The effect of mobilizing large numbers of CD34 + cells (super-mobilizers) on the engraftment and survival in patients undergoing autologous stem cell transplantation.

With the use of plerixafor in addition to growth factor for peripheral blood stem cell mobilization, the yield of autologous stem cell harvest has been higher while the length of apheresis days has become shorter. There is still debate whether higher cell collection efficacy in autologous stem cell transplant (ASCT) affect outcomes. In this retrospective study, we defined two groups of patients, group 1, super-mobilizers, with more than double the target cell dose collected (n = 15), while group 2 included all other patients (n = 75). Multiple myeloma (MM) and lymphoma patients were combined. Patients with chemo-mobilization, those needed more than one day apheresis, or with less than 100 days after ASCT were excluded. Correlations were performed between cell collection efficacy and post thaw CD34 cell viability (by 7AAD flow cytometry method), product HCT, and engraftment of neutrophils and platelets. We performed multiple linear regression using the above variables in addition to age, sex and disease type. We used Kaplan Meier's curves to show effect of cell collection efficacy on 1-year overall survival (OS). Our results show that all super-mobilizers received plerixafor in addition to G-CSF, while 83% did in group 2. Correlations between cell collection efficacy and neutrophil and platelet engraftment in group 1 and 2 was modest and better in group 1 (R=0.449 Vs 0.233 for neutrophils; R=0.464 Vs 0.110 for platelets, respectively). However, multiple linear regression showed statistically significant association between cell collection, as a continuous variable, with disease type (P < 0.001), product HCT (P < 0.001), post thaw viability (P = 0.003), and age (P = 0.013). MM patients were more likely to be super-mobilizers, while the product HCT was higher in the super-mobilizers. No significant effect of cell collection efficacy was found on engraftment of neutrophils or platelets. With relatively short post ASCT follow up, 6 patients in group 2 died of any cause while no deaths were recorded in the super-mobilizers group (P = 0.1892 by log-rank test). In conclusion, stem cell collection efficacy in ASCT is more frequent in MM than lymphoma patients, but is not predictive of faster engraftment. On the other hand, 1-year OS was 100% in the super-mobilizers group versus 93% in the other group.

Postautologous stem cell transplantation engraftment syndrome: Improved treatment and outcomes.

Postautologous stem cell transplantation (ASCT) engraftment syndrome (ES) is a well-known clinical complication; however, many aspects remain largely controversial. In this retrospective study, we reviewed records of 156 ASCTs done over 2 years in our institution. Our results show that 45 (34 multiple myeloma/amyloidosis and 11 lymphoma) of 156 adult patients (29%) were diagnosed with ES. Patients with ES were significantly more likely to have fever, rash, and diarrhea upon engraftment. Risk factors for ES included shorter time from diagnosis to ASCT (P = .029), and lower number of pretransplant treatment regimens (0.012). Post-ASCT, patients with ES had significantly lower absolute lymphocyte count on first engraftment day (P = .014). Most ES patients received treatment with steroids. Initial dose of prednisone/methylprednisolone was 2 mg/kg (n = 34), 1 mg/kg (n = 7), while four patients received 1000 mg IV with median length of therapy 7.5 days. One ES patient with inadequate steroid therapy died of ES complications, while another developed respiratory failure requiring intubation but had full recovery with steroids treatment. In conclusion, patients with shorter time from diagnosis to ASCT and with less prior therapy are more likely to develop ES. Overall survival of ES patients has improved with greater awareness of the diagnosis and earlier use of steroids.

Poor peripheral blood stem cell mobilization affects long-term outcomes in multiple myeloma patients undergoing autologous stem cell transplantation.

BACKGROUND: Peripheral blood stem cell (PBSC) mobilization is routinely undertaken prior to autologous stem cell transplantation (ASCT) in patients with multiple myeloma (MM). A number of studies have identified risk factors for poor PBSC mobilization, however, little data exists to correlate mobilization with disease-specific outcomes in this patient population. Prospective work in MM has demonstrated similar outcomes in a homogenous patient population. METHODS: In this single institution analysis, we retrospectively studied the impact of poor PBSC mobilization on progression free survival (PFS) and OS in MM patients undergoing PBSC mobilization. Poor mobilizers are defined as patients that collected < 4 × 106 CD34+ cells/kg over maximum of 5 apheresis days, or those that required ≥2 mobilization cycles to achieve this target. RESULTS: We confirm that poor PBSC mobilization is significantly associated with a shortened PFS (P = .0012) and OS (P = .0005) compared with good mobilizers. Our univariate analysis also shows that independent risk factors for poor mobilization include male gender, higher ideal body weight, and a greater median number of lines of chemotherapy prior to PBSC mobilization. However, by multivariate analysis, only number of prior lines of chemotherapy remains significantly predictive of poor mobilization (Odds ratio 1.857, P = .0095). The use of high-dose G-CSF (> 10 mcg/kg/day) and/or plerixafor can significantly improve mobilization and ASCT chances in this population. DISCUSSION: These data indicate that poor mobilization can be predictable and is associated with more aggressive disease biology and worse outcomes, warranting intensive post-ASCT management.

Ex vivo virotherapy with myxoma virus does not impair hematopoietic stem and progenitor cells.

BACKGROUND: Relapsing disease is a major challenge after hematopoietic cell transplantation for hematological malignancies. Myxoma virus (MYXV) is an oncolytic virus that can target and eliminate contaminating cancer cells from auto-transplant grafts. The aims of this study were to examine the impact of MYXV on normal hematopoietic stem and progenitor cells and define the optimal treatment conditions for ex vivo virotherapy. METHODS: Bone marrow (BM) and mobilized peripheral blood stem cells (mPBSCs) from patients with hematologic malignancies were treated with MYXV at various time, temperature and incubation media conditions. Treated BM cells from healthy normal donors were evaluated using flow cytometry for MYXV infection, long-term culture-initiating cell (LTC-IC) assay and colony-forming cell (CFC) assay. RESULTS: MYXV initiated infection in up to 45% of antigen-presenting monocytes, B cells and natural killer cells; however, these infections were uniformly aborted in >95% of all cells. Fresh graft sources showed higher levels of MYXV infection initiation than cryopreserved specimens, but in all cases less than 10% of CD34(+) cells could be infected after ex vivo MYXV treatment. MYXV did not impair LTC-IC colony numbers compared with mock treatment. CFC colony types and numbers were also not impaired by MYXV treatment. MYXV incubation time, temperature or culture media did not significantly change the percentage of infected cells, LTC-IC colony formation or CFC colony formation. CONCLUSIONS: Human hematopoietic cells are non-permissive for MYXV. Human hematopoietic stem and progenitor cells were not infected and thus unaffected by MYXV ex vivo treatment.

Effect of melphalan 140 mg/m(2) vs 200 mg/m(2) on toxicities and outcomes in multiple myeloma patients undergoing single autologous stem cell transplantation-a single center experience.

Although melphalan at a dose of 140 mg/m(2) (MEL140) is an acceptable conditioning regimen for autologous stem cell transplantation (ASCT) in multiple myeloma (MM) patients, very few studies compared it to the most commonly used dose of 200 mg/m(2) (MEL200). A retrospective review of records of MM patients (2001-2010) identified 33 patients who received MEL140 and 96 patients who received MEL200. As expected, significantly higher percentage of patients in the MEL140 arm were >65 years or had cardiac ejection fraction <50%, had Karnofsky score <80, or had creatinine >2 at the time of ASCT (P≤.01). There were no significant differences in incidence of treatment related mortality and morbidity. At a median follow-up of 74 months from ASCT, there were no significant differences in relapse free survival (RFS) and overall survival (OS) between the two groups. Similar proportion had myeloma status improve to ≥VGPR at 3 months post-ASCT. Usage of post-ASCT maintenance was similar. In multivariate cox proportional hazards model, only disease status of ≥VGPR at the time of ASCT significantly improved RFS (P=.024), but not OS (P=.104). In conclusion, MM patients who received MEL140 had similar long-term outcomes to MEL200 patients despite their older age and co-morbidities.

New cancers after autotransplantations for multiple myeloma.

We describe baseline incidence and risk factors for new cancers in 4161 persons receiving autotransplants for multiple myeloma in the United States from 1990 to 2010. Observed incidence of invasive new cancers was compared with expected incidence relative to the US population. The cohort represented 13,387 person-years at-risk. In total, 163 new cancers were observed, for a crude incidence rate of 1.2 new cancers per 100 person-years and cumulative incidences of 2.6% (95% confidence interval [CI], 2.09 to 3.17), 4.2% (95% CI, 3.49 to 5.00), and 6.1% (95% CI, 5.08 to 7.24) at 3, 5, and 7 years, respectively. The incidence of new cancers in the autotransplantation cohort was similar to age-, race-, and gender-adjusted comparison subjects with an observed/expected (O/E) ratio of 1.00 (99% CI, .81 to 1.22). However, acute myeloid leukemia and melanoma were observed at higher than expected rates with O/E ratios of 5.19 (99% CI, 1.67 to 12.04; P = .0004), and 3.58 (99% CI, 1.82 to 6.29; P < .0001), respectively. Obesity, older age, and male gender were associated with increased risks of new cancers in multivariate analyses. This large data set provides a baseline for comparison and defines the histologic type specific risk for new cancers in patients with MM receiving postautotransplantation therapies, such as maintenance.

Lenalidomide after stem-cell transplantation for multiple myeloma.

BACKGROUND: Data are lacking on whether lenalidomide maintenance therapy prolongs the time to disease progression after autologous hematopoietic stem-cell transplantation in patients with multiple myeloma. METHODS: Between April 2005 and July 2009, we randomly assigned 460 patients who were younger than 71 years of age and had stable disease or a marginal, partial, or complete response 100 days after undergoing stem-cell transplantation to lenalidomide or placebo, which was administered until disease progression. The starting dose of lenalidomide was 10 mg per day (range, 5 to 15). RESULTS: The study-drug assignments were unblinded in 2009, when a planned interim analysis showed a significantly longer time to disease progression in the lenalidomide group. At unblinding, 20% of patients who received lenalidomide and 44% of patients who received placebo had progressive disease or had died (P<0.001); of the remaining 128 patients who received placebo and who did not have progressive disease, 86 crossed over to lenalidomide. At a median follow-up of 34 months, 86 of 231 patients who received lenalidomide (37%) and 132 of 229 patients who received placebo (58%) had disease progression or had died. The median time to progression was 46 months in the lenalidomide group and 27 months in the placebo group (P<0.001). A total of 35 patients who received lenalidomide (15%) and 53 patients who received placebo (23%) died (P=0.03). More grade 3 or 4 hematologic adverse events and grade 3 nonhematologic adverse events occurred in patients who received lenalidomide (P<0.001 for both comparisons). Second primary cancers occurred in 18 patients who received lenalidomide (8%) and 6 patients who received placebo (3%). CONCLUSIONS: Lenalidomide maintenance therapy, initiated at day 100 after hematopoietic stem-cell transplantation, was associated with more toxicity and second cancers but a significantly longer time to disease progression and significantly improved overall survival among patients with myeloma. (Funded by the National Cancer Institute; ClinicalTrials.gov number, NCT00114101.).

Myxoma and vaccinia viruses bind differentially to human leukocytes.

Myxoma virus (MYXV) and vaccinia virus (VACV), two distinct members of the family Poxviridae, are both currently being developed as oncolytic virotherapeutic agents. Recent studies have demonstrated that ex vivo treatment with MYXV can selectively recognize and kill contaminating cancerous cells from autologous bone marrow transplants without perturbing the engraftment of normal CD34(+) hematopoietic stem and progenitor cells. However, the mechanism(s) by which MYXV specifically recognizes and eliminates the cancer cells in the autografts is not understood. While little is known about the cellular attachment factor(s) exploited by MYXV for entry into any target cells, VACV has been shown to utilize cell surface glycosaminoglycans such as heparan sulfate (HS), the extracellular matrix protein laminin, and/or integrin ß1. We have constructed MYXV and VACV virions tagged with the Venus fluorescent protein and compared their characteristics of binding to various human cancer cell lines as well as to primary human leukocytes. We report that the binding of MYXV or VACV to some adherent cell lines could be partially inhibited by heparin, but laminin blocked only VACV binding. In contrast to cultured fibroblasts, the binding of MYXV and VACV to a wide spectrum of primary human leukocytes could not be competed by either HS or laminin. Additionally, MYXV and VACV exhibited very different binding characteristics against certain select human leukocytes, suggesting that the two poxviruses utilize different cell surface determinants for the attachment to these cells. These results indicate that VACV and MYXV can exhibit very different oncolytic tropisms against some cancerous human leukocytes.

Salvage second hematopoietic cell transplantation in myeloma.

Autologous hematopoietic cell transplantation (AHCT) as initial therapy of patients with multiple myeloma (MM) improves survival. However, data to support this approach for relapsed/progressive disease after initial AHCT (AHCT1) are limited. Using Center for International Blood and Marrow Transplant Research data, we report the outcomes of 187 patients who underwent a second AHCT (AHCT2) for the treatment of relapsed/progressive MM. Planned tandem AHCT was excluded. Median age at AHCT2 was 59 years (range, 28 to 72), and median patient follow-up was 47 months (range, 3 to 97). Nonrelapse mortality after AHCT2 was 2% at 1 year and 4% at 3 years. Median interval from AHCT1 to relapse/progression was 18 months, and median interval between transplantations was 32 months. After AHCT2, the incidence of relapse/progression at 1 and 3 years was 51% and 82%, respectively. At 3 years after AHCT2, progression-free survival was 13%, and overall survival was 46%. In multivariate analyses, those relapsing ≥36 months after AHCT1 had superior progression-free (P = .045) and overall survival (P = .019). Patients who underwent AHCT2 after 2004 had superior survival (P = .026). AHCT2 is safe and feasible for disease progression after AHCT1. In this retrospective study, individuals relapsing ≥36 months from AHCT1 derived greater benefit from AHCT2 compared with those with a shorter disease-free interval. Storage of an adequate graft before AHCT1 will ensure that the option of a second autologous transplantation is retained for patients with relapsed/progressive MM.

Selective purging of human multiple myeloma cells from autologous stem cell transplantation grafts using oncolytic myxoma virus.

Autologous stem cell transplantation and novel therapies have improved overall survival of patients with multiple myeloma; however, most patients relapse and eventually succumb to their disease. Evidence indicates that residual cancer cells contaminate autologous grafts and may contribute to early relapses after autologous stem cell transplantation. Here, we demonstrate that ex vivo treatment with an oncolytic poxvirus called myxoma virus results in specific elimination of human myeloma cells by inducing rapid cellular apoptosis while fully sparing normal hematopoietic stem and progenitor cells. The specificity of this elimination is based on strong binding of the virus to myeloma cells coupled with an inability of the virus to bind or infect CD34(+) hematopoietic stem and progenitor cells. These 2 features allow myxoma to readily identify and distinguish even low levels of myeloma cells in complex mixtures. This ex vivo rabbit-specific oncolytic poxvirus called myxoma virus treatment also effectively inhibits systemic in vivo engraftment of human myeloma cells into immunodeficient mice and results in efficient elimination of primary CD138(+) myeloma cells contaminating patient hematopoietic cell products. We conclude that ex vivo myxoma treatment represents a safe and effective method to selectively eliminate myeloma cells from hematopoietic autografts before reinfusion.

Regulation of in vitro human T cell development through interleukin-7 deprivation and anti-CD3 stimulation.

BACKGROUND: The role of IL-7 and pre-TCR signaling during T cell development has been well characterized in murine but not in human system. We and others have reported that human BM hematopoietic progenitor cells (HPCs) display poor proliferation, inefficient double negative (DN) to double positive (DP) transition and no functional maturation in the in vitro OP9-Delta-like 1 (DL1) culture system. RESULTS: In this study, we investigated the importance of optimal IL-7 and pre-TCR signaling during adult human T cell development. Using a modified OP9-DL1 culture ectopically expressing IL-7 and Fms-like tyrosine kinase 3 ligand (Flt3L), we demonstrated enhanced T cell precursor expansion. IL-7 removal at various time points during T cell development promoted a slight increase of DP cells; however, these cells did not differentiate further and underwent cell death. As pre-TCR signaling rescues DN cells from programmed cell death, we treated the culture with anti-CD3 antibody. Upon pre-TCR stimulation, the IL-7 deprived T precursors differentiated into CD3+TCRαß+DP cells and further matured into functional CD4 T cells, albeit displayed a skewed TCR Vß repertoire. CONCLUSIONS: Our study establishes for the first time a critical control for differentiation and maturation of adult human T cells from HPCs by concomitant regulation of IL-7 and pre-TCR signaling.

Severe neutropenia following a prolonged course of vancomycin that progressed to agranulocytosis with drug reexposure.

OBJECTIVE: To report a case of severe neutropenia after discontinuing prolonged treatment with vancomycin that progressed to agranulocytosis with vancomycin reexposure. CASE SUMMARY: A 78-year-old woman presented with severe neutropenia (absolute neutrophil count [ANC] 37 cells/µL) and hypocellular bone marrow with absence of myeloid elements 8 weeks after discontinuing a 3-week treatment course of vancomycin 750 mg every 12 hours. Filgrastim 300 µg daily was started for neutropenia and vancomycin 750 mg every 12 hours and aztreonam 1 g every 8 hours were initiated for catheter-related acute thrombophlebitis of the upper extremity. The patient's ANC decreased to 10 cells/µL within 3 days of starting vancomycin. We suspected an autoimmune process, potentially related to vancomycin exposure, and began treatment with methylprednisolone 1 mg/kg daily. The ANC precipitously dropped to 0 cells/µL despite treatment with steroids and an increased filgrastim dose of 480 µg/day. All antibiotics were discontinued on the fifth day of hospitalization. Within 48 hours, her neutrophil count showed recovery (white blood cell count 500 cells/µL; 10% neutrophils). DISCUSSION: Idiosyncratic drug-induced agranulocytosis is an uncommon phenomenon but is often associated with serious consequences such as sepsis. We believe this case is unique because of the unusually late neutropenia discovered several weeks after finishing a prolonged course of vancomycin. Furthermore, agranulocytosis developed after unintentional rechallenge with vancomycin. According to the Naranjo probability scale, this case illustrates a probable adverse event caused by vancomycin. CONCLUSIONS: This case demonstrates a serious adverse event potentially associated with vancomycin use, and calls attention to the safety of rechallenging with vancomycin during a possible drug-induced neutropenia.

Lactate Dehydrogenase B and Pyruvate Oxidation Pathway Associated With Carfilzomib-Related Cardiotoxicity in Multiple Myeloma Patients: Result of a Multi-Omics Integrative Analysis.

Multiple myeloma (MM) is the second most frequent hematologic cancer in the United States. Carfilzomib (CFZ), an irreversible proteasome inhibitor being used to treat relapsed and refractory MM, has been associated with cardiotoxicity, including heart failure. We hypothesized that a multi-omics approach integrating data from different omics would provide insights into the mechanisms of CFZ-related cardiovascular adverse events (CVAEs). Plasma samples were collected from 13 MM patients treated with CFZ (including 7 with CVAEs and 6 with no CVAEs) at the University of Florida Health Cancer Center. These samples were evaluated in global metabolomic profiling, global proteomic profiling, and microRNA (miRNA) profiling. Integrative pathway analysis was performed to identify genes and pathways differentially expressed between patients with and without CVAEs. The proteomics analysis identified the up-regulation of lactate dehydrogenase B (LDHB) [fold change (FC) = 8.2, p = 0.01] in patients who experienced CVAEs. The metabolomics analysis identified lower plasma abundance of pyruvate (FC = 0.16, p = 0.0004) and higher abundance of lactate (FC = 2.4, p = 0.0001) in patients with CVAEs. Differential expression analysis of miRNAs profiling identified mir-146b to be up-regulatein (FC = 14, p = 0.046) in patients with CVAE. Pathway analysis suggested that the pyruvate fermentation to lactate pathway is associated with CFZ-CVAEs. In this pilot multi-omics integrative analysis, we observed the down-regulation of pyruvate and up-regulation of LDHB among patients who experienced CVAEs, suggesting the importance of the pyruvate oxidation pathway associated with mitochondrial dysfunction. Validation and further investigation in a larger independent cohort are warranted to better understand the mechanisms of CFZ-CVAEs.

Melflufen and Dexamethasone in Heavily Pretreated Relapsed and Refractory Multiple Myeloma.

PURPOSE: Melphalan flufenamide (melflufen) is a first-in-class peptide-drug conjugate that targets aminopeptidases and rapidly and selectively releases alkylating agents into tumor cells. The phase II HORIZON trial evaluated the efficacy of melflufen plus dexamethasone in relapsed and refractory multiple myeloma (RRMM), a population with an important unmet medical need. PATIENTS AND METHODS: Patients with RRMM refractory to pomalidomide and/or an anti-CD38 monoclonal antibody received melflufen 40 mg intravenously on day 1 of each 28-day cycle plus once weekly oral dexamethasone at a dose of 40 mg (20 mg in patients older than 75 years). The primary end point was overall response rate (partial response or better) assessed by the investigator and confirmed by independent review. Secondary end points included duration of response, progression-free survival, overall survival, and safety. The primary analysis is complete with long-term follow-up ongoing. RESULTS: Of 157 patients (median age 65 years; median five prior lines of therapy) enrolled and treated, 119 patients (76%) had triple-class-refractory disease, 55 (35%) had extramedullary disease, and 92 (59%) were refractory to previous alkylator therapy. The overall response rate was 29% in the all-treated population, with 26% in the triple-class-refractory population. In the all-treated population, median duration of response was 5.5 months, median progression-free survival was 4.2 months, and median overall survival was 11.6 months at a median follow-up of 14 months. Grade ≥ 3 treatment-emergent adverse events occurred in 96% of patients, most commonly neutropenia (79%), thrombocytopenia (76%), and anemia (43%). Pneumonia (10%) was the most common grade 3/4 nonhematologic event. Thrombocytopenia and bleeding (both grade 3/4 but fully reversible) occurred concomitantly in four patients. GI events, reported in 97 patients (62%), were predominantly grade 1/2 (93%); none were grade 4. CONCLUSION: Melflufen plus dexamethasone showed clinically meaningful efficacy and a manageable safety profile in patients with heavily pretreated RRMM, including those with triple-class-refractory and extramedullary disease.

Genome-wide Association Study Identified Chromosome 8 Locus Associated with Medication-Related Osteonecrosis of the Jaw.

Medication-related osteonecrosis of the jaw (MRONJ) is a rare but serious drug-related adverse event. To identify pharmacogenomic markers of MRONJ associated with bisphosphonate therapy, we conducted a genomewide association study (GWAS) meta-analysis followed by functional analysis of 5,008 individuals of European ancestry treated with bisphosphonates, which includes the largest number of MRONJ cases to date (444 cases and 4,564 controls). Discovery GWAS was performed in randomly selected 70% of the patients with cancer and replication GWAS was performed in the remaining 30% of the patients with cancer treated with intravenous bisphosphonates followed by meta-analysis of all 3,639 patients with cancer. GWAS was also performed in 1,369 patients with osteoporosis treated with oral bisphosphonates. The lead single-nucleotide polymorphism (SNP), rs2736308 on chromosome 8, was associated with an increased risk of MRONJ with an odds ratio (OR) of 2.71 and 95% confidence interval (CI) of 1.90-3.86 (P = 3.57*10-8 ) in the meta-analysis of patients with cancer. This SNP was validated in the MRONJ GWAS in patients with osteoporosis (OR: 2.82, 95% CI: 1.55-4.09, P = 6.84*10-4 ). The meta-analysis combining patients with cancer and patients with osteoporosis yielded the same lead SNP rs2736308 on chromosome 8 as the top SNP (OR: 2.74, 95% CI: 2.09-3.39, P = 9.65*10-11 ). This locus is associated with regulation of the BLK, CTSB, and FDFT1 genes, which had been associated with bone mineral density. FDFT1 encodes a membrane-associated enzyme, which is implicated in the bisphosphonate pathway. This study provides insights into the potential mechanism of MRONJ.

Obesity does not preclude safe and effective myeloablative hematopoietic cell transplantation (HCT) for acute myelogenous leukemia (AML) in adults.

The incidence of excessive adiposity is increasing worldwide, and is associated with numerous adverse health outcomes. We compared outcomes by body mass index (BMI) for adult patients with acute myelogenous leukemia (AML) who underwent autologous (auto, n = 373), related donor (RD, n = 2041), or unrelated donor (URD, n = 1801) allogeneic myeloablative hematopoietic cell transplantation (HCT) using bone marrow or peripheral blood stem cells reported to the Center for International Blood and Marrow Transplant Research (CIBMTR) from 1995 to 2004. Four weight groups by BMI (kg/m(2)) were defined: underweight <18 kg/m(2); normal 18-25 kg/m(2); overweight >25-30 kg/m(2); and obese >30 kg/m(2). Multivariable analysis referenced to the normal weight group showed an increased risk of death for underweight patients in the RD group (relative risk [RR], 1.92; 95% confidence interval [CI], 1.28-2.89; P = .002), but not in the URD group. There were no other differences in outcomes among the other weight groups within the other HCT groups. Overweight and obese patients enjoyed a modest decrease in relapse incidence, although this did not translate into a survival benefit. Small numbers of patients limit the ability to better characterize the adverse outcomes seen in the underweight RD but not the underweight URD allogeneic HCT patients. Obesity alone should not be considered a barrier to HCT.

Ex vivo development, expansion and in vivo analysis of a novel lineage of dendritic cells from hematopoietic stem cells.

Dendritic cells (DCs) play a key role in innate and adaptive immunity but the access to sufficient amount of DCs for basic and translational research has been limited.We established a novel ex vivo system to develop and expand DCs from hematopoietic stem/progenitor cells (HPCs). Both human and mouse HPCs were expanded first in feeder culture supplemented with c-Kit ligand (KL, stem cell factor, steel factor or CD117 ligand), Flt3 ligand (fms-like tyrosine kinase 3, Flt3L, FL), thrombopoietin (TPO), IL-3, IL-6, and basic fibroblast growth factor (bFGF), and then in a second feeder culture ectopically expressing all above growth factors plus GM-CSF and IL-15.In the dual culture system, CD34+ HPCs differentiated toward DC progenitors (DCPs), which expanded more than five orders of magnitude. The DCPs showed myeloid DC surface phenotype with up-regulation of transcription factors PU.1 and Id2, and DC-related factors homeostatic chemokine ligand 17 (CCL17) and beta-chemokine receptor 6 (CCR6). Multiplex ELISA array and cDNA microarray analyses revealed that the DCPs shared some features of IL-4 and IL-15 DCs but displayed a pronounced proinflammatory phenotype. DCP-derived DCs showed antigen-uptake and immune activation functions analogous to that of the peripheral blood-derived DCs. Furthermore, bone marrow HPC-derived DCP vaccines of tumor-bearing mice suppressed tumor growth in vivo.This novel approach of generating DCP-DCs, which are different from known IL-4 and IL-15 DCs, overcomes both quantitative and qualitative limitations in obtaining functional autologous DCs from a small number of HPCs with great translational potential.

Diverse T-cell differentiation potentials of human fetal thymus, fetal liver, cord blood and adult bone marrow CD34 cells on lentiviral Delta-like-1-modified mouse stromal cells.

Human haematopoietic progenitor/stem cells (HPCs) differentiate into functional T cells in the thymus through a series of checkpoints. A convenient in vitro system will greatly facilitate the understanding of T-cell development and future engineering of therapeutic T cells. In this report, we established a lentiviral vector-engineered stromal cell line (LSC) expressing the key lymphopoiesis regulator Notch ligand, Delta-like 1 (DL1), as feeder cells (LSC-mDL1) supplemented with Flt3 ligand (fms-like tyrosine kinase 3, Flt3L or FL) and interleukin-7 for the development of T cells from CD34(+) HPCs. We demonstrated T-cell development from human HPCs with various origins including fetal thymus (FT), fetal liver (FL), cord blood (CB) and adult bone marrow (BM). The CD34(+) HPCs from FT, FL and adult BM expanded more than 100-fold before reaching the beta-selection and CD4/CD8 double-positive T-cell stage. The CB HPCs, on the other hand, expanded more than 1000-fold before beta-selection. Furthermore, the time required to reach beta-selection differed for the various HPCs, 7 days for FT, 14 days for FL and CB, and 35 days for adult BM. Nevertheless, all of the T cells developed in vitro were stalled at the double-positive or immature single-positive stage with the exception that some CB-derived T cells arrived at a positive selection stage. Consequently, the LSC-mDL1 culture system illustrated diverse T-cell development potentials of pre- and post-natal and adult human BM HPCs. However, further modification of this in vitro T-cell development system is necessary to attain fully functional T cells.