Mark C. Walters

Matched-related donor transplantation for sickle cell disease: report from the Center for International Blood and Transplant Research

We report outcomes after myeloablative haematopoietic cell transplantation (HCT) from human leucocyte antigen (HLA)‐matched sibling donors in 67 patients with sickle cell disease transplanted between 1989 and 2002. The most common indications for transplantation were stroke and recurrent vaso‐occlusive crisis in 38% and 37% of patients respectively. The median age at transplantation was 10u2003years and 67% of patients had received >10 red blood cell transfusions before HCT. Twenty‐seven percent of patients had a poor performance score at transplantation. Ninety‐four percent received busulfan and cyclophosphamide‐containing conditioning regimens and bone marrow was the predominant source of donor cells. Most patients achieved haematopoietic recovery and no deaths occurred during the early post‐transplant period. Rates of acute and chronic graft‐versus‐host disease were 10% and 22% respectively. Sixty‐four of 67 patients are alive with 5‐year probabilities of disease‐free and overall survival of 85% and 97% respectively. Nine patients had graft failure with recovery of sickle erythropoiesis, eight of who had recurrent sickle‐related events. This report confirms and extends earlier reports that HCT from HLA‐matched related donors offers a very high survival rate, with few transplant‐related complications and the elimination of sickle‐related complications in the majority of patients who undergo this therapy.

One-Unit versus Two-Unit Cord-Blood Transplantation for Hematologic Cancers

BACKGROUNDnUmbilical-cord blood has been used as the source of hematopoietic stem cells in an estimated 30,000 transplants. The limited number of hematopoietic cells in a single cord-blood unit prevents its use in recipients with larger body mass and results in delayed hematopoietic recovery and higher mortality. Therefore, we hypothesized that the greater numbers of hematopoietic cells in two units of cord blood would be associated with improved outcomes after transplantation.nnnMETHODSnBetween December 1, 2006, and February 24, 2012, a total of 224 patients 1 to 21 years of age with hematologic cancer were randomly assigned to undergo double-unit (111 patients) or single-unit (113 patients) cord-blood transplantation after a uniform myeloablative conditioning regimen and immunoprophylaxis for graft-versus-host disease (GVHD). The primary end point was 1-year overall survival.nnnRESULTSnTreatment groups were matched for age, sex, self-reported race (white vs. nonwhite), performance status, degree of donor-recipient HLA matching, and disease type and status at transplantation. The 1-year overall survival rate was 65% (95% confidence interval [CI], 56 to 74) and 73% (95% CI, 63 to 80) among recipients of double and single cord-blood units, respectively (P=0.17). Similar outcomes in the two groups were also observed with respect to the rates of disease-free survival, neutrophil recovery, transplantation-related death, relapse, infections, immunologic reconstitution, and grade II-IV acute GVHD. However, improved platelet recovery and lower incidences of grade III and IV acute and extensive chronic GVHD were observed among recipients of a single cord-blood unit.nnnCONCLUSIONSnWe found that among children and adolescents with hematologic cancer, survival rates were similar after single-unit and double-unit cord-blood transplantation; however, a single-unit cord-blood transplant was associated with better platelet recovery and a lower risk of GVHD. (Funded by the National Heart, Lung, and Blood Institute and the National Cancer Institute; ClinicalTrials.gov number, NCT00412360.).

Bone marrow transplantation in sickle cell anemia.

Hematopoietic cell transplantation (HCT) is a treatment with curative potential for sickle cell disease (SCD). The experience of HCT for persons with β-thalassemia major has been successfully extended to SCD. Currently, the event-free survival rate after allogeneic-matched sibling HCT for SCD is 82%. However, short-term and long-term transplant-related complications remain substantial barriers to HCT, particularly in older patients with life-long complications of SCD. Novel conditioning regimens that minimize transplant-associated toxicity have been developed and show promise for wider application of HCT. Alternative stem cell sources may also expand the availability of HCT for selected patients with SCD.

INTERPRETATION OF THE COMPLETE BLOOD COUNT

The authors impression is that the CBC provides much more information than is routinely used. When anemia is present, the CBC contains considerable information regarding its cause, which can assist in formulating a differential diagnosis and directing further evaluation. White blood cell and platelet count levels may similarly direct practitioners to consider or dismiss underlying conditions. This article assists the pediatrician in optimizing use of this familiar diagnostic tool.

Indications and Results of HLA-Identical Sibling Hematopoietic Cell Transplantation for Sickle Cell Disease.

Although a number of published trials exist of HLA-identical sibling hematopoietic cell transplantation (HCT) for sickle cell disease (SCD) that span 2xa0decades, when and for whom this therapy should be pursued is a subject of debate. Assessments of the risks of transplant-related complications that include infertility and debilitating graft-versus-host disease and long-term quality of life after successful HCT are difficult to perform without prospective trials in transplant and nontransplant cohorts. However, it is possible to assess the risk of mortality and to compare published rates of survival in individuals with SCD treated and not treated by HCT. In this brief review, projections about mortality risk based on recent published reports are reviewed and summarized. The published data show overall survival and event-free survival rates of 95% and 92%, respectively, in children treated by HLA-identical sibling HCT. The overall survival rates in the Center for International Blood and Marrow Transplant Research (Nxa0=xa0412) and European Blood and Marrow Transplant (Nxa0=xa0487) registries were 91% and 95%, respectively. These results provide broad support for the therapeutic value of HLA-identical sibling HCT for children with SCD and serve as the basis for a strong recommendation in favor of the option of HCT when a suitable donor is available. The experience of HLA-identical sibling HCT in adults with SCD is limited but appears to be similar to results in children. These preliminary observations, however, warrant further investigation.

Gene Therapy in Patients with Transfusion-Dependent β-Thalassemia

Background Donor availability and transplantation‐related risks limit the broad use of allogeneic hematopoietic‐cell transplantation in patients with transfusion‐dependent β‐thalassemia. After previously establishing that lentiviral transfer of a marked β‐globin (βA‐T87Q) gene could substitute for long‐term red‐cell transfusions in a patient with β‐thalassemia, we wanted to evaluate the safety and efficacy of such gene therapy in patients with transfusion‐dependent β‐thalassemia. Methods In two phase 1–2 studies, we obtained mobilized autologous CD34+ cells from 22 patients (12 to 35 years of age) with transfusion‐dependent β‐thalassemia and transduced the cells ex vivo with LentiGlobin BB305 vector, which encodes adult hemoglobin (HbA) with a T87Q amino acid substitution (HbAT87Q). The cells were then reinfused after the patients had undergone myeloablative busulfan conditioning. We subsequently monitored adverse events, vector integration, and levels of replication‐competent lentivirus. Efficacy assessments included levels of total hemoglobin and HbAT87Q, transfusion requirements, and average vector copy number. Results At a median of 26 months (range, 15 to 42) after infusion of the gene‐modified cells, all but 1 of the 13 patients who had a non–β0/β0 genotype had stopped receiving red‐cell transfusions; the levels of HbAT87Q ranged from 3.4 to 10.0 g per deciliter, and the levels of total hemoglobin ranged from 8.2 to 13.7 g per deciliter. Correction of biologic markers of dyserythropoiesis was achieved in evaluated patients with hemoglobin levels near normal ranges. In 9 patients with a β0/β0 genotype or two copies of the IVS1‐110 mutation, the median annualized transfusion volume was decreased by 73%, and red‐cell transfusions were discontinued in 3 patients. Treatment‐related adverse events were typical of those associated with autologous stem‐cell transplantation. No clonal dominance related to vector integration was observed. Conclusions Gene therapy with autologous CD34+ cells transduced with the BB305 vector reduced or eliminated the need for long‐term red‐cell transfusions in 22 patients with severe β‐thalassemia without serious adverse events related to the drug product. (Funded by Bluebird Bio and others; HGB‐204 and HGB‐205 ClinicalTrials.gov numbers, NCT01745120 and NCT02151526.)

Collection of sibling donor cord blood for children with thalassemia.

Bone marrow transplantation has curative potential for patients with thalassemia major who have a matched sibling marrow donor, but usefulness of alternative stem cell sources is undergoing investigation. Cord blood (CB) from a sibling has different characteristics from marrow and has potential advantages and disadvantages as a stem cell source. Whereas many families caring for a child with thalassemia major (or other transplant-treatable condition) experience an additional pregnancy, most give birth at hospitals without the infrastructure needed to collect and process the new infants CB. To address this, and with funding from the National Institutes of Health, we have developed the first noncommercial CB program, operating across the United States, designed specifically to facilitate medically indicated CB collections from sibling donors. Using a case-management model, we have collected CB for 25 thalassemia families in eight states. Three of these CB units have now been used for transplantation; two others are human leukocyte antigen-identical and contain adequate nucleated cell dose to perform transplantation in their intended recipient. We conclude that a CB bank focused on sibling donations may be a useful stem cell resource and that families with specific medical need, such as a child with thalassemia, should consider preserving CB from siblings.

c-Jun inhibits NF-E2 transcriptional activity in association with p18/maf in Friend erythroleukemia cells

We have reported previously that antisense c-jun overcomes a block of Friend erythroleukemia cells to differentiation suggesting that the factor c-Jun may be an important negative regulator of erythroid differentiation. The recently described erythroid transcription factor NF-E2 plays an important role in the regulation of the transcription of globin genes and recognizes a sequence containing an AP-1 site. NF-E2 is a complex of two bZip proteins, p45 and p18/Maf. In order to determine whether c-Jun can interact with NF-E2/AP-1 sites to regulate transcriptional activation from them, we have compared the activity of AP-1 and NF-E2 in transient transcriptional assays, in erythroid and non-erythroid cells in the presence of c-jun sense and antisense expression vectors. In non-erythroid cells, c-Jun activates and NF-E2p18 inhibits both AP-1 and NF-E2 activities, suggesting that NF-E2/AP-1 sites function as AP-1 binding sites in these cells. In contrast, NF-E2p18 is a positive regulator of NF-E2 activity in erythroid cells. c-Jun alone is also a positive regulator of NF-E2 activity in erythroid cells but in association with NF-E2p18 inhibits this activity. Moreover antisense c-jun increases endogenous NF-E2 activity in erythroid cells. These results suggest that c-Jun could act as a repressor of NF-E2 transcriptional activity by forming inactive c-Jun/NF-E2p18 heterocomplexes which interfer with the transcription of globin genes in Friend erythroleukemia cells.

New approaches to hematopoietic cell transplantation for hematological diseases in children

Hematopoietic cell transplantation (HCT) has been used for more 30 years for the treatment of selected malignant and nonmalignant diseases. Traditionally, HCT for hematological disorders has relied on myeloablative conditioning before HLA-identical sibling bone marrow transplantation to correct the underlying hematological defect. Most children with hematological diseases who are referred to HCT have features that portend significant morbidity and early mortality. Among SAA patients who have HLA-identical sibling donors, younger patients with profound pancytopenia might be considered early for HCT. For others who lack sibling donors, patients who receive HCT from alternate sources have generally failed one or more courses of intensive immunosuppressive therapy and remain transfusion-dependent, some with hemosiderosis, red cell alloimmunization, and platelet transfusion refractoriness [44,46,48]. Currently, HCT for SCD is generally restricted to those who have experienced a significant sickle-related complication such as stroke, recurrent acute chest syndrome, or recurrent painful episodes [7,13]. In contrast, most reserve HCT in thalassemia for younger, Lucarelli class I, good-risk patients who have HLA-identical sibling donors, and veer away from older, high-risk thalassemics for whom transplantation is a riskier clinical intervention. For groups such as young adults with thalassemia major, HCT might become more widely applicable if its toxicity was reduced. Several approaches undergoing development include reduced-intensity conditioning and attempts to prevent GVHD. New methods to reduce the intensity and toxicity of conditioning as well as to use highly purified stem cells with the reduction in graft versus host disease may allow for the use of matched unrelated donors or haploidentical donors. This would serve to provide potentially more children who could benefit from stem cell transplantation with donors. These advances will hopefully lead to benefits for the majority of children who lack HLA-identical donors.

CRISPR-Cas9 interrogation of a putative fetal globin repressor in human erythroid cells.

Sickle Cell Disease and ß-thalassemia, which are caused by defective or deficient adult ß-globin (HBB) respectively, are the most common serious genetic blood diseases in the world. Expression of the fetal ß-like globin, also known as γ-globin, can ameliorate both disorders by serving in place of the adult ß-globin. Here we use CRISPR-Cas9 gene editing to explore a putative γ-globin silencer region identified by comparison of naturally-occurring deletion mutations associated with up-regulated γ-globin. We find that deletion of a 1.7 kb consensus element or select 350 bp sub-regions from bulk populations of cells increases levels of fetal hemoglobin (HbF) or γ-globin. Screening of individual sgRNAs in one sub-region revealed three single guides that caused mild increases in γ-globin expression. However, clonal cell lines with the 1.7 kb region deleted did not up-regulate γ-globin and neither did lines with either of two of sub-regions identified in the screen deleted. These data suggest that the region is not an autonomous γ-globin silencer, and thus by itself is not a suitable therapeutic target in the ß-hemoglobinopathies.