Massive clonal expansion of medulloblastoma-specific T cells during adoptive cellular therapy.

In both human and murine systems, we have developed an adoptive cellular therapy platform against medulloblastoma and glioblastoma that uses dendritic cells pulsed with a tumor RNA transcriptome to expand polyclonal tumor-reactive T cells against a plurality of antigens within heterogeneous brain tumors. We demonstrate that peripheral TCR Vß repertoire analysis after adoptive cellular therapy reveals that effective response to adoptive cellular therapy is concordant with massive in vivo expansion and persistence of tumor-specific T cell clones within the peripheral blood. In preclinical models of medulloblastoma and glioblastoma, and in a patient with relapsed medulloblastoma receiving adoptive cellular therapy, an early and massive expansion of tumor-reactive lymphocytes, coupled with prolonged persistence in the peripheral blood, is observed during effective therapeutic response to immunotherapy treatment.

Umbilical cord blood donation: public or private?

Umbilical cord blood (UCB) is a graft source for patients with malignant or genetic diseases who can be cured by allogeneic hematopoietic cell transplantation (HCT), but who do not have an appropriately HLA-matched family or volunteer unrelated adult donor. Starting in the 1990s, unrelated UCB banks were established, accepting donations from term deliveries and storing UCB units for public use. An estimated 730 000 UCB units have been donated and stored to date and ~35 000 UCB transplants have been performed worldwide. Over the past 20 years, private and family banks have grown rapidly, storing ~4 million UCB units for a particular patient or family, usually charging an up-front and yearly storage fee; therefore, these banks are able to be financially sustainable without releasing UCB units. Private banks are not obligated to fulfill the same regulatory requirements of the public banks. The public banks have released ~30 times more UCB units for therapy. Some countries have transitioned to an integrated banking model, a hybrid of public and family banking. Today, pregnant women, their families, obstetrical providers and pediatricians are faced with multiple choices about the disposition of their newborn's cord blood. In this commentary, we review the progress of UCB banking technology; we also analyze the current data on pediatric and adult unrelated UCB, including the recent expansion of interest in transplantation for hemoglobinopathies, and discuss emerging studies on the use of autologous UCB for neurologic diseases and regenerative medicine. We will review worldwide approaches to UCB banking, ethical considerations, criteria for public and family banking, integrated banking ideas and future strategies for UCB banking.

Newborns of obese parents have altered DNA methylation patterns at imprinted genes.

BACKGROUND: Several epidemiologic studies have demonstrated associations between periconceptional environmental exposures and health status of the offspring in later life. Although these environmentally related effects have been attributed to epigenetic changes, such as DNA methylation shifts at imprinted genes, little is known about the potential effects of maternal and paternal preconceptional overnutrition or obesity. OBJECTIVE: We examined parental preconceptional obesity in relation to DNA methylation profiles at multiple human imprinted genes important in normal growth and development, such as: maternally expressed gene 3 (MEG3), mesoderm-specific transcript (MEST), paternally expressed gene 3 (PEG3), pleiomorphic adenoma gene-like 1 (PLAGL1), epsilon sarcoglycan and paternally expressed gene 10 (SGCE/PEG10) and neuronatin (NNAT). METHODS: We measured methylation percentages at the differentially methylated regions (DMRs) by bisulfite pyrosequencing in DNA extracted from umbilical cord blood leukocytes of 92 newborns. Preconceptional obesity, defined as BMI ⩾30 kg m(-2), was ascertained through standardized questionnaires. RESULTS: After adjusting for potential confounders and cluster effects, paternal obesity was significantly associated with lower methylation levels at the MEST (ß=-2.57; s.e.=0.95; P=0.008), PEG3 (ß=-1.71; s.e.=0.61; P=0.005) and NNAT (ß=-3.59; s.e.=1.76; P=0.04) DMRs. Changes related to maternal obesity detected at other loci were as follows: ß-coefficient was +2.58 (s.e.=1.00; P=0.01) at the PLAGL1 DMR and -3.42 (s.e.=1.69; P=0.04) at the MEG3 DMR. CONCLUSION: We found altered methylation outcomes at multiple imprint regulatory regions in children born to obese parents, compared with children born to non-obese parents. In spite of the small sample size, our data suggest a preconceptional influence of parental life-style or overnutrition on the (re)programming of imprint marks during gametogenesis and early development. More specifically, the significant and independent association between paternal obesity and the offspring's methylation status suggests the susceptibility of the developing sperm for environmental insults. The acquired imprint instability may be carried onto the next generation and increase the risk for chronic diseases in adulthood.

Sequential myeloablative autologous stem cell transplantation and reduced intensity allogeneic hematopoietic cell transplantation is safe and feasible in children, adolescents and young adults with poor-risk refractory or recurrent Hodgkin and non-Hodgkin lymphoma.

The outcome of children, adolescents and young adults (CAYA) with poor-risk recurrent/refractory lymphoma is dismal (⩽30%). To overcome this poor prognosis, we designed an approach to maximize an allogeneic graft vs lymphoma effect in the setting of low disease burden. We conducted a multi-center prospective study of myeloablative conditioning (MAC) and autologous stem cell transplantation (AutoSCT), followed by a reduced intensity conditioning (RIC) and allogeneic hematopoietic cell transplantation (AlloHCT) in CAYA, with poor-risk refractory or recurrent lymphoma. Conditioning for MAC AutoSCT consisted of carmustine/etoposide/cyclophosphamide, RIC consisted of busulfan/fludarabine. Thirty patients, 16 Hodgkin lymphoma (HL) and 14 non-Hodgkin lymphoma (NHL), with a median age of 16 years and median follow-up of 5years, were enrolled. Twenty-three patients completed both MAC AutoSCT and RIC AlloHCT. Allogeneic donor sources included unrelated cord blood (n=9), unrelated donor (n=8) and matched siblings (n=6). The incidence of transplant-related mortality following RIC AlloHCT was only 12%. In patients with HL and NHL, 10 year EFS was 59.8% and 70% (P=0.613), respectively. In summary, this approach is safe, and long-term EFS with this approach is encouraging considering the poor-risk patient characteristics and the use of unrelated donors for RIC AlloHCT in the majority of cases.

Associations between antibiotic exposure during pregnancy, birth weight and aberrant methylation at imprinted genes among offspring.

OBJECTIVES: Low birth weight (LBW) has been associated with common adult-onset chronic diseases, including obesity, cardiovascular disease, type II diabetes and some cancers. The etiology of LBW is multi-factorial. However, recent evidence suggests exposure to antibiotics may also increase the risk of LBW. The mechanisms underlying this association are unknown, although epigenetic mechanisms are hypothesized. In this study, we evaluated the association between maternal antibiotic use and LBW and examined the potential role of altered DNA methylation that controls growth regulatory imprinted genes in these associations. METHODS: Between 2009-2011, 397 pregnant women were enrolled and followed until delivery. Prenatal antibiotic use was ascertained through maternal self-report. Imprinted genes methylation levels were measured at differentially methylated regions (DMRs) using bisulfite pyrosequencing. Generalized linear models were used to examine associations among antibiotic use, birth weight and DMR methylation fractions. RESULTS: After adjusting for infant gender, race/ethnicity, maternal body mass index, delivery route, gestational weight gain, gestational age at delivery, folic acid intake, physical activity, maternal smoking and parity, antibiotic use during pregnancy was associated with 138 g lower birth weight compared with non-antibiotic use (ß-coefficient=-132.99, s.e.=50.70, P=0.008). These associations were strongest in newborns of women who reported antibiotic use other than penicillins (ß-coefficient=-135.57, s.e.=57.38, P=0.02). Methylation at five DMRs, IGF2 (P=0.05), H19 (P=0.15), PLAGL1 (P=0.01), MEG3 (P=0.006) and PEG3 (P=0.08), was associated with maternal antibiotic use; among these, only methylation at the PLAGL1 DMR was also associated with birth weight. CONCLUSION: We report an inverse association between in utero exposure to antibiotics and lower infant birth weight and provide the first empirical evidence supporting imprinted gene plasticity in these associations.

Pre-engraftment syndrome after myeloablative dual umbilical cord blood transplantation: risk factors and response to treatment.

High fevers and/or rashes prior to neutrophil engraftment are frequently observed after umbilical cord blood (UCB) transplantation, and the condition is referred to as pre-engraftment syndrome (PES). Few studies have evaluated the risk factors for and treatment response to PES. Therefore, we retrospectively characterized PES in 57 consecutive engrafted patients (≥ 12 years old) who received myeloablative dual UCB transplantation. All patients received TBI (≥ 13.2 Gy)-based myeloablative conditioning. Tacrolimus (n=35) or CYA (n=22) combined with mycophenolate mofetil was used as GVHD prophylaxis. PES was defined as the presence of non-infectious fever (≥ 38.5 °C) and/or rash prior to or on the day of neutrophil engraftment. The incidence (95% confidence interval) of PES was 77% (66-88%). The incidence of PES was significantly higher in patients who received CYA as a GVHD prophylaxis than those who received tacrolimus (P<0.001), and this association was confirmed in the multivariate analysis. The occurrence of PES did not impact OS or tumor relapse, although it may have increased non-relapse mortality (P=0.071). The incidence of acute GHVD or treatment-related mortality was not influenced by the choice to use corticosteroids to treat PES. This study suggests that use of CYA for GVHD prophylaxis increases the risk of PES following dual UCB transplantation.

Rescuing the neonatal brain from hypoxic injury with autologous cord blood.

Brain injury resulting from perinatal hypoxic-ischemic encephalopathy (HIE) is a major cause of acute mortality in infants and chronic neurologic disability in surviving children. Recent multicenter clinical trials demonstrated the effectiveness of hypothermia initiated within the first 6 postnatal hours to reduce the risk of death or major neurological disabilities among neonates with HIE. However, in these trials, approximately 40% of cooled infants died or survived with significant impairments. Therefore, adjunct therapies are required to improve the outcome in neonates with HIE. Cord blood (CB) is a rich source of stem cells. Administration of human CB cells in animal models of HIE has generally resulted in improved outcomes and multiple mechanisms have been suggested including anti-inflammation, release of neurotrophic factors and stimulation of endogenous neurogenesis. Investigators at Duke are conducting studies of autologous CB infusion in neonates with HIE and in children with cerebral palsy. These pilot studies indicate no added risk from the regimens used, but results of ongoing placebo-controlled trials are needed to assess efficacy. Meanwhile, further investigations are warranted to determine the best strategies, that is, timing, dosing, route of delivery, choice of stem cells and ex vivo modulations, to attain long-term benefits of CB stem cell therapy.

Extensive haplotype diversity in African American mothers and their cord blood units.

HLA-A, -B, -C, -DRB1, -DQB1 assignments were obtained for 374 pairs of African American mothers and their umbilical cord blood units (CBU) by DNA sequencing. An algorithm developed by the National Marrow Donor Program was used to assign 1122 haplotypes by segregation. Seventy percent of the haplotypes carried assignments at all five loci. In the remainder, alleles at various loci, most often DQB1 in 48% of the haplotypes with a missing assignment, could not be assigned due to sharing of both alleles by mother and CBU. There were 652 haplotypes carrying a unique combination of alleles at the five loci; the majority (74%) were singletons. Novel B∼C and DRB1~DQB1 associations were observed. The results show the genetic diversity in this population and provide validation for a publically available tool for pedigree analysis. Our observations underscore the need for procurement of increased numbers of units in the national cord blood inventory in order to identify matching donors for all patients requiring hematopoietic stem cell transplantation.

Allogeneic hematopoietic SCT for alpha-mannosidosis: an analysis of 17 patients.

Alpha-mannosidosis is a rare lysosomal storage disease. Hematopoietic SCT (HSCT) is usually recommended as a therapeutic option though reports are anecdotal to date. This retrospective multi institutional analysis describes 17 patients that were diagnosed at a median of 2.5 (1.1-23) years and underwent HSCT at a median of 3.6 (1.3-23.1) years. In all, 15 patients are alive (88%) after a median follow-up of 5.5 (2.1-12.6) years. Two patients died within the first 5 months after HSCT. Of the survivors, two developed severe acute GvHD (>=grade II) and six developed chronic GvHD. Three patients required re-transplantation because of graft failure. All 15 showed stable engraftment. The extent of the patients' developmental delay before HSCT varied over a wide range. After HSCT, patients made developmental progress, although normal development was not achieved. Hearing ability improved in some, but not in all patients. We conclude that HSCT is a feasible therapeutic option that may promote mental development in alpha-mannosidosis.

The effects of depression and use of antidepressive medicines during pregnancy on the methylation status of the IGF2 imprinted control regions in the offspring.

In utero exposures to environmental factors may result in persistent epigenetic modifications affecting normal development and susceptibility to chronic diseases in later life. We explored the relationship between exposure of the growing fetus to maternal depression or antidepressants and DNA methylation at two differentially methylated regions (DMRs) of the imprinted Insulin-like Growth Factor 2 (IGF2) gene. Aberrant DNA methylation at the IGF2 and neighboring H19 DMRs has been associated with deregulated IGF2 expression, childhood cancers and several chronic diseases during adulthood. Our study population is comprised of pregnant mothers and their newborns (n = 436), as part of the Newborn Epigenetics Study (NEST). A standardized questionnaire was completed and medical record data were abstracted to ascertain maternal depression and antidepressive drug use. DMR methylation levels in umbilical cord blood leukocytes were quantified using pyrosequencing. From the 436 newborns, laboratory data were obtained for 356 individuals at the IGF2 DMRs, and for 411 individuals at the H19 DMRs; about half of each group was African American or Caucasian. While overall no association between depression and methylation profiles was found, we observed a significant hypermethylation of the H19 DMRs in newborns of African American (n = 177) but not Caucasian (n = 168) mothers who reported the use of antidepressive drugs during pregnancy (ß = +6.89, p = 0.01). Of note, our data reveal a race-independent association between smoking during pregnancy and methylation at the IGF2 DMR (+3.05%, p = 0.01). In conclusion, our findings suggest a race-dependent response related to maternal use of antidepressants at one of the IGF2 DMRs in the offspring.

Umbilical cord blood transplantation for non-malignant diseases.

Many factors, including lower risk of GVHD, rapid availability of 4/6-6/6 matched cord blood (CB) units and incremental gains in the outcomes, have led to an increasing use of CB transplantation (CBT) to treat many patients who lack fully matched adult BM donors. A large electronically searchable worldwide inventory of publicly banked CB units allows for quicker donor identification and selection. In this review, we examine the current status and cumulative experience of related and unrelated donor CBT for the treatment of non-malignant diseases, including hemoglobinopathies, BM failure syndromes, primary immunodeficiency diseases (PIDs) and inherited metabolic disorders (IMDs), and conclude that CBT offers a promising and effective therapy for these diseases. Future strategies to facilitate earlier diagnosis and to decrease transplant-related risks should further improve the short- and long-term outcomes. Every effort should be made to perform transplantation early in the course of disease before extensive damage to various tissues and organs ensues.

Diffusion tensor imaging detects abnormalities in the corticospinal tracts of neonates with infantile Krabbe disease.

BACKGROUND AND PURPOSE: It is not possible to determine if neonates diagnosed with Krabbe disease through statewide neonate screening programs will develop the disease as infants, juveniles, or adults. The only available treatment for this fatal neurodegenerative condition is unrelated umbilical cord transplantation, but this treatment is only effective before clinical symptoms appear. Therefore, a marker of disease progression is needed. The purpose of this study was to evaluate the use of diffusion tensor imaging (DTI) with fiber tracking in identifying early changes in major motor tracts of asymptomatic neonates with infantile Krabbe disease. MATERIALS AND METHODS: Six neonates with infantile Krabbe disease identified because of family history underwent brain MR imaging within the first 4 weeks of life. Six-direction DTI and quantitative tractography of the corticospinal tracts were performed. Hypothesis tests, 1 for each hemisphere, were used to determine whether the fractional anisotropy (FA) ratio of the neonates with infantile Krabbe disease was significantly different from that of 45 age- and sex-matched controls. RESULTS: The average FA ratio for patients with Krabbe disease was 0.89 and 0.87 for left and right tracts, respectively (P = .002 and < .001). After adjusting for gestational age, gestational age at birth, birth weight, sex, and race, the 6 patients with Krabbe disease had significantly lower FA values than the controls (P < .001). CONCLUSIONS: DTI with quantitative tractography detected significant differences in the corticospinal tracts of asymptomatic neonates who had the early-onset form of Krabbe disease. Once standardized and validated, this tool has the potential to be used as a marker of disease progression in neonates diagnosed through statewide neonate screening programs.

Unintended changes in cognition, mood, and behavior arising from cell-based interventions for neurological conditions: ethical challenges.

The prospect of using cell-based interventions (CBIs) to treat neurological conditions raises several important ethical and policy questions. In this target article, we focus on issues related to the unique constellation of traits that characterize CBIs targeted at the central nervous system. In particular, there is at least a theoretical prospect that these cells will alter the recipients' cognition, mood, and behavior-brain functions that are central to our concept of the self. The potential for such changes, although perhaps remote, is cause for concern and careful ethical analysis. Both to enable better informed consent in the future and as an end in itself, we argue that early human trials of CBIs for neurological conditions must monitor subjects for changes in cognition, mood, and behavior; further, we recommend concrete steps for that monitoring. Such steps will help better characterize the potential risks and benefits of CBIs as they are tested and potentially used for treatment.

Isolation of oligodendrocyte-like cells from human umbilical cord blood.

BACKGROUND: As human umbilical cord blood (UCB) is known to be a rich source of progenitor cells, the prospect of isolating a subset of these cells that could differentiate into cells of non-hematopoietic lineages suggests a therapeutic use for patients with inherited lysosomal and peroxisomal storage diseases currently treated with UCB transplantation. METHODS: Oligodendrocyte-like cells were isolated from UCB by density-gradient centrifugation and expanded using selective media. We then characterized this population of cells using standard immunohistochemical staining methods for neural cell proteins and polymerase chain reaction (PCR) to detect RNA sequences for myelin basic protein (MBP). We also developed a functional assay demonstrating myelination of neurons in vitro. RESULTS: Cells with oligodendrocyte-like morphology were reproducibly cultured ex vivo from fresh human UCB. Cells stained positively for multiple oligodendria cell markers (O1, MBP and CNPase) via immunohistochemical staining and flow cytometry. PCR confirmed the presence of MBP and CNPase mRNA. A further in vitro functional assay demonstrated the myelination of mature neuronal cells from the brain of a myelin-deficient murine model co-cultured with the oligodendrocyte-like cells. DISCUSSION: After human UCB transplant, donor-derived cells have been noted to migrate to the brain over time. Although is not known whether these cells solely deliver enzyme replacement or a subset engrafts and differentiates into mature neural cells, the clinical improvements noted in these patients suggest a potential role for targeted cellular therapy. Oligodendrocyte-like cells isolated ex vivo and expanded from human UCB could provide a potential cellular therapy for patients with demyelinating or dismyelinating diseases.

Cell-based interventions for neurologic conditions: ethical challenges for early human trials.

BACKGROUND: Attempts to translate basic stem cell research into treatments for neurologic diseases and injury are well under way. With a clinical trial for one such treatment approved and in progress in the United States, and additional proposals under review, we must begin to address the ethical issues raised by such early forays into human clinical trials for cell-based interventions for neurologic conditions. METHODS: An interdisciplinary working group composed of experts in neuroscience, cell biology, bioethics, law, and transplantation, along with leading disease researchers, was convened twice over 2 years to identify and deliberate on the scientific and ethical issues raised by the transition from preclinical to clinical research of cell-based interventions for neurologic conditions. RESULTS: While the relevant ethical issues are in many respects standard challenges of human subjects research, they are heightened in complexity by the novelty of the science, the focus on the CNS, and the political climate in which the science is proceeding. CONCLUSIONS: Distinctive challenges confronting US scientists, administrators, institutional review boards, stem cell research oversight committees, and others who will need to make decisions about work involving stem cells and their derivatives and evaluate the ethics of early human trials include evaluating the risks, safety, and benefits of these trials, determining and evaluating cell line provenance, and determining inclusion criteria, informed consent, and the ethics of conducting early human trials in the public spotlight. Further study and deliberation by stakeholders is required to move toward professional and institutional policies and practices governing this research.

Emerging trends in transplantation of inherited metabolic diseases.

Allogeneic hematopoietic stem cell transplantation (HSCT) can prolong life and improve its quality in patients with inherited metabolic diseases. HSCT offers a permanent source of enzyme replacement therapy and also might mediate nonhematopoietic cell regeneration or repair. Unrelated cord blood is an exciting newer graft source for treatment of patients with these fatal disorders, providing increased access to donors and significant clinical efficacy, particularly when transplantation is performed in early stages. Pre-transplant performance status is highly predictive of overall survival.

Modification of a commercial cell sorter to support efficient and reliable preparation of ALDH-bright cells for clinical use.

BACKGROUND: Cell populations manufactured by conventional commercial cell sorters have been safely infused into patients, but reliably sterilizing these instruments remains challenging. We are developing clinical protocols involving use of ALDH bright cells manufactured by cell sorting in patients. However, we encountered problems when we attempted to reliably sterilize the FACSAria cell sorter using standard methods. RESULTS: We have identified and modified potential sources of microbial contamination in several FACSAria systems. We added new filter systems to the sheath and sample air lines, to the wet cart fluid supply, and to the sample line. Sheath was provided from an external sterile, disposable bag through sterile disposable tubing sets. The plenum reservoirs were modified in several ways to allow efficient decontamination of internal surfaces. A new bubble filter assembly was added and one valve was eliminated from the sample pathway to improve flow cell sterilization. A new cleaning and sterilization protocol was developed and validated. All cell products manufactured using the modified instrument and validated cleaning protocol have met lot release criteria for prevention of microbial contamination and safe clinical use. DISCUSSION: The instrument modification and cleaning protocol described enable reliable manufacture of ALDH bright cell populations that are suitable for clinical trials. We have manufactured nineteen consecutive samples that meet all clinical release criteria in an on-going Phase 1 human trial.

Isolation of early hematopoietic cells, including megakaryocyte progenitors, in the ALDH-bright cell population of cryopreserved, banked UC blood.

BACKGROUND: ALDH-bright (ALDH(br)) cell populations sorted from freshly collected umbilical cord blood (UCB) on the basis of their high aldehyde dehydrogenase (ALDH) activity are highly enriched for HPC. HPC with low ALDH activity (ALDH(dim)) are primarily short-term progenitors, whereas progenitors that initiate long-term cultures or establish long-term grafts in xenograft models are ALDH(br). We examined the multilineage hematopoietic and platelet progenitor activities of ALDH(br) cells recovered from cryopreserved UCB units typically employed in the practice of clinical transplantation. METHODS: Frozen UCB units were thawed, washed, immunomagnetically depleted of cells expressing glycophorin A and CD14, reacted for flow cytometric detection of ALDH, and sorted to yield ALDH(br) and ALDH(dim) populations. We measured surface Ag expression and viability of cells in the ALDH(br) and ALDH(dim) populations by flow cytometry and hematopoietic (CFC-H) and megakaryocytic (CFC-Mk) colony-forming cells in each population. RESULTS: ALDH(br) populations isolated from thawed UCB cells were highly enriched for CD34(+) and CD133(+) cells. Flow-sorted ALDH(br) populations were enriched 1116-fold in CFC-H, 10-fold in multilineage GEMM colonies and 2015-fold in CFC-Mk compared with the ALDH(dim) population. All progenitors giving rise to large Mk colonies were derived from ALDH(br) populations. DISCUSSION: ALDH(br) populations recovered from thawed, banked UCB with the method we describe have HPC activity and may be useful in the clinic to facilitate reconstitution of erythroid, myeloid and megakaryocytic blood elements.

Hematopoietic cell transplantation for Chediak-Higashi syndrome.

We reviewed outcomes after allogeneic hematopoietic cell transplantation (HCT) in 35 children with Chediak-Higashi syndrome (CHS). Twenty-two patients had a history of the life-threatening accelerated phase of CHS before HCT and 11 were in accelerated phase at transplantation. Thirteen patients received their allograft from an human leukocyte antigen (HLA)-matched sibling, 10 from an alternative related donor and 12 from an unrelated donor. Eleven recipients of HLA-matched sibling donor, three recipients of alternative related donor and eight recipients of unrelated donor HCT are alive. With a median follow-up of 6.5 years, the 5-year probability of overall survival is 62%. Mortality was highest in those with accelerated phase disease at transplantation and after alternative related donor HCT. Only four of 11 patients with active disease at transplantation are alive. Seven recipients of alternative related donor HCT had active disease at transplantation and this may have influenced the poor outcome in this group. Although numbers are limited, HCT appears to be effective therapy for correcting and preventing hematologic and immunologic complications of CHS, and an unrelated donor may be a suitable alternative for patients without an HLA-matched sibling. Early referral and transplantation in remission after accelerated phase disease may improve disease-free survival.