Challenge to Predict Mobilized Peripheral Blood Stem Cells on the Fourth Day of Granulocyte Colony-Stimulating Factor Treatment in Healthy Donors: Predictive Value of Basal CD34+ Cell and Platelet Counts

A longitudinal, prospective, observational, single-center cohort study on healthy donors was designed to identify predictors of CD34⁺ cell mobilization on day 4 after granulocyte colony-stimulating factor (G-CSF) administration. As potential predictors of mobilization, age, sex, body weight, height, blood volume, WBC count, peripheral blood (PB) mononuclear cell count, platelet (Plt) count, and hematocrit and hemoglobin levels were considered. Two different evaluations of CD34⁺ cell counts were determined for each donor: baseline (before G-CSF administration) and in PB on day 4 after G-CSF administration. One hundred twenty-two consecutive healthy donors with a median age of 47.5 years were enrolled. The median value of CD34⁺ on day 4 was 43 cells/µL (interquartile range, 23 to 68), and 81.1% of donors had ≥20 cells/µL. Basal WBC count, Plt count, and CD34⁺ were significantly higher for the subjects with CD34⁺ levels over median values on day 4. A multivariate quartile regression analysis, adjusted by sex, age, basal CD34⁺, and basal Plt count, showed a progressively stronger relationship between baseline CD34⁺ and Plt levels and the CD34⁺ levels on day 4. The basal CD34⁺ cut-off level to predict the levels of CD34⁺ on day 4 was either ≤2 cells/μL or ≥3 cells/μL and that of basal Plt count was ≤229 × 10⁹/L or ≥230 × 10⁹/L, respectively, to determine whether mobilization therapy should or should not be attempted. PB stem cell mobilization with G-CSF was highly effective on day 4, and herein we describe a model for predicting the probability of performing PB stem cell collection after a short course of G-CSF.     

Plerixafor Plus Granulocyte Colony-Stimulating Factor versus Placebo Plus Granulocyte Colony-Stimulating Factor for Mobilization of CD34(+) Hematopoietic Stem Cells in Patients with Multiple Myeloma and Low Peripheral Blood CD34(+) Cell Count: Results of a Subset Analysis of a Randomized Trial

Preapheresis peripheral blood (PB) CD34(+) cell count is a strong predictor of hematopoietic stem cell (HSC) mobilization and is routinely used to optimize the timing, cost, and success of HSC collection in patients with multiple myeloma. However, a uniform PB CD34(+) cell count that predicts mobilization failure has not been defined, resulting in the development of institute-specific algorithms for mobilization, particularly regarding the decision of when to use the novel stem cell mobilization agent plerixafor. In this post hoc analysis, we evaluated the mobilization efficacy of plerixafor plus granulocyte colony-stimulating factor (G-CSF) versus placebo plus G-CSF in patients with multiple myeloma, stratified by preapheresis PB CD34(+) cell count: <10, <15, <20, and ≥20 cells/μL. Regardless of the PB CD34(+) cell count, the total yield of CD34(+) cells from apheresis was significantly higher in the plerixafor group than in the placebo group, and significantly more patients in the plerixafor group collected the minimum (≥2?×?10(6) cells/kg) and optimum (≥6?×?10(6) cells/kg) stem cell yields on each day of apheresis. As a corollary, the greater stem cell collection in plerixafor-treated patients resulted in the need for significantly fewer days of apheresis to reach minimum and optimum cell doses across all cell count groups. For all CD34(+) cell count groups, the proportion of patients proceeding to transplantation and the median time to platelet and neutrophil engraftment were similar in the plerixafor and placebo groups. Our findings demonstrate that in patients with multiple myeloma who might be predicted to fail mobilization based on low PB CD34(+) cell count, the addition of plerixafor to G-CSF allows for collection of the minimal and optimal cell doses in a greater proportion of patients compared with G-CSF alone. In addition, plerixafor plus G-CSF significantly improves the likelihood of optimal HSC collection in patients with higher preapheresis PB CD34(+) cell counts (≥20 cells/μL) compared with placebo plus G-CSF. Collectively, this analysis of predicted poor mobilizers validates the superiority of plerixafor plus G-CSF compared with G-CSF alone, which had been demonstrated previously in the overall patient population.     

Induction of Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) and Granulocyte Colony-Stimulating Factor (G-CSF) Expression in Bone Marrow and Fractionated Marrow Cell Populations by Interleukin 3 (IL-3): IL-3-Mediated Positive Feedback Mechanisms of Granulopoiesis

Abstract

Interleukin 3 (IL-3) induces proliferation and differentiation of mast cell progenitors in vitro, whereas it induces granulocytosis in vivo. In this paper, a positive feedback mechanism of granulopoiesis was studied in order to elucidate the granulocytosis induced by IL-3 in mouse. IL-3 induced expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) in total bone marrow cells and a marrow adherent cell population. In fractionated marrow cell populations, a different expression pattern of induction by IL-3 stimulation was observed; GM-CSF was expressed in macrophages and fraction 1 (P<1.061) and 2(1.061<P<1.074) of bone marrow cells fractionated by equilibrium density centrifugation, G-CSF was expressed in macrophages and fraction 2 and 3 (1.074<P<1.097), and interleukin 6 (IL-6) in macrophages and fraction 1 to 3. These results indicate a hierarchical regulation of cytokine production and the existence of a positive feedback mechanism in granulopoiesis. IL-6, induced by IL-3, stimulates stem cells into cycle and induces stem cells to respond to IL-3. The stem cells differentiate to granulocyte-macrophage colony-forming cells by the combined effect of IL-3 and IL-6. IL-3 also induces GM- and G-CSF expression which in turn makes granulocyte-macrophage colony-forming cells differentiate to granulocytes. These factors organize a cytokine network in granulopoiesis.     

Plerixafor Plus Granulocyte Colony-Stimulating Factor Improves the Mobilization of Hematopoietic Stem Cells in Patients with Non-Hodgkin Lymphoma and Low Circulating Peripheral Blood CD34+ Cells

Many institutions have adopted algorithms based on preapheresis circulating CD34+ cell counts to optimize the use of plerixafor. However, a circulating peripheral blood CD34+ cell threshold that predicts mobilization failure has not been defined. The superiority of plerixafor + granulocyte colony-stimulating factor (G-CSF) over placebo + G-CSF for hematopoietic stem cell mobilization and collection was shown for patients with non-Hodgkin lymphoma in a phase III, prospective, randomized, controlled study. The question remains as to which patients may benefit most from the use of plerixafor. In this post hoc retrospective analysis, mobilization outcomes were compared between the 2 treatment arms in patients stratified by peripheral blood CD34+ cell count (<5, 5 to 9, 10 to 14, 15 to 19, or ≥20 cells/μL) obtained before study treatment and apheresis. Compared with placebo plus G-CSF, plerixafor plus G-CSF significantly increased the peripheral blood CD34+ cells/μL over prior day levels in all 5 stratified groups. The probability of subsequent transplantation without a rescue mobilization was far greater in the plerixafor-treated patients for the lowest initial (day 4) peripheral blood CD34+ cells/μL groups (<5, 5 to 9, or 10 to 14). Engraftment and durability were the same for the 2 treatment groups for all strata, but the effect in the lower strata could be altered by the addition of cells from rescue mobilizations. These findings may provide insight into the optimal use of plerixafor in all patients undergoing stem cell mobilization.     

RasGRP1 Transgenic Mice Develop Cutaneous Squamous Cell Carcinomas in Response to Skin Wounding : Potential Role of Granulocyte Colony-Stimulating Factor

Models of epidermal carcinogenesis have demonstrated that Ras is a critical molecule involved in tumor initiation and progression. Previously, we have shown that RasGRP1 increases the susceptibility of mice to skin tumorigenesis when overexpressed in the epidermis by a transgenic approach, related to its ability to activate Ras. Moreover, RasGRP1 transgenic mice develop spontaneous papillomas and cutaneous squamous cell carcinomas, some of which appear to originate in sites of injury, suggesting that RasGRP1 may be responding to signals generated during the wound-healing process. In this study, we examined the response of the RasGRP1 transgenic animals to full-thickness incision wounding of the skin, and demonstrated that they respond by developing tumors along the wounded site. The tumors did not present mutations in the H-ras gene, but Rasgrp1 transgene dosage correlated with tumor susceptibility and size. Analysis of serum cytokines showed increased levels of granulocyte colony-stimulating factor in transgenic animals after wounding. Furthermore, in vitro experiments with primary keratinocytes showed that granulocyte colony-stimulating factor stimulated Ras activation, although RasGRP1 was dispensable for this effect. Since granulocyte colony-stimulating factor has been recently associated with proliferation of skin cancer cells, our results may help in the elucidation of pathways that activate Ras in the epidermis during tumorigenesis in the absence of oncogenic ras mutations.The role of Ras activation in non-melanoma skin cancer is well-documented, both from analysis of human squamous cell carcinomas (SCC)¹ as well as from studies using mouse models of skin carcinogenesis.^2,3 In particular, the multistage carcinogenesis protocol on mouse skin has identified activating mutations in the ras proto-oncogene as the initiation event in skin neoplasms.⁴ Interestingly, whereas Ras mutations are prevalent in mouse models of skin carcinogenesis, they have only been identified in 12% to 46% of sporadic human SCC samples, despite the fact that Ras is activated in the majority of the human SCC.^1,5 This suggests that other mechanisms of Ras activation play a role in the human disease. For example, epidermal growth factor receptor overexpression by amplification is known to occur in human SCC,^6,7 and epidermal growth factor receptor could lead to the biochemical stimulation of Ras. In animal models, overexpression of epidermal growth factor receptor ligands like transforming growth factor-α,⁸ or a dominant form of the Ras exchange factor Sos of seven less (SOS),⁹ act as an initiation event in the epidermis, further demonstrating that alterations in Ras upstream signals could lead to tumorigenesis in the skin via wild-type Ras activation.Biochemical activation of Ras in keratinocytes can be triggered by various extracellular stimuli, but in all cases it requires the participation of exchange factors that catalyze the GDP–GTP exchange. The best studied GDP–GTP exchange factor is SOS1, which is activated in response to receptor tyrosine kinase activation.^10,11 In recent years, we have identified a new GDP–GTP exchange factor in epidermal keratinocytes, RasGRP1, and demonstrated that it can transduce the activation of Ras by the diacylglycerol analog and potent skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA).¹² The initial studies prompted us to investigate the role of RasGRP1 in the tumor promotion effects of phorbol esters in the multistage carcinogenesis protocol using a transgenic mouse model for overexpression of RasGRP1 under the keratin 5 promoter (K5.RasGRP1). Surprisingly, RasGRP1 overexpression did not increase the susceptibility to TPA in animals initiated by the carcinogen 7, 12-Dimethylbenz[a]anthracene (DMBA), but caused more malignant tumors than those observed in the wild-type mice, suggesting a role of RasGRP1 in tumor progression.¹³ This effect appeared strongly related to increases in active Ras, as RasGRP1 overexpression clearly leads to elevated Ras^GTP levels in mouse epidermal keratinocytes, both under basal as well as TPA stimulated conditions.¹³ Interestingly, the K5.RasGRP1 mice also produced tumors in response to TPA alone, implying that RasGRP1 overexpression can act as an initiation event. In addition to the response to chemical carcinogens and tumor promoters, we observed that the RasGRP1 transgenic colony developed spontaneous skin tumors over time, more frequently seen in animals housed in groups or with previous episodes of skin abrasion or injury.¹⁴ This response resembled that of the Tg.Ac mice, which express v-H-ras under the ζ-globin promoter and generate tumors in response to tumor promoting stimuli like wounding and phorbol esters in absence of carcinogenic/initiation events.^15,16The in vivo studies with the K5.RasGRP1 mice have implicated RasGRP1 as a novel link for Ras activation in epidermal keratinocytes and skin cancer. However, the signaling mechanisms contributing to RasGRP1 activation and spontaneous tumor formation, as well as the direct evidence for a role of skin wounding as a tumor promoter stimulus in the context of RasGRP1 overexpression, remained to be established. To address these questions, we have now characterized the tumorigenic response of the K5.RasGRP1 to full-thickness incision wounding of the skin. Our findings show that RasGRP1 overexpression in the epidermis conferred sensitivity to wounding-induced promotional stimuli. Furthermore, upon wounding, there was a significant increase in the levels of granulocyte colony-stimulating factor (G-CSF) in the circulation of transgenic mice compared with the wild-type animals. In vitro, G-CSF was able to stimulate Ras activation in keratinocytes in a very rapid fashion, although RasGRP1 appeared dispensable for this effect. Since G-CSF has been associated to the growth and progression of skin carcinoma cells, the data presented here may have implications for the understanding of RasGRP1-Ras signaling in skin tumor biology.     

Platelet Engraftment in Patients with Hematologic Malignancies following Unmanipulated Haploidentical Blood and Marrow Transplantation: Effects of CD34+ Cell Dose and Disease Status

Unmanipulated haploidentical blood and marrow transplantation has been developed as an alternative transplantation strategy for patients without an HLA-matched related or unrelated donor. In this transplantation setting, factors associated with hematopoietic recovery have not been defined completely. The aim of this study was to investigate the effects of donor and recipient characteristics on neutrophil and platelet engraftment after unmanipulated HSCT. The study group comprised 348 patients who underwent unmanipulated haploidentical blood and marrow transplantation to treat hematologic malignancy at a single institution between 2002 and 2007. Factors correlating with neutrophil and platelet engraftment posttransplantation were analyzed retrospectively. All patients achieved an absolute neutrophil count ANC of 500/μL in a median of 13 days (range, 9 to 49 days). Of the 348 patients, 331 (95.11%) achieved an untransfused platelet count of > 20,000/μL in a median of 16 days (range, 7 to 356 days). Multivariate analysis showed that the amount of CD34⁺ cells infused (CD34⁺ cells ≥ 2.19 × 10⁶/kg recipient weight vs < 2.19 ×10⁶/kg recipient weight; hazard ratio [HR] = 1.695; 95% confidence interval [CI] = 1.361 to 2.112; P < .0001), and disease stage (advanced vs early; HR = 0.724; 95% CI = 0.577 to 0.907; P = .005) were independently associated with increased risk of platelet engraftment. Our results suggest that low numbers of CD34⁺ cells in allografts and advanced-stage disease may be critical factors associated with delayed platelet engraftment after unmanipulated haploidentical transplantation.     

Granulocyte Colony-Stimulating Factor Use after Autologous Peripheral Blood Stem Cell Transplantation: Comparison of Two Practices

Administration of granulocyte colony-stimulating factor (G-CSF) after autologous peripheral blood stem cell transplantation (PBSCT) is generally recommended to reduce the duration of severe neutropenia; however, data regarding the optimal timing of G-CSFs post-transplantation are limited and conflicting. This retrospective study was performed at NewYork-Presbyterian/Weill Cornell Medical Center between November 5, 2013, and August 9, 2016, of adult inpatient autologous PBSCT recipients who received G-CSF empirically starting on day +5 (early) versus on those who received G-CSF on day +12 only if absolute neutrophil count (ANC) was <0.5?×?10⁹/L (ANC-driven). G-CSF was dosed at 300?µg in patients weighing <75?kg and 480?µg in those weighing ≥75?kg. One hundred consecutive patients underwent autologous PBSCT using either the early (n?=?50) or ANC-driven (n?=?50) G-CSF regimen. Patient and transplantation characteristics were comparable in the 2 groups. In the ANC-driven group, 24% (n?=?12) received G-CSF on day +12 and 60% (n?=?30) started G-CSF earlier due to febrile neutropenia or at the physician's discretion, 6% (n?=?3) started after day +12 at the physician's discretion, and 10% (n?=?5) did not receive any G-CSF. The median start day of G-CSF therapy was day +10 in the ANC-driven group versus day +5 in the early group (P?<?.0001). For the primary outcome, the median time to neutrophil engraftment was 12 days (interquartile range [IQR] 11-13 days) in the early group versus 13 days (IQR, 12-14 days) in the ANC-driven group (P?=?.07). There were no significant between-group differences in time to platelet engraftment, 1-year relapse rate, or 1-year overall survival. The incidence of febrile neutropenia was 74% in the early group versus 90% in the ANC-driven group (P?=?.04); however, there was no significant between-group difference in the incidence of positive bacterial cultures or transfer to the intensive care unit. The duration of G-CSF administration until neutrophil engraftment was 6 days in the early group versus 3 days in the ANC-driven group (P?<?.0001). The median duration of post-transplantation hospitalization was 15 days (IQR, 14-19 days) in the early group versus 16 days (IQR, 15-22 days) in the ANC-driven group (P?=?.28). Our data show that early initiation of G-CSF (on day +5) and ANC-driven initiation of G-CSF following autologous PBSCT were associated with a similar time to neutrophil engraftment, length of stay post-transplantation, and 1-year overall survival.     

The Role of the Different CD34 Epitopes in Detection and Positive Selection of CD34+ Bone Marrow and Peripheral Blood Stem Cells

Positive selection of CD34⁺ cells is an attractive approach to reduce tumour cell contamination in bone marrow (BM) and peripheral blood progenitor cell (PBPC) autografts in malignancies not expressing CD34. All current selection methods use monoclonal antibodies (MoAbs) specific for the class I or class II CD34 epitopes, while for detection most investigators use class III MoAbs. Since the distribution of the different CD34 epitopes on haematopoietic progenitors differs, we studied their significance in CD34⁺ selection procedures. Testing MoAbs against class I, II and III CD34 epitopes on normal BM we observed that ± 23% of class III positive cells was class I negative. A higher expression of the class III epitope compared with classes I or II was observed on the KG1 cell line, whereas no differences in binding capability were found. The class III epitope anti-CD34, 561, was compared with the class I epitope anti-CD34, BI-3C5, both coupled to M450 Dynabeads. The yield of CD34⁺ cells obtained with the 561 beads was 1.7% of the mononuclear cells versus 0.95% using the class I epitope, a 1.95-fold increase (1.3–2.7), whereas the purity was similar (96% in both cases). The absolute number of CD34⁺ cells was therefore twofold higher after 561 selection, including cells with a more mature phenotype. In single cell assay comparable numbers of highly proliferative progenitors but higher numbers of differentiated colonies per phenotypic subfraction were measured. In conclusion, M450 beads coated with the 561 anti-class III CD34 epitope are more efficient in isolating CD34⁺ cells from bone marrow, probably due to a broader distribution of the class III epitope.     

Impact of a Low CD34+ Cell Dose on Allogeneic Peripheral Blood Stem Cell Transplantation

Although the CD34⁺ cell dose in allogeneic peripheral blood stem cell transplantation (PBSCT) is considered to be associated with transplantation outcomes, a lower acceptable threshold has not been defined. We retrospectively analyzed 2919 adult patients with hematologic malignancies who underwent related PBSCT in Japan between 2001 and 2014. According to the number of CD34⁺ cells in the graft, we categorized 2494 patients in the standard group (2 to 5 × 10⁶ cells/kg), 377 patient in the low group (1 to 2?×?10⁶ cells/kg), and 48 patients in the very low group (<1?×?10⁶ cells/kg). Compared with the standard group, the low and very low groups showed delayed neutrophil recovery (93.8%, 89.5%, and 78.3%, respectively at day +28; P?<?.001) and platelet recovery (69.3%, 53.0%, and 45.5%, respectively at day +28; P?<?.001). The 2-year overall survival (OS) in the 3 groups was 45.5%, 45.3%, and 29.8%, respectively, with inferior survival in the very low group. However, a higher percentage of high-risk patients may account for the inferior survival in the very low group, and no significant difference in OS was found in a multivariate analysis. There were no differences in relapse, nonrelapse mortality, or the development of graft-versus-host disease among the 3 groups. In conclusion, allogeneic PBSCT with low CD34⁺ cell doses of 1 to 2?×?10⁶ cells/kg gives acceptable results, whereas further investigations are needed to evaluate the effects of lower doses of <1?×?10⁶ cells/kg owing to the smaller number and the higher percentage of patients with adverse prognostic factors in this cohort.     

No Impact of Total or Myeloid Cd34+ Cell Numbers on Neutrophil Engraftment and Transplantation-Related Mortality after Allogeneic Pediatric Bone Marrow Transplantation

Although the influence of transplanted bone marrow (BM) CD34+ cells on neutrophil engraftment (NE) and transplantation outcomes has been discussed controversially, thresholds between 2 and 4 × 10⁶/kg CD34+ cells are commonly accepted. This has substantial consequences for a donor in terms of BM volume to be collected, which frequently covers up to 15 to 20 mL/kg. As the BM CD34+ compartment contains varying fractions of CD34+/CD19+ B lymphoid progenitors, we tested the hypothesis that the infused CD34+/CD45dim/CD19-/CD10- myeloid stem cells might reliably predict NE in 94 children who received BM from 37 HLA-identical sibling donors (MSD) and 57 matched unrelated donors after myeloablative conditioning. The grafts contained a median of 3.6 × 10⁶/kg total CD34+ cells, which consisted of a median of 73% myeloid CD34+ cells and 27% B lymphoid progenitors. Grafts from donors <15 years old yielded significantly lower myeloid fractions compared with grafts from older donors (P < .001). All patients achieved sustained NE after median 20 (range, 11 to 40) days. By multivariate analysis, neither the number of total CD34+ cells (P = .605) nor of myeloid CD34+ cells (P = .981) correlated with NE, whereas transplantation from MSD (hazard ratio [HR] 3.51; P = .019) and the administration of granulocyte colony–stimulating factor (HR 2.24; P = .002) remained independent factors associated with earlier NE. Furthermore, neither total nor myeloid CD34+ cell quantities were associated with incidences of severe infections before NE (P = .271 and P = .132) or transplantation-related mortality (TRM) at day +100 (P = .294 and P = .490). Taking into account that the number of transplanted total CD34+ or myeloid CD34+ cells does not seem to have a relevant impact on time to NE, sepsis rates, or TRM, the need of certain threshold cell numbers should be revisited, at least for pediatric MSD.     

Differences in Cellular Composition of Peripheral Blood Stem Cell Grafts from Healthy Stem Cell Donors Mobilized with Either Granulocyte Colony-Stimulating Factor (G-CSF) Alone or G-CSF and Plerixafor

This study was conducted to characterize and compare peripheral blood stem cell grafts from healthy donors who underwent granulocyte colony-stimulating factor (G-CSF) mobilization and subsequently received 1 dose of plerixafor after insufficient stem cell yields were achieved at the first apheresis. Aliquots from 35 donors were collected from the first apheresis after mobilization with G-CSF alone and from the second apheresis after additional plerixafor administration. Samples were freshly analyzed for cellular subsets by 8-color flow cytometry. Leukapheresis samples mobilized with additional plerixafor showed a significant increase of total nucleated cells, including B cells, CD4⁺ and CD8⁺ T cells, and CD34⁺ hematopoietic stem and progenitor cells. Absolute numbers of plasmacytoid dendritic cells were also significantly increased, whereas no changes were detected for myeloid dendritic cells. Furthermore, absolute numbers of regulatory T cells increased, with naive CD45RA⁺ regulatory T cells showing the highest rise. Finally, strikingly higher numbers of myeloid-derived suppressor cells were detected in the plerixafor and G-CSF–mobilized graft. The mobilization of peripheral stem cells in healthy donors with G-CSF and plerixafor led to a significant difference in cellular graft composition compared with G-CSF alone. The clinical impact of the different cell composition for the graft recipient warrants further clinical investigation.     

Mobilization of Hematopoietic Progenitors from Normal Donors Using the Combination of Granulocyte-Macrophage Colony-Stimulating Factor and Granulocyte Colony-Stimulating Factor Results in Fewer Plasmacytoid Dendritic Cells in the Graft and Enhanced Donor T Cell Engraftment with Th1 Polarization: Results from a Randomized Clinical Trial

Granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) both mobilize CD34⁺ stem cells into the blood when administered before apheresis but have distinct effects on dendritic cell (DC) differentiation. We previously demonstrated that the combination of GM+G-CSF results in fewer plasmacytoid DCs (pDCs) when used to mobilize peripheral blood stem cells for autologous transplantation. To test the hypothesis that the content of pDCs in an allograft can be modulated with the cytokines used for mobilization, we randomized the human leukocyte antigen–matched sibling donors of 50 patients with hematological malignancies to a mobilization regimen of either GM+G-CSF (n = 25) or G-CSF alone (n = 25). Primary and secondary endpoints included the cellular constituents of the mobilized grafts, the kinetics of posttransplantation immune reconstitution, and clinical outcomes of the transplantation recipients. Grafts from donors receiving GM+G-CSF contained equivalent numbers of CD34⁺ cells with fewer pDCs and T cells, with a higher fraction of Th1-polarized donor T cells than G-CSF mobilized grafts. Immune recovery was enhanced among recipients of GM+G-CSF. Survival was not significantly different between transplantation recipients in the two arms. The use of GM+G-CSF modulates immune function and recovery after allogeneic transplantation and should be explored in larger studies powered to evaluate clinical outcomes.     

Distinct Biomarker Profiles in Ex Vivo T Cell Depletion Graft Manipulation Strategies: CD34+ Selection versus CD3+/19+ Depletion in Matched Sibling Allogeneic Peripheral Blood Stem Cell Transplantation

Various approaches have been developed for ex vivo T cell depletion in allogeneic stem cell transplantation to prevent graft-versus-host disease (GVHD). Direct comparisons of T cell depletion strategies have not been well studied, however. We evaluated cellular and plasma biomarkers in 2 different graft manipulation strategies, CD3⁺CD19⁺ cell depletion (CD3/19D) versus CD34⁺ selection (CD34S), and their associations with clinical outcomes. Identical conditions, including the myeloablative preparative regimen, HLA-identical sibling donor, GVHD prophylaxis, and graft source, were used in the 2 cohorts. Major clinical outcomes were similar in the 2 groups in terms of overall survival, nonrelapse mortality, and cumulative incidence of relapse; however, the cumulative incidence of acute GVHD trended to be higher in the CD3/19D cohort compared with the CD34S cohort. A distinct biomarker profile was noted in the CD3/19D cohort: higher levels of ST2, impaired Helios^? FoxP3⁺Treg reconstitution, and rapid reconstitution of naïve, Th2, and Th17 CD4 cells in the early post-transplantation period. In vitro graft replication studies confirmed that CD3/19D disproportionately depleted Tregs and other CD4 subset repertoires in the graft. This study confirms the utility of biomarker monitoring, which can be directly correlated with biological consequences and possible future therapeutic indications.     

Influence of Age on Acute and Chronic GVHD in Children Undergoing HLA-Identical Sibling Bone Marrow Transplantation for Acute Leukemia: Implications for Prophylaxis

Relapse remains the major cause of mortality after hematopoietic cell transplantation (HCT) for pediatric acute leukemia. Previous research has suggested that reducing the intensity of calcineurin inhibitor-based graft-versus-host disease (GVHD) prophylaxis may be an effective strategy for abrogating the risk of relapse in pediatric patients undergoing matched sibling donor (MSD) HCT. We reasoned that the benefits of this strategy could be maximized by selectively applying it to those patients least likely to develop GVHD. We conducted a study of risk factors for GVHD, to risk-stratify patients based on age. Patients age <18 years with leukemia who received myeloablative, T cell-replete MSD bone marrow transplantation and calcineurin inhibitor-based GVHD prophylaxis between 2000 and 2013 and were entered into the Center for International Blood and Marrow Transplant Research registry were included. The cumulative incidence of grade II-IV acute GVHD (aGVHD) was 19%, that of grade II-IV aGVHD 7%, and that of chronic GVHD (cGVHD) was 16%. Compared with age 13 to 18 years, age 2 to 12 years was associated with a lower risk of grade II-IV aGVHD (hazard ratio [HR], .42; 95% confidence interval [CI], .26 to .70; P?=?.0008), grade II-IV aGVHD (HR, .24; 95% CI, .10 to .56; P?=?.001), and cGVHD (HR, .32; 95% CI, .19 to .54; P?<?.001). Compared with 2000-2004, the risk of grade II-IV aGVHD was lower in children undergoing transplantation in 2005-2008 (HR, .36; 95% CI, .20 to .65; P?=?.0007) and in 2009-2013 (HR, .24; 95% CI. .11 to .53; P?=?.0004). Similarly, the risk of grade III-IV aGVHD was lower in children undergoing transplantation in 2005-2008 (HR, .23; 95% CI, .08 to .65; P?=?.0056) and 2009-2013 (HR, .16; 95% CI, .04 to .67; P?=?.0126) compared with those doing so in 2000-2004. We conclude that aGVHD rates have decreased significantly over time, and that children age 2 to 12 years are at very low risk for aGVHD and cGVHD. These results should be validated in an independent analysis, because these patients with high-risk malignancies may be good candidates for trials of reduced GVHD prophylaxis.     

流式细胞术CD34+细胞计数在骨髓增生异常综合征分型诊断中的作用

为探讨流式细胞术(FCM)CD34+细胞计数在骨髓增生异常综合征(MDS)分型诊断中的应用价值.采用多参数FCM设门技术检测MDS患者骨髓CD34+细胞计数,与形态学分型诊断比较.结果显示:对照组和MDS组形态学原始细胞计数分别为0.43±0.64%和2.50±3.68%,FCM CD34+细胞计数分别为0.45±0....     

大鼠骨髓基质细胞表面抗原CD71、CD44和CD45的检测

目的研究表面抗原CD71、CD44和CD45在体外培养的大鼠骨髓基质细胞的表达,了解骨髓基质细胞的生物学特性。方法采用全骨髓法分离培养大鼠骨髓基质细胞,待原代培养的细胞达70%以上汇合时,用含胰酶和EDTA的消化液消化细胞,进行传代培养。传至第3代(P3)的骨髓基质细胞采用流式细胞术检测表面抗原CD71、CD44和CD45的表达,专用配套软件计算细胞表面抗原阳性%率。结果骨髓基质细胞呈纺锤形、成纤维细胞样生长,漩涡状分布。传至第3代的细胞形态趋于一致。流式细胞仪检测结果显示,CD71、CD44和CD45阳性细胞百分比分别为99.07±7.6%、52.81±7.3%和10.85±5.0%。结论P3的骨髓基质细胞具有较强的增殖特性。     

Evaluation of Cord Blood Total Nucleated and CD34+ Cell Content,Cell Dose,and 8-Allele HLA Match by Patient Ancestry

How cord blood (CB) CD34⁺ cell content and dose and 8-allele HLA match vary by patient ancestry is unknown. We analyzed cell content, dose, and high-resolution HLA-match of units selected for CB transplantation (CBT) by recipient ancestry. Of 544 units (286 infused, 258 next-best backups) chosen for 144 racially diverse adult patients (median weight, 81 kg), the median total nucleated cell (TNC) and CD34⁺cell ⁺contents were higher for Europeans than for non-Europeans: 216 × 10⁷versus 197 × 10⁷ (P = .002) and 160 × 10⁵ versus 132 × 10⁵ (P = .007), respectively. There were marked cell content disparities among ancestry groups, with units selected for Africans having the lowest TNC (189 × 10⁷) and CD34⁺ cell (122 × 10⁵) contents. Units for non-Europeans were also more HLA-mismatched (P = .017). When only the 286 transplanted units were analyzed, the adverse effect of reduced cell content was exacerbated by the higher weights in some groups. For example, northwestern Europeans (high patient weight, high unit cell content) had the best-dosed units, and Africans (high weight, low unit cell content) had the lowest. In Asians, low cell content was partially compensated for by lower weight. Marked differences in 8-allele HLA-match distribution were also observed by ancestry group; for example, 23% of units for northwestern Europeans were 3/8 to 4/8 HLA-matched, compared with 40% for southern Europeans, 46% for white Hispanics, and 51% for Africans. During the study period, 20 additional patients (17 non-Europeans; median weight, 98 kg) did not undergo CBT owing to the lack of a suitable graft. CB extends transplantation access to most patients, but racial disparities exist in cell content, dose, and HLA match.