis was performed by Cutler neic PBSCs may be improved modestly but only in those et al39 summarizing 15 Phase 2 and Phase 3 trials as- patients with more advanced hematologic malignancies.
sessing the risk of GVHD in recipients of allogeneic Although no study has correlated rates of either acute PBSC and BM. This analysis demonstrated a modest or chronic GVHD with the number of CD3 cells/kg in increased relative risk of acute GVHD (relative risk 1.2) the stem cell products, several studies have suggested and a significant increased risk of developing cGVHD that CD34 cells in excess of 8 × 106/kg found in alloge- (relative risk 1.8) in recipients of allogeneic PBSCs. It neic PBSC products are associated with a greater risk also demonstrated a modest reduction in relative risk of relapse in recipients of allogeneic PBSCs comparedto BM (relative risk 0.8).
Impact of G-CSF Mobilization on Graft Content
Champlin et al and the IBMTR40 performed a ret- and Immune Reconstitution
rospective case controlled study comparing the out- Very little data exist on the relative impact of alloge- comes of recipients of allogeneic PBSCs and BM. Me- neic PBSCs versus BM on immunologic reconstitution dian follow-up was 1 year, and this study focused on 1- after allogeneic stem cell transplantation. Storek et al42 year outcomes. A total of 288 HLA-identical sibling analyzed the incidence of documented and suspected PBSC recipients was compared with 536 case control infections after transplantation of mobilized allogeneic allogeneic BM recipients. All patients received T-re- PBSCs and BM in the randomized trial carried out by plete stem cell products. There was no significant dif- Bensinger et al.38 The cumulative incidence of infec- ference in the incidence of grades II-IV acute GVHD tions was higher in the allogeneic BM group (120 ver- (40% for PB and 35% for BM; P = NS) or grades III- sus 90 at 1 year). Since rates of acute GVHD after allo- IV acute GVHD (13% for PB and 19% for BM; P = geneic PBSC infusions are similar to BM in spite of NS). There was less variability in recovery times of these products having 10- to 50-fold increased CD3 both platelets and neutrophils after allogeneic PBSC cells/kg over allogeneic BM products, a number of in- compared to BM and statistically faster neutrophil and vestigators have tried to understand the reason for this.
platelet recovery after allogeneic PBSC compared to To date, no studies have clearly shown a difference in allogeneic BM. Treatment-related mortality (TRM), either B-cell or T-cell recovery after allogeneic PBSC disease-free survival, and overall survival were similar transplantation compared to BM transplantation.
A number of reports have emphasized the role of sors to mature DC2 cells overexpressing costimulatory cytokines as mediators of GVHD. Cytokines produced molecules such as CD80 and CD86 restoring their abil- by both CD4 and CD8 T cells can be segregated into ity to induce a proliferative response to naïve CD4+/ two patterns: type I cytokines such as interferon-γ and CD45RA allogeneic T cells. Incubation of naïve allo- IL-2 and type 2 cytokines, such as IL-4 and IL-10. Type geneic T cells with DC1 resulted in polarization of these 1 cytokines are proinflammatory and type 2 cytokines T cells toward the Th1 phenotype as measured by are considered anti-inflammatory. Multiple studies have restimulation of these T cells with PMA and ionomycin shown that T cells that elaborate type 1 cytokines (Th1 and detecting primarily IL-2 and interferon-γ as the cells) mediate GVHD whereas those T cells that elabo- major intracellular cytokines produced after rate type 2 cytokines (Th2 cells) inhibit GVHD. Pan et restimulation. In contrast, incubation of naïve T cells al43 demonstrated that splenocytes from mice mobilized with DC2 cells results in the polarization of these T with G-CSF were polarized toward the Th2 phenotype.
cells toward the Th2 phenotype as noted by the intra- Those mice who received splenocytes from G-CSF- cellular accumulation of IL-4 and IL-10 after mobilized donor mice demonstrated significantly longer restimulation in vitro with PMA and ionomycin. In con- survival and less GVHD that those allogeneic trans- clusion, G-CSF mobilization results in stem cell prod- plant recipient mice who were infused with splenocytes ucts with 10- to 50-fold more T cells and 4- to 6-fold from naïve unmobilized donor mice. T cells from G- more DC2 cells. The increased numbers of DC2 in G- CSF treated mice showed a significant increase in IL-4 mobilized products may reduce the relative risk of acute production with a simultaneous decrease in IL-2 and GVHD as seen in preclinical murine allogeneic trans- interferon-γ production. This polarization persisted in plant studies described above and observed in the ini- secondary mixed lymphocyte reactions (MLR) despite tial Phase 2 and 3 clinical trials in humans comparing the absence of G-CSF during in vitro MLR.
BM versus mobilized PBSCs as sources of allogeneic Arpinati et al hypothesized that G-CSF-mobilized stem cells. It is of interest that cord blood stem cell PBSC contained antigen-presenting cells which prime products, which are associated with a low risk of severe T cells to produce Th2 cytokines.44 Two distinct lin- acute GVHD, contain primarily DC2 and negligible eages of dendritic cells (DC) have been described in DC1.45 Consistent with this notion, Waller found a strong humans. DC1 cells or myeloid DCs express HLA-DR, inverse correlation with the number of precursor DC2 CD11c, CD13, and CD33 and require GM-CSF for their infused in allogeneic bone marrow and the incidence of survival. These cells are negative for both myeloid and both cGVHD and, more importantly, relapse.46 lymphoid specific markers (Lin–), produce high levelsof IL-12 when stimulated with tumor necrosis factor Alternative Allogeneic PBSC Mobilization Regimens
(TNF) or CD40 ligand and drive the differentiation of Although other cytokines, in addition to G-CSF, have naïve T cells into the Th1 phenotype. DC2 or lymphoid been used to mobilized autologous PBSCs from humans DC are HLA-DR+/CD11c–/CD4+/IL-3Ra+ express high including GM-CSF, Flt-3 ligand, stem cell factor (SCF), levels of T-cell receptor α chain and depend on IL-3, Daniplestim (IL-3 agonist), thrombopoietin agonists, and not GM-CSF, for their survival and differentiation.
chimeric cytokines including Leridistim (IL-3 agonist- After appropriate activation, they can induce T-cell dif- G-CSF chimeric molecule) and Progenipoietin-1 (Flt- ferentiation into Th2 cells. These investigators studied 3 ligand-G-CSF chimeric molecule), peg-filgrastim the effects of G-CSF mobilization (10-16 µg/kg/day for (NeulastaTM) and SDF-1 antagonist (AMD 3100), only 5 days) on DC content in the peripheral blood in these G-CSF and GM-CSF have been approved by the FDA allogeneic donors. G-CSF treatment was found to mo- and only G-CSF has been studied extensively for the bilize DC2 but not DC1. Although the numbers of do- mobilization of allogeneic PBSCs in humans.
nors and controls studied were very small, the median We have performed several sequential nonrandom- number of DC1 per liter in the G-CSF group was not ized trials to determine the comparative effects of allo- different (11 versus 10 × 106/liter; P = .52) than in the geneic PBSCs mobilized with G-CSF at 10 µg/kg/day control premobilization group. In contrast, the numbers (n = 96), G-CSF (10 µg/kg/day) combined with GM- of DC2 were significantly increased in the G-CSF mo- CSF at 5 µg/kg/day (n = 102) or GM-CSF alone (10 or bilization group compared with control (median 24.8 15 µg/kg/day (n = 32). A comparison of the various versus 4.9 × 106/liter; P = .0009). As expected, a pro- allogeneic PBSC mobilization regimens is shown in liferative response of naïve allogeneic T cells could be Table 2. All donors underwent leukapheresis (20 liter)
detected in vitro to fresh DC1 but not to fresh DC2.
on the fifth day of cytokine administration. The target Activation of DC2 in vitro with TNF, GM-CSF, and CD34+ content was 5.0 × 106 CD34/kg with a mini- IL-3 resulted in the rapid maturation of these precur- mum of 2 × 106/kg. The data shown in Table 2 are ex-
pressed as mean +/- SD. PBSCs mobilized with G/GM will need to be performed to more accurately dissect resulted in collection of grafts with similar CD34 con- the phenotypic differences in grafts mobilized by dif- tent compared with G alone. Grafts obtained following ferent cytokines/chemokines or combinations and to mobilization with GM alone contained significantly more accurately assess the impact on important end- fewer CD34+ cells, but sufficient numbers for rapid en- points such as multilineage engraftment, disease-free graftment. Grafts mobilized with GM or G/GM con- survival, overall survival, GVHD, and relapse.
tained significantly fewer T and NK cells. There were Chemokines such as IL-8 have been used to in- no obvious differences in donor toxicities including duce the egress of hematopoietic stem cells into the bone pain. All recipients of GM mobilized cells en- peripheral blood of mice and nonhuman primates. This grafted with kinetics similar to recipients of G and G/ effect is rapid (30 minutes-4 hours) and may result from GM, although neutrophil recovery was delayed about the ability of these chemokines to induce the release of 1 day. Rates of neutrophil and platelet recovery and a protease from mature myeloid cells resulting in a de- cGVHD for the G and GM groups are shown in Table
crease in the intramedullary concentration of SDF-1, 3. In 30 evaluable recipients of peripheral blood grafts
the ligand for the receptor CXCR4, which is expressed mobilized with GM alone, the actuarial risk of grades in many cells including hematopoietic stem cells.47,48 2-4 acute GVHD was only 0.13 ± 0.05 and 0.00 (0/31) Increasing evidence points to the critical role of the for grades 3-4 acute GVHD. In a multivariate analysis CXCR4/SDF-1 axis in both murine and human stem including patient and donor age, sex mismatching, con- cell mobilization. The bicyclam molecular AMD 3100 ditioning regimen received, CD3+ cell dose and CD34+ was first clinically developed for its potent and selec- cell dose, only the receipt of PBSCs mobilized with tive inhibition of HIV type 1 and 2 replication through GM-CSF alone correlated with a lower risk of grades binding to the chemokine receptor, CXCR4. Initial clini- 2-4 acute GVHD. These data suggest that altering the cal trials in AIDS patients demonstrated that AMD-3100 mobilization regimen and cytokines used may alter the induced a rapid (within 1 hour) increase in both WBC functional aspects of the graft thereby modifying out- and circulating progenitor cells. Broxmeyer and col- comes such as GVHD. Randomized Phase 3 studies leagues demonstrated a 40-fold increase in the mobili- Table 2. Comparison of G versus G/GM versus GM mobilization on allograft content.
G vs G/GM: < .0001; G vs GM: < .001 G vs G/GM: < .0001; G vs GM: < .001 G vs G/GM: < .001; G vs GM: < .0001 Table 3. Effect of cytokine mobilization regimen on graft-versus-host disease (GVHD)/survival.
G-CSF and
G vs GM: .003GM vs G/GM: < .0001G vs G/GM: .11 Abbreviations: G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophagecolony-stimulating factor; NS, not significant zation of hematopoietic progenitors within 1 hour of 12. Morrison SJ, Wandycz AM, Akashi K, Globerson A, AMD-3100 injection in mice.49,50 Studies in human vol- Weissman IL. The aging of hematopoietic stem cells. NatMed. 1996;2:1011-1016.
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consistent and rapid impact on stem cell mobilization 14. Ramshaw HS, Crittenden RB, Dooner M, Peters SO, Rao SS, Quesenberry PJ. High levels of engraftment with a single when given alone and a synergistic effect on CD34+ infusion of bone marrow cells into normal unprepared mice.
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Bundì e buin Carnevâl a duç oms, feminas e frus. Un an a l’è già pasât e come che i vi vevi prometût i soi tornât. Cun dut il cûr i vi àuguri un bon doimilesièt, ma cul gnûf governo no sin partis cul pit drèt. In veretât i stenti a crodi che a capo dal governo al seti inchiamò Romano Prodi. A lu àn sopranomenât mortadèla, ma mi par che al samei plui a una sanganèla: al meri


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