Abstract Engraftment failure is a rare but life-threatening complication of hematopoietic cell transplantation (HCT). Newer approaches to HCT, including use of haploidentical donors, umbilical cord blood (UCB) transplant, nonmyeloablative and reduced-intensity conditioning regimens, appear to have an increased risk of graft failure. Multipotent mesenchymal stromal cells (MSCs) are essential bone marrow components that have the potential to differentiate in vitro into tissues along mesenchymal lineages, including bone marrow stroma. This regenerative potential, coupled with the capability to secrete cytokines and growth factors, suggests that MSCs would facilitate and promote hematopoiesis. Moreover, MSCs have immu-noregulatory properties and thus could have an additional application in the setting of HCT by reducing both graft rejection and graft-versus-host disease. Initial trials have demonstrated the safety and feasibility of infusion of ex vivo-expanded autolo-gous and allogeneic MSCs. Results from these early trials suggested MSCs may enhance hematopoiesis when infused at the time of HCT; however, subsequent trials have not yet provided con fi rmation that MSCs accelerate hematopoietic recovery when given shortly after HCT. Ongoing research initiatives include use of MSC infusions for patients who have some evidence of regenerating marrow but have delayed or incomplete hematopoiesis.
P. F. Caimi , M.D. • H. M. Lazarus , M.D., F.A.C.P . (*) Department of Medicine , University Hospitals Case Medical Center, Case Comprehensive Cancer Center, Case Western Reserve University , Cleveland , OH 44106 , USA e-mail: [email protected]
Chapter 24 MSCs for Enhancement of Hematopoietic Progenitor Cell Engraftment and Poor Graft Function
Paolo F. Caimi and Hillard M. Lazarus
444 P.F. Caimi and H.M. Lazarus
Introduction
Hematopoietic cell transplantation (HCT) is a potentially curative therapy for a variety of benign and malignant hematologic diseases. In benign disorders, such as bone marrow failure syndromes, hemoglobinopathies, immunode fi ciencies, and enzyme de fi ciencies, infusion of the normal donor-derived hematopoietic progeni-tor cells (HPCs) can reconstitute normal hematopoiesis and provide immunocom-petent and metabolically intact progenitor cells. In malignant disorders, the autologous or allogeneic HPCs rescue patients from the myeloablative effects of escalated doses of chemotherapy; further, allogeneic immunocompetent cells may confer the added bene fi t of a “graft-versus-malignancy” (allogeneic) effect. The full clinical effectiveness of HCT is hampered by several barriers that relate both to the choice of the graft source as well as the type of conditioning regimen. In the autolo-gous HCT setting, delayed, poor, or absent engraftment may result from infusion of insuf fi cient numbers of HPCs. Umbilical cord blood (UCB) represents a valuable source of HPCs in patients who do not have available sibling-matched or matched unrelated donors, but slow engraftment of red cells, neutrophils, and platelets often is the norm. Other strategies include use of haploidentical donors (parents or chil-dren of the patient) that may be associated with an increased risk of engraftment failure [ 1 ] . In these situations, prolonged neutropenia carries a markedly increased risk of opportunistic infection. A prolonged time to restore production of red blood cells and platelets is associated with the dangers of increased alloimmunization, iron overload, and transmission of infections, in addition to the inconvenience of frequently receiving blood products and the associated signi fi cant fi nancial cost.
Myeloablative conditioning regimens are fraught with added danger when administered to frail or elderly patients and to those with signi fi cant comorbidities. Further, graft-versus-host disease (GVHD) with or without opportunistic infection remains a signi fi cant complication of the posttransplant state. Reduced-intensity conditioning (RIC) and nonmyeloablative conditioning have been used with increased frequency to facilitate allogeneic HCT in patients who cannot tolerate the signi fi cantly higher-dose myeloablative chemotherapy. These approaches rely pre-dominantly upon the donor effector cells in the graft to exert the therapeutic alloge-neic or immune effect. Graft failure, however, is an important complication of RIC and nonmyeloablative allogeneic HCT, with rates reported between 18 and 42%, depending on the regimen used [ 2 ] . Finally, in vitro T cell depletion of the graft, for a variety of reasons, may be associated with engraftment failure states [ 3 ] .
Multipotent Mesenchymal Stromal Cells (MSCs)
Multipotent mesenchymal stromal cells (MSCs) are essential components of the bone marrow microenvironment. These cells provide a supporting physical matrix and elaborate a variety of cytokines and other factors that have been shown in vitro and in vivo to support hematopoiesis [ 4 ] . As a result, over the last two decades,
44524 MSCs for Enhancement of Hematopoietic Progenitor Cell Engraftment…
MSCs have been the object of intensive research [ 5, 6 ] . These cells have the capacity to differentiate in vitro along mesenchymal lineages and give rise to multiple tis-sues, including bone, cartilage, adipose tissue, and bone marrow stroma; they also exhibit intense paracrine activity, secreting bioactive molecules with trophic and immunomodulatory capacity [ 7 ] . When infused, MSCs home to tissue sites of active in fl ammation [ 8 ] and participate in tissue repair [ 7 ] . The tissue regenerative poten-tial has prompted interest in using MSCs to provide stability and restore function in organs such as the heart and nervous system, using intravenous as well as intrathe-cally administered MSCs [ 9– 11 ] ; the immunomodulatory or immunosuppressive activity of MSCs has led to novel research initiatives in autoimmune conditions [ 12 ] and in the treatment of GVHD. The combination of these characteristics, e.g., the capacity to enhance regeneration of the hematopoietic process and the bone marrow stroma, the immunomodulatory properties [ 13 ] , and the secretion of bioactive agents [ 14 ] , has led to the study of the role of MSCs in facilitating HPC engraftment and the prevention and treatment of graft failure after HCT.
Biologic Properties of MSCs
First identi fi ed by Friedenstein and colleagues in postnatal bone marrow more than 40 years ago [ 5, 15 ] , MSCs have been isolated from a wide variety of adult organs, including periosteum, muscle connective tissue, perichondrium, and adipose tissue [ 16– 19 ] . These cells also are present in fetal bone marrow, lung, liver, and spleen [ 20, 21 ] . Amniotic fl uid and placenta are rich sources of MSCs, the latter containing both maternal and fetal MSCs [ 22, 23 ] . Their isolation from UCB has been reported with variable success [ 24 ] . The proportion of MSCs in bone marrow is small and varies among species, representing 0.001–0.01% of all nucleated marrow cells. Moreover, their number decreases with age, ranging from 1 MSC per 10,000 nucle-ated marrow cells in newborns to 1 MSC per 250,000 nucleated marrow cells in adults [ 25 ] . This paucity of MSCs in bone marrow and other tissues, along with the lack of speci fi c markers, has made study of directly isolated cells dif fi cult, and thus, little is known about the primary progenitor cell in vivo. On the other hand, MSCs have a remarkable capacity to undergo expansion in ex vivo culture settings, which has led to two important developments. First, most of the information regarding MSC function and phenotype is based on cells expanded in vitro. Secondly, as a small bone marrow sample can be expanded to yield hundreds of millions of cells, clinical use of culture-expanded MSCs has been made possible, and thus, clinical studies have been performed before a reliable preclinical animal model was readily available [ 26 ] .
The wide variety of tissues from which MSCs can be harvested and the multiple available methods for isolation and expansion of cells prompted the International Society for Cellular Therapy (ISCT) to de fi ne minimal criteria for the de fi nition of MSCs [ 6 ] . These criteria include the adherence to plastic under standard culture conditions; a speci fi c phenotype ( ³ 95% of the cells expressing CD105, CD73, and
446 P.F. Caimi and H.M. Lazarus
CD90 and £ 2% of cells expressing CD45, CD34, CD14, CD11b, CD79a, or CD19 or HLA Class II); and the capacity to differentiate into osteoblasts, chondrocytes, or adipocytes under standard in vitro differentiating conditions. The population of plastic-adherent cells, however, is heterogeneous and only a small proportion of these cells can generate fi broblast colonies in vitro [ 27 ] . In 2005 the ISCT clari fi ed the nomenclature for “mesenchymal stem cells,” a term popularized in early 1990s by Caplan [ 25 ] , to the current “multipotent mesenchymal stromal cells.” This des-ignation conveys the multipotentiality and tissue-regenerating capacity without ascribing to them the homogeneous quality of stem cells [ 25 ] .
The range and mechanisms of the immunomodulatory properties of MSCs have not yet been fully elucidated. MSCs have been shown to suppress T cell proliferation and cytokine production in response to alloantigens and nonspeci fi c mitogens [ 28, 29 ] . These cells appear to exert their in fl uence both by soluble fac-tors (IFN-gamma and nitric oxide appear to have a central role [ 30, 31 ] ) as well as by direct cell-cell interactions [ 32 ] . MSCs also inhibit the proliferation, differen-tiation, and cytokine secretion of dendritic cells [ 33– 35 ] and natural killer (NK) cells leading to decreased cell-mediated cytotoxic activity [ 36 ] . B cell prolifera-tion, chemotaxis, antibody production, and terminal differentiation also are sup-pressed by MSCs [ 37– 39 ] .
In addition to their immunomodulatory properties, MSCs appear to be immuno-logically privileged cells [ 40 ] . Culture-expanded human MSCs express major histo-compatibility complex (MHC) class I proteins, are negative for MHC class II proteins, and appear to lack expression of the costimulatory molecules CD80, CD86, and CD40 [ 41– 44 ] . MHC class I expression and upregulation in the presence of increased interferon-gamma make MSCs less susceptible to NK cell lysis. MSCs can suppress the proliferation of CD4+ and CD8+ T lymphocytes [ 32 ] while selec-tively promoting proliferation of CD4+ CD25+ T regulatory lymphocytes [ 45 ] . When cocultured with allogeneic lymphocytes, MSCs did not induce lymphocyte proliferation [ 46 ] ; and in animal models, infusion of allogeneic mismatched MSCs did not induce an immune response [ 8 ] . Moreover, the immunosuppressive effect of MSCs appears to be independent from MHC compatibility status [ 29 ] . Such data provide a scienti fi c basis for undertaking investigations of infusing MSCs obtained from unrelated or mismatched donors for immunomodulation and promotion of engraftment in the setting of HCT.
Clinical Studies
Early Studies
As a result of differences in the immunoregulatory properties of MSCs between species, no adequate preclinical animal models are available to predict the in vivo function of MSCs in the HCT setting [ 47 ] . The establishment of human MSC expan-sion methods, however, has facilitated their clinical use. The consequence has been
44724 MSCs for Enhancement of Hematopoietic Progenitor Cell Engraftment…
a unique situation in which clinical trials of MSC infusion were initiated before complete understanding of the in vivo properties of these cells [ 48 ] .
In 1995 Lazarus and colleagues [ 49 ] published the fi rst clinical trial using MSCs. This pilot study examined collection, ex vivo expansion, and reinfusion of autolo-gous bone marrow-derived MSCs obtained from 23 hematologic malignancy patients. The procedure, demonstrated to be safe and feasible, was followed by a phase I–II trial of autologous, culture-expanded bone marrow-derived MSC infu-sion after high-dose chemotherapy and autologous peripheral blood progenitor cell transplantation in 28 breast cancer patients [ 50 ] . No toxic effects directly attribut-able to MSC infusion were observed and hematologic recovery was rapid. Blood neutrophil engraftment (neutrophils > 500/ m L) occurred at a median of 8 days and untransfused platelet count exceeded 20,000/ m L in a median of 8.5 days. Unfortunately, no fi rm conclusions regarding the bene fi t in hematopoietic engraft-ment could be drawn due to the nonrandomized nature of this trial.
Frassoni and coworkers [ 51 ] retrospectively examined a matched-pair analysis of 31 hematologic malignancy patients undergoing HLA-identical, sibling-matched HCT procedures at the US and European centers. Patients received culture-expanded bone marrow-derived MSCs harvested from the HLA-identical HPC donors. Compared with historic controls, study patients had a statistically signi fi cant lower incidence of acute and chronic GVHD and superior survival rates after 6 months of follow-up [ 51 ] . No observations were reported regarding engraftment rates. These initial trials spearheaded the use of human MSCs, demonstrating the safety and feasibility of their use, both in the autologous and HLA-identical allogeneic setting, and suggested these cells may have therapeutic potential in the HCT setting, as engraftment enhancers or prophylaxis of GVHD.
Infusion of MSCs for Enhancement of Hematopoietic Engraftment
Recently, several trials have been undertaken to evaluate the coadministration of bone marrow-derived, culture-expanded MSCs along with varied sources of HPCs during allogeneic HCT for the purpose of correcting or preventing engraftment failure. The Karolinska University clinical group [ 52 ] reported seven patients who underwent cotransplantation of HPCs and MSCs. Three patients were treated for a previous graft failure or rejection. The remaining four were part of a pilot study designed to enhance hematopoietic engraftment in which haploidentical MSCs were given. Despite remarkable variability in the patient population, source of HPCs and MSCs, and HLA compatibility status of donors, all patients had hematopoietic engraftment, with median time to neutrophil engraftment (ANC > 0.5 × 10 9 /L) of 12 days (range 10–28 days) and median time to platelet engraftment (platelets > 30 × 10 9 /L) of 12 days (range 8–36 days). While these are encouraging results, the patient popula-tion, underlying diagnoses and transplant settings were markedly heterogeneous, making dif fi cult a generalization of the fi ndings on the effects of MSCs.
448 P.F. Caimi and H.M. Lazarus
Ball and colleagues [ 53 ] cotransplanted MSCs and HPCs obtained from haploidentical related donors into 14 children who had hematologic malignancies, immune de fi ciencies, and nonmalignant disorders. HPC engraftment was demon-strated in all patients. Mean MSC dose was 1.6 × 10 6 MSC/kg (range 1–3.3 × 10 6 MSC/kg). When compared to historic controls ( n = 47), the 14 study patients had comparable platelet and neutrophil recoveries yet faster attainments of a total blood leukocyte count > 1 × 10 9 /L. Acute and chronic GVHD rates were comparable with historic controls.
Macmillan and colleagues [ 54 ] infused ex vivo-expanded MSCs in conjunction with an UCB transplant. Fifteen children with hematologic malignancies received UCB grafts; 8 subjects also received parental haploidentical MSCs infusions on the day of UCB transplant, while 3 received repeat MSC infusions 21 days later. A second MSC infusion could not be given to the remaining fi ve patients secondary to insuf fi cient growth of parental MSCs. The median MSC dose on day 0 was 2.1 × 10 6 MSC/kg (range 0.9–5 × 10 6 MSC/kg). Haploidentical MSC infusions at the time of UCB transplant were shown to be safe, but tempo to recovery of neutrophil and platelet engraftment as well as the rates of acute GVHD were similar to those of historic controls.
A recent multicenter European trial explored the coinfusion of culture-expanded parental MSCs in 13 children who received UCB grafts obtained from related or unrelated donors [ 55 ] . Median MSC dose was 1.9 × 10 6 MSC/kg (range 1–3.9 × 10 6 MSC/kg). The incidence of graft failure or the rate of neutro-phil and platelet engraftment did not differ statistically from that observed in 39 historic controls matched for the diagnosis and type of UCB donor. Overall sur-vival rates also did not differ. On the other hand, there were no cases of severe acute GVHD (grades III–IV) in patients who received MSC coinfusion, com-pared to 10 (grade III, 7 cases and grade IV, 3 cases) of 39 patients in the historic controls. The authors postulated that in the UCB transplant setting, graft failure is more a function of the low numbers of infused HPCs rather than an immune-mediated mechanism; hence, MSC coinfusion may not be justi fi ed in the UCB transplant setting. However, MSCs may enhance hematopoietic engraftment via additional, non-immune- mediated mechanisms, such as cytokine release and reconstitution of the bone marrow stroma, effects that may be of particular importance in cases with borderline hematopoiesis.
Gonzalo-Daganzo et al. reported [ 56 ] a phase I–II study in which nine hemato-logic malignancy patients received third-party MSCs 1–24 h after coinfusion of UCB grafts and third-party HPCs (UCB/HPC). The median (range) MSC dose was 1.18 (1.04–2.22) × 10 6 MSC/kg. No adverse effects of MSC infusion were observed. Hematopoietic engraftment and achievement of full UCB chimerism appeared to be delayed in the study group when compared with 46 controls that received UCB/HPC coinfusion alone, although the differences were not statistically signi fi cant. The incidence of acute GVHD also did not differ statistically from that observed in control patients. Two patients who received additional subsequent MSC infusions for treatment of corticosteroid-refractory GVHD attained complete responses. This study showed that MSC infusion at the time of cotransplantation of UCB/HPC is
44924 MSCs for Enhancement of Hematopoietic Progenitor Cell Engraftment…
safe and well tolerated, but the small number of patients included precludes further conclusions from being drawn regarding the effect of MSCs on engraftment and GVHD incidence.
Finally, Baron and colleagues [ 57 ] reported a recent study using cotransplan-tation of MSCs and HLA-mismatched MSCs after nonmyeloablative condition-ing. Twenty hematologic malignancy patients were compared to 16 historic controls who also received nonmyeloablative conditioning and HLA-mismatched HCT. MSC cotransplantation was associated with a decrease in 1-year nonre-lapse mortality (HR = 0.2, 95% CI 0.04–0.9, p = 0.03) and 1-year overall mortal-ity (HR = 0.4, 95% CI 0.1–0.9, p = 0.03). Severe, acute GVHD was low in the cotransplantation group, without an increased relapse risk, suggesting that GVHD was ameliorated without abrogating the graft-versus-tumor effect. Engraftment was prompt in both groups but there was no discernible enhance-ment of engraftment with MSC coinfusion; one patient receiving MSCs experi-enced primary graft failure. This study shows that MSC cotransplantation is feasible in the setting of nonmyeloablative HLA-mismatched HCT, where the risk of GVHD and graft rejection is higher, but no clear engraftment bene fi t was reported.
In summary, studies of MSC infusion at the time of HCT aimed at enhancement of engraftment, and prevention of HPC graft failure have not yielded conclusive results. While safety of MSC infusion largely has been demonstrated, the studies assessing the effect of MSC infusion on engraftment have been limited to small, nonrandomized studies that utilize historic controls. Results have been variable and several studies have failed to demonstrate engraftment enhancement with MSCs. Larger, randomized studies are needed to settle whether the use of these cells is justi fi ed.
Infusion of MSCs for Treatment of Graft Failure
Primary graft failure after HCT represents a rare but serious, life-threatening com-plication of the HCT procedure. Management strategies include use of recombinant hematopoietic growth factors, modi fi cations of immunosuppressant regimens, infu-sion of “backup” autologous HPCs, or second allogeneic HCT. Outcomes remain dismal, even after second allogeneic HCT, as evidenced by a recent observational study from the Center for International Blood and Marrow Transplant Research (CIBMTR) [ 58 ] . Such patients with prolonged bone marrow failure are at high risk of succumbing to infection and hemorrhage.
In addition to development of strategies for early identi fi cation of those patients at high risk of engraftment failure, new therapeutic alternatives are needed for this complication. MSCs may prove of value in this setting, with their bone marrow stromal regenerative potential and the additional capacity to ameliorate graft rejec-tion without signi fi cantly increasing risk of infection.
450 P.F. Caimi and H.M. Lazarus
Fouillard and colleagues [ 59 ] reported a case of a 40-year-old woman with AML in complete remission who exhibited primary graft failure after autologous HCT. Partial recovery of her counts was achieved with administration of granulocyte colony-stimulating factor and erythropoietin therapy administered three times a week. Three years after HCT she received culture-expanded MSCs harvested from her HLA-mismatched brother. MSC dose was 2.78 × 10 6 /kg. Rapid and sustained recovery of her neutrophil and platelet counts was observed, while no effect on hemoglobin concentration was observed. Granulocyte-macrophage colony-forming units (CFU-GM) and colony-forming unit fi broblasts (CFU-F) were increased 1 month and 1 year after MSC infusion. There were no side effects or adverse reac-tions to the MSC infusion and no GVHD was observed. Studies of MSC engraft-ment in the recipient 1 month post MSC infusion showed male DNA was detected at a frequency of 10 −5 per cell, whereas 1 year after infusion, it was no longer detect-able, results that are compatible with very low levels of MSC engraftment observed in animal studies. This report suggests that MSCs can potentially be used for treat-ment of engraftment failure, although the mechanism mediating their bene fi t is not yet elucidated.
Fang and colleagues [ 60 ] reported two pediatric patients with severe aplastic anemia (SAA) who presented with graft failure after receiving HLA-identical sibling peripheral blood HCT. The fi rst case, an 11-year-old girl, had relapse of SAA after her second HLA-identical sibling HCT. Haploidentical culture-expanded adipose tissue MSCs harvested from the patient’s mother were infused after a third infusion of HPCs from her HLA-identical sister. Neutrophil count reached 0.6 × 10 9 /L by 16 days after HCT, while the platelet count apparently recovered by 20 days, although the details were not provided by the authors. No acute or chronic GVHD was observed. The second case was a 12-year-old boy with SAA in relapse after HLA-identical sibling HCT. A second HCT from the same donor was followed by infusion of culture-expanded adipose tissue MSCs harvested from his mother. Neutrophil recovery occurred by 15 days while self-sustaining platelet count was achieved by 19 days. Two months after transplant the patient experienced “grade alpha” (apparently grade I) acute GVHD of the skin that completely responded to corticosteroid therapy. Both patients had sus-tained hematopoietic function beyond 2 years at the time of publication. Several reports have documented the safety and feasibility of infusion of culture-expanded MSCs harvested from adipose tissue [ 61, 62 ] . These reports suggest that coinfusion with HPCs may improve the rates of graft failure. Although referred to as equivalent to marrow-derived MSCs, experience with this source of cells is still limited and it is possible that there are subtle biologic and immu-nologic differences not yet identi fi ed. These positive results warrant proceeding with larger studies of MSC infusion and coinfusion with HPCs for treatment of engraftment failure.
Meuleman and coauthors [ 63 ] recently reported the results of a pilot clinical trial of infusion of culture-expanded MSCs without coinfusion of HPCs. Patients included had received HCT and achieved full donor chimerism but with poor engraftment. The authors de fi ned the latter as persistent posttransplant
45124 MSCs for Enhancement of Hematopoietic Progenitor Cell Engraftment…
pancytopenia, with blood neutrophils < 1 × 10 9 /L and platelets < 50 × 10 9 /L at 30 days after HCT and despite treatment with a minimum of 10 days of granulo-cyte colony-stimulating factor. Patients with complete engraftment failure or rejection were excluded from this study. Six patients were included, all received myeloablative, allogeneic, mobilized peripheral blood HCT; three patients received HLA-identical related donor and three haploidentical related donor hematopoietic grafts. Although full donor chimerism was observed in all cases, bone marrow examination showed hypoplasia and all patients had varying degrees of pancytopenia. MSCs were obtained from bone marrow aspiration of the origi-nal HPC donors. The MSC dose was 1 × 10 6 MSC/kg. No acute side effects to MSC infusion were observed. Two patients manifested hematologic recovery after MSC infusion; both had received HLA-identical sibling HCTs and were in fi rst complete remission, in comparison to the more heavily pretreated other sub-jects. One patient presented early CMV infection (day 12) and subsequently died several months later from repeat CMV infection. The relationship between the viral infection and MSC infusion is unclear, as MSCs have not been shown to affect virus-speci fi c T cell function [ 64, 65 ] . This study demonstrates the feasibil-ity of MSC infusion without HPCs and suggests MSCs may aid in hematopoietic recovery in those patients who have residual hematopoiesis, likely through a com-bination of stromal reconstruction, cytokines, and modulation of rejection. Therefore, heavily pretreated patients or those with profound pancytopenia may bene fi t from larger doses of MSCs and possibly with coinfusion with HPCs as other investigators [ 52 ] have observed.
Conclusions
Although MSCs have been the subject of intensive laboratory and clinical research over the last 15 years, their in vivo properties and effects after administration in the clinical setting of HCT have not yet been fully established. Their biologic proper-ties, including constitutive secretion of bioactive molecules, capacity to differenti-ate into bone marrow stroma, and remarkable immunomodulatory capacity, have suggested that these cells may have a role to facilitate hematopoietic engraftment and prevent development of GVHD.
Small pilot studies demonstrated the safety of infusion of HLA-compatible MSCs and suggested a possible bene fi t in hematopoietic engraftment rates. Subsequent studies have demonstrated the feasibility of HLA-haploidentical and HLA-unmatched MSCs in a variety of settings, including coinfusion with HLA-identical and haploidentical peripheral blood HPCs as well as with UCB grafts. These studies, however, have not conclusively demonstrated that MSCs provide an advantage in terms of hematopoietic engraftment rates. Larger, randomized studies have been performed to evaluate the effect of MSCs for treatment and prophylaxis of GVHD. In the next few years, we anticipate the design and conduct of trials to evaluate the effect of MSC infusion on hematopoietic engraftment.
452 P.F. Caimi and H.M. Lazarus
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