Combined haploidentical and cord blood transplantation for refractory severe aplastic anaemia and hypoplastic myelodysplastic syndrome.

Umbilical cord blood (UCB) transplantation is a potentially curative treatment for patients with refractory severe aplastic anaemia (SAA), but has historically been associated with delayed engraftment and high graft failure and mortality rates. We conducted a prospective phase 2 trial to assess outcome of an allogeneic transplant regimen that co-infused a single UCB unit with CD34+ -selected cells from a haploidentical relative. Among 29 SAA patients [including 10 evolved to myelodysplastic syndrome (MDS)] who underwent the haplo cord transplantation (median age 20 years), 97% had neutrophil recovery (median 10 days), and 93% had platelet recovery (median 32 days). Early myeloid engraftment was from the haplo donor and was gradually replaced by durable engraftment from UCB in most patients. The cumulative incidences of grade II-IV acute and chronic graft-versus-host disease (GVHD) were 21% and 41%, respectively. With a median follow-up of 7·5 years, overall survival was 83% and GVHD/relapse-free survival was 69%. Patient- and transplant-related factors had no impact on engraftment and survival although transplants with haplo-versus-cord killer-cell immunoglobulin-like receptor (KIR) ligand incompatibility had delayed cord engraftment. Our study shows haplo cord transplantation is associated with excellent engraftment and long-term outcome, providing an alternative option for patients with refractory SAA and hypoplastic MDS who lack human leucocyte antigen (HLA)-matched donors.

Differences in the phenotype, cytokine gene expression profiles, and in vivo alloreactivity of T cells mobilized with plerixafor compared with G-CSF.

Plerixafor (Mozobil) is a CXCR4 antagonist that rapidly mobilizes CD34(+) cells into circulation. Recently, plerixafor has been used as a single agent to mobilize peripheral blood stem cells for allogeneic hematopoietic cell transplantation. Although G-CSF mobilization is known to alter the phenotype and cytokine polarization of transplanted T cells, the effects of plerixafor mobilization on T cells have not been well characterized. In this study, we show that alterations in the T cell phenotype and cytokine gene expression profiles characteristic of G-CSF mobilization do not occur after mobilization with plerixafor. Compared with nonmobilized T cells, plerixafor-mobilized T cells had similar phenotype, mixed lymphocyte reactivity, and Foxp3 gene expression levels in CD4(+) T cells, and did not undergo a change in expression levels of 84 genes associated with Th1/Th2/Th3 pathways. In contrast with plerixafor, G-CSF mobilization decreased CD62L expression on both CD4 and CD8(+) T cells and altered expression levels of 16 cytokine-associated genes in CD3(+) T cells. To assess the clinical relevance of these findings, we explored a murine model of graft-versus-host disease in which transplant recipients received plerixafor or G-CSF mobilized allograft from MHC-matched, minor histocompatibility-mismatched donors; recipients of plerixafor mobilized peripheral blood stem cells had a significantly higher incidence of skin graft-versus-host disease compared with mice receiving G-CSF mobilized transplants (100 versus 50%, respectively, p = 0.02). These preclinical data show plerixafor, in contrast with G-CSF, does not alter the phenotype and cytokine polarization of T cells, which raises the possibility that T cell-mediated immune sequelae of allogeneic transplantation in humans may differ when donor allografts are mobilized with plerixafor compared with G-CSF.

Peripheral blood stem cell transplant-related Plasmodium falciparum infection in a patient with sickle cell disease.

BACKGROUND: Although transmission of Plasmodium falciparum (Pf) infection during red blood cell (RBC) transfusion from an infected donor has been well documented, malaria parasites are not known to infect hematopoietic stem cells. We report a case of Pf infection in a patient 11 days after peripheral blood stem cell transplant for sickle cell disease. STUDY DESIGN AND METHODS: Malaria parasites were detected in thick blood smears by Giemsa staining. Pf HRP2 antigen was measured by enzyme-linked immunosorbent assay on whole blood and plasma. Pf DNA was detected in whole blood and stem cell retention samples by real-time polymerase chain reaction using Pf species-specific primers and probes. Genotyping of eight Pf microsatellites was performed on genomic DNA extracted from whole blood. RESULTS: Pf was not detected by molecular, serologic, or parasitologic means in samples from the recipient until Day 11 posttransplant, coincident with the onset of symptoms. In contrast, Pf antigen was retrospectively detected in stored plasma collected 3 months before transplant from the asymptomatic donor. Pf DNA was detected in whole blood from both the donor and the recipient after transplant, and genotyping confirmed shared markers between donor and recipient Pf strains. Lookback analysis of RBC donors was negative for Pf infection. CONCLUSIONS: These findings are consistent with transmission by the stem cell product and have profound implications with respect to the screening of potential stem cell donors and recipients from malaria-endemic regions.

A pilot study evaluating the safety and CD34+ cell mobilizing activity of escalating doses of plerixafor in healthy volunteers.

This study evaluated the safety and CD34+ cell mobilizing activity of escalating doses of plerixafor in healthy volunteers. Three cohorts of six subjects received two different doses of plerixafor separated by at least 2 weeks to allow for adequate pharmacodynamic wash-out. The following dosing cohorts were evaluated: 0·24 and 0·32 mg/kg (Cohort 1); 0·32 and 0·40 mg/kg (Cohort 2); and 0·40 and 0·48 mg/kg (Cohort 3). Circulating CD34+ cells were measured 0, 2, 4, 6, 8, 10, 12, 14, 18 and 24 h after each dose. Blood colony-forming units were measured at baseline and 6 h after each dose. Common adverse events were diarrhoea, injection site erythema, perioral numbness, sinus tachycardia, headache, nausea, abdominal distention and injection site pain. No dose limiting toxicities occurred. When higher doses of plerixafor were administered, there was a trend towards higher peak CD34+ counts and CD34+ area under the curves, although these differences did not achieve statistical significance, perhaps due to intra-subject variability. Together, these data show that the higher doses of plerixafor evaluated in this study are reasonably safe and suggest that a larger study should be performed to definitively answer whether increased numbers of CD34+ cell are mobilized with higher doses of plerixafor.

A pilot study of consolidative immunotherapy in patients with high-risk pediatric sarcomas.

PURPOSE: Patients with metastatic or recurrent Ewing's sarcoma family of tumors and alveolar rhabdomyosarcoma have <25% 5-year survival in most studies. This study administered a novel immunotherapy regimen aimed at consolidating remission in these patients. EXPERIMENTAL DESIGN: Fifty-two patients with translocation positive, recurrent, or metastatic Ewing's sarcoma family of tumors or alveolar rhabdomyosarcoma underwent prechemotherapy cell harvest via apheresis for potential receipt of immunotherapy. Following completion of standard multimodal therapy, 30 patients ultimately initiated immunotherapy and were sequentially assigned to three cohorts. All cohorts received autologous T cells, influenza vaccinations, and dendritic cells pulsed with peptides derived from tumor-specific translocation breakpoints and E7, a peptide known to bind HLA-A2. Cohort 1 received moderate-dose recombinant human interleukin-2 (rhIL-2), cohort 2 received low-dose rhIL-2, and cohort 3 did not receive rhIL-2. RESULTS: All immunotherapy recipients generated influenza-specific immune responses, whereas immune responses to the translocation breakpoint peptides occurred in 39%, and only 25% of HLA-A2(+) patients developed E7-specific responses. Toxicity was minimal. Intention-to-treat analysis revealed a 31% 5-year overall survival for all patients apheresed (median potential follow-up 7.3 years) with a 43% 5-year overall survival for patients initiating immunotherapy. CONCLUSIONS: Consolidative immunotherapy is a scientifically based and clinically practical approach for integrating immunotherapy into a multimodal regimen for chemoresponsive cancer. Patients receiving immunotherapy experienced minimal toxicity and favorable survival. The robust influenza immune responses observed suggest that postchemotherapy immune incompetence will not fundamentally limit this approach. Future studies will seek to increase efficacy by using more immunogenic antigens and more potent dendritic cells.

Donor demographic and laboratory predictors of allogeneic peripheral blood stem cell mobilization in an ethnically diverse population.

A reliable estimate of peripheral blood stem cell (PBSC) mobilization response to granulocyte colony-stimulating factor (G-CSF) may identify donors at risk for poor mobilization and help optimize transplantation approaches. We studied 639 allogeneic PBSC collections performed in 412 white, 75 black, 116 Hispanic, and 36 Asian/Pacific adult donors who were prescribed G-CSF dosed at either 10 or 16 microg/kg per day for 5 days followed by large-volume leukapheresis (LVL). Additional LVL (mean, 11 L) to collect lymphocytes for donor lymphocyte infusion (DLI) and other therapies was performed before G-CSF administration in 299 of these donors. Day 5 preapheresis blood CD34(+) cell counts after mobilization were significantly lower in whites compared with blacks, Hispanics, and Asian/Pacific donors (79 vs 104, 94, and 101 cells/microL, P < .001). In addition, donors who underwent lymphapheresis before mobilization had higher CD34(+) cell counts than donors who did not (94 vs 79 cells/microL, P < .001). In multivariate analysis, higher post-G-CSF CD34(+) cell counts were most strongly associated with the total amount of G-CSF received, followed by the pre-G-CSF platelet count, pre-G-CSF mononuclear count, and performance of prior LVL for DLI collection. Age, white ethnicity, and female gender were associated with significantly lower post-G-CSF CD34(+) cell counts.

Evaluation of gene expression profiles of immature dendritic cells prepared from peripheral blood mononuclear cells.

BACKGROUND: Dendritic cells (DCs) generated ex vivo from peripheral blood monocytes or mobilized CD34+ cells and intended for clinical immunotherapy are typically characterized by morphologic, phenotypic, and functional assays. Assay results are highly dependent on conditions used to prepare the cells, so there is no standard assay battery for clinical DC products. This study evaluated gene expression profiling for characterization of immature DCs prepared from monocytes that had been elutriated from normal donor peripheral blood mononuclear cells (PBMNCs) immediately after collection or after storage at 4 degrees C for 48 hours. STUDY DESIGN AND METHODS: RNA was isolated from fresh and 48-hour-stored PBMNCs, elutriated monocytes, elutriated lymphocytes, and immature DCs from five healthy subjects and was analyzed with a cDNA gene expression microarray with 17,500 genes. RESULTS: Unsupervised hierarchical clustering separated the 40 products into four groups: one with all 10 immature DCs, one with all 10 elutriated lymphocytes, one with 7 PBMNCs, and one with 10 elutriated monocytes and 3 PBMNCs. Within each of the four groups, however, fresh and stored products, or products derived from fresh or stored products, clustered together. Comparison of genes differentially expressed by fresh versus stored products (paired t tests, p < 0.005) found 273 genes that differed between fresh and stored PBMCs, 429 between lymphocytes elutriated from fresh versus stored PBMNCs, 711 between monocytes elutriated from fresh versus stored PBMNCs, and 3 between immature DCs prepared from monocytes elutriated from fresh versus stored PBMCs. CONCLUSIONS: This study demonstrates the potential utility of gene expression profiling for characterization of cell therapy products.

Sterility testing of cell therapy products: parallel comparison of automated methods with a CFR-compliant method.

BACKGROUND: Automated blood culture systems are not FDA-approved for sterility testing of human cells, tissues, or cellular- or tissue-based products. It was previously demonstrated that BacT/ALERT (bioMérieux) and Bactec (Becton Dickinson) were superior to the manual CFR method described in the general biologics regulations, in rates of detection and time to detection of organisms seeded into mock mononuclear cell products with a variety of background media and antibiotics. In this study, the two automated systems were compared to the CFR method for sterility testing of actual cell therapy products manufactured in our facility. STUDY DESIGN AND METHODS: Over a 36-month period, in-process and final product samples from all cell therapy products manufactured in our facility were tested for sterility both by the CFR method and by either BacT/ALERT or Bactec. Products were categorized according to collection and processing variables for analysis of results. RESULTS: For 1617 samples of a broad range of cell therapy products, rates of true-positive tests were comparable for the automated and CFR methods (2.3% vs. 2.1%), but the CFR method had higher rates of false-positive results (7.3% vs. 0.2%). For automated systems, time to detection of organisms was equivalent to, or faster than, the CFR method. CONCLUSION: Compared to the CFR method, both BacT/ALERT and Bactec are more sensitive, faster in time to detection, less prone to false-positive results, and less labor-intensive. Both of these automated systems are suitable for sterility testing of cell therapy products after site-specific validation has been performed.

Magnetic resonance imaging and confocal microscopy studies of magnetically labeled endothelial progenitor cells trafficking to sites of tumor angiogenesis.

UNLABELLED: AC133 cells, a subpopulation of CD34+ hematopoietic stem cells, can transform into endothelial cells that may integrate into the neovasculature of tumors or ischemic tissue. Most current imaging modalities do not allow monitoring of early migration and incorporation of endothelial progenitor cells (EPCs) into tumor neovasculature. The goals of this study were to use magnetic resonance imaging (MRI) to track the migration and incorporation of intravenously injected, magnetically labeled EPCs into the blood vessels in a rapidly growing flank tumor model and to determine whether the pattern of EPC incorporation is related to the time of injection or tumor size. MATERIALS AND METHODS: EPCs labeled with ferumoxide-protamine sulfate (FePro) complexes were injected into mice bearing xenografted glioma, and MRI was obtained at different stages of tumor development and size. RESULTS: Migration and incorporation of labeled EPCs into tumor neovasculature were detected as low signal intensity on MRI at the tumor periphery as early as 3 days after EPC administration in preformed tumors. However, low signal intensities were not observed in tumors implanted at the time of EPC administration until tumor size reached 1 cm at 12 to 14 days. Prussian blue staining showed iron-positive cells at the sites corresponding to low signal intensity on MRI. Confocal microscopy showed incorporation into the neovasculature, and immunohistochemistry clearly demonstrated the transformation of the administered EPCs into endothelial cells. CONCLUSION: MRI demonstrated the incorporation of FePro-labeled human CD34+/AC133+ EPCs into the neovasculature of implanted flank tumors.

Outcomes and risks of granulocyte colony-stimulating factor in patients with coronary artery disease.

OBJECTIVES: Cytokine mobilization of progenitor cells from bone marrow may promote myocardial neovascularization with relief of ischemia. BACKGROUND: Patients with coronary artery disease (CAD) have low numbers of endothelial progenitor cells compared with healthy subjects. METHODS: Granulocyte colony-stimulating factor (G-CSF), 10 microg/kg/day for five days, was administered to 16 CAD patients. Progenitor cells were measured by flow cytometry; ischemia was assessed by exercise stress testing and by dobutamine stress cardiac magnetic resonance imaging. RESULTS: Granulocyte colony-stimulating factor increased CD34+/CD133+ cells in the circulation from 1.5 +/- 0.2 microl to 52.4 +/- 10.4 microl (p < 0.001), similar to the response observed in 15 healthy subjects (75.1 +/- 12.6 microl, p = 0.173). Indices of platelet and coagulation activation were not changed by treatment, but C-reactive protein increased from 4.5 +/- 1.3 mg/l to 8.6 +/- 1.3 mg/l (p = 0.017). Two patients experienced serious adverse events: 1) non-ST-segment elevation myocardial infarction (MI) 8 h after the fifth G-CSF dose, and 2) MI and death 17 days after treatment. At 1 month after treatment, there was no improvement from baseline values (i.e., reduction) in wall motion score (from 25.7 +/- 2.1 to 28.3 +/- 1.9, p = 0.196) or segments with abnormal perfusion (7.6 +/- 1.1 to 7.7 +/- 1.1, p = 0.916) and a trend towards a greater number of ischemic segments (from 4.5 +/- 0.6 to 6.1 +/- 1.0, p = 0.068). There was no improvement in exercise duration at 1 month (p = 0.37) or at 3 months (p = 0.98) versus baseline. CONCLUSIONS: Granulocyte colony-stimulating factor administration to CAD patients mobilizes cells with endothelial progenitor potential from bone marrow, but without objective evidence of cardiac benefit and with the potential for adverse outcomes in some patients.

Granulocyte colony-stimulating factor mobilizes functional endothelial progenitor cells in patients with coronary artery disease.

OBJECTIVE: Endothelial progenitor cells (EPCs) that may repair vascular injury are reduced in patients with coronary artery disease (CAD). We reasoned that EPC number and function may be increased by granulocyte colony-stimulating factor (G-CSF) used to mobilize hematopoietic progenitor cells in healthy donors. METHODS AND RESULTS: Sixteen CAD patients had reduced CD34(+)/CD133(+) (0.0224+/-0.0063% versus 0.121+/-0.038% mononuclear cells [MNCs], P<0.01) and CD133(+)/VEGFR-2(+) cells, consistent with EPC phenotype (0.00033+/-0.00015% versus 0.0017+/-0.0006% MNCs, P<0.01), compared with 7 healthy controls. Patients also had fewer clusters of cells in culture, with out-growth consistent with mature endothelial phenotype (2+/-1/well) compared with 16 healthy subjects at high risk (13+/-4/well, P<0.05) or 14 at low risk (22+/-3/well, P<0.001) for CAD. G-CSF 10 microg/kg per day for 5 days increased CD34(+)/CD133(+) cells from 0.5+/-0.2/microL to 59.5+/-10.6/microL and CD133(+)/ VEGFR-2(+) cells from 0.007+/-0.004/microL to 1.9+/-0.6/microL (both P<0.001). Also increased were CD133(+) cells that coexpressed the homing receptor CXCR4 (30.4+/-8.3/microL, P<0.05). Endothelial cell-forming clusters in 10 patients increased to 27+/-9/well after treatment (P<0.05), with a decline to 9+/-4/well at 2 weeks (P=0.06). CONCLUSIONS: Despite reduced EPCs compared with healthy controls, patients with CAD respond to G-CSF with increases in EPC number and homing receptor expression in the circulation and endothelial out-growth in culture. Endothelial progenitor cells (EPCs) are reduced in coronary artery disease. Granulocyte colony-stimulating factor (CSF) administered to patients increased: (1) CD133+/VEGFR-2+ cells consistent with EPC phenotype; (2) CD133+ cells coexpressing the chemokine receptor CXCR4, important for homing of EPCs to ischemic tissue; and (3) endothelial cell-forming clusters in culture. Whether EPCs mobilized into the circulation will be useful for the purpose of initiating vascular growth and myocyte repair in coronary artery disease patients must be tested in clinical trials.

Characteristics of photopheresis treatments for the management of rejection in heart and lung transplant recipients.

Photopheresis has been used in the management of rejection of heart and/or lung transplants. Although its mechanism of action remains unknown, irradiated T-helper cell-induced immunosuppression is the main theory. Since transplant recipients are often lymphopenic and lymphocytes are the target cells in phototherapy, we performed this study to determine which factors affect the cellular yield to undergo irradiation. We reviewed the records of all photophereses performed in our institution between July 1998 and April 2000 using the UVAR (first generation) or XTS (second generation) instruments (Therakos, Exton, PA). Our data included patient's blood volume, absolute lymphocyte count and hematocrit, catheter type, flow rate of collection cycles and centrifuge bowl size, as well as volume, hematocrit, and lymphocyte count of the cell suspension. With a mixed model multivariate analysis we sought to determine which variables predicted the lymphocyte yield. A total of 406 procedures in 25 adult patients was analyzed. There was no significant difference between the lymphocyte yield among the procedures performed with the first- and the second-generation instruments. The patient's absolute lymphocyte count was the only parameter, which positively correlated with the total number of lymphocytes collected for irradiation (P < 0.0001). Indeed, based on the mixed model, the total number of lymphocytes for irradiation can be predicted from the pre-procedure lymphocyte count. Additional studies are necessary to correlate the number of treated cells with patient outcome.