Hyo-Seop Ahn

Immunogenicity and safety of a 23-valent pneumococcal polysaccharide vaccine in healthy children and in children at increased risk of pneumococcal infection.

Splenectomized children as well as those suffering from nephrotic syndrome or recurrent asthmatic bronchitis have an increased susceptibility to systemic pneumococcal infections compared to healthy children. To determine the immunogenicity and safety of a 23-valent pneumococcal polysaccharide vaccine (PPV), 119 children (21 healthy, 26 splenectomized children, 48 with nephrotic syndrome and 24 with recurrent asthmatic bronchitis), aged 2-18 years, received one subcutaneous injection of a 23-valent PPV. Anti-capsular antibodies (Ab) to types 6B, 9V, 14, 18C, 19F and 23F were measured by ELISA before and 4 weeks after immunization. In all cases the adverse reactions were mild and transient, consisting of local pain and/or erythema or swelling in 41% and fever above 38.5 degrees C in 2% of the children. The healthy children responded well to vaccination with a mean fold increase (FI) of 2.6 in postvaccination Ab titers compared to prevaccination titers. The combined geometric mean Ab concentrations in the high-risk children were significantly lower than those of healthy children both before and after vaccination. However, the combined geometric mean FI were not significantly different between high-risk and healthy children. These results indicate that PPV is immunogenic and safe in high-risk as well as in healthy Korean children.

Effect of iron overload and iron-chelating therapy on allogeneic hematopoietic SCT in children.

Iron overload is known to increase complications of hematopoietic SCT (HSCT). We investigated the association of pre-transplant ferritin level with complications and survival after allogeneic HSCT, and evaluated the efficacy of iron-chelating therapy before HSCT. We retrospectively reviewed 101 patients who underwent allogeneic HSCT and divided these patients into three groups: F>1000, patients with ferritin level above 1000u2009ng/ml at the time of HSCT; F<1000, patients whose ferritin levels were maintained below 1000u2009ng/ml before HSCT without iron-chelating therapy; IC, patients with ferritin level decreased to less than 1000u2009ng/ml after iron-chelating therapy before HSCT. In the comparison between the F>1000 group and the F<1000 group, hyperbilirubinemia and treatment-related mortality (TRM) were significantly higher in the F>1000 group. The F>1000 group also showed decreased OS and EFS. In the comparison of the F<1000 and IC groups, there was no significant difference in complications and survival. When compared with the F>1000 group, the IC group showed lower TRM and higher survival. Elevated serum ferritin level was associated with increased TRM and decreased survival, and the analysis of the IC group suggested the benefit of iron-chelating therapy to improve the outcome of HSCT.

Hepatic veno-occlusive disease in children after hematopoietic stem cell transplantation: incidence, risk factors, and outcome

Four hundred and sixty-seven hematopoietic stem cell transplantations (HSCTs) (217 autologous and 250 allogeneic HSCT) were performed in 374 children at four pediatric HSCT centers in Korea from January 2005 to December 2007. Among 467 transplants, veno-occlusive disease (VOD) developed in 72 transplants (15.4%) at a median of 10 days after HSCT. Multivariate analysis showed that BU or TBI-containing regimen (P=0.002), VOD prophylaxis without lipo-prostaglandin E1 (PGE1) (P=0.012), number of previous HSCT (P=0.014), and pretransplant serum ferritin (P=0.018) were independent risk factors for developing VOD. Mean serum ferritin levels were significantly higher in HSCT with VOD (2109.6±2842.5u2009ng/ml) than in HSCT without VOD (1315.9±1094.4u2009ng/ml) (P<0.001). The relative risk of death within 100 days of HSCT in transplants with VOD compared with transplants without VOD was 3.39 (confidence interval: 1.78–6.45). Our results suggest that lipo-PGE1 might have a protective effect against the development of VOD, and pretransplant serum ferritin could act as a risk factor for VOD. A larger prospective study is needed to confirm a possible role of lipo-PGE1 and iron chelation therapy in reducing the incidence of VOD.

Fludarabine, cyclophosphamide plus thymoglobulin conditioning regimen for unrelated bone marrow transplantation in severe aplastic anemia.

Summary:Antithymocyte globulin (ATG) has been used in severe aplastic anemia (SAA) as a part of the conditioning regimen. Among the many kinds of ATG preparations, thymoglobulin had been found to be more effective in preventing GVHD and rejection of organ transplants. As the fludarabine-based conditioning regimens without total body irradiation have been reported to be promising for bone marrow transplantation (BMT) from alternative donors in SAA, thymoglobulin was added to fludarabine and cyclophosphamide conditioning to reduce GVHD and to allow good engraftment in unrelated BMT. Five patients underwent BMT with cyclophosphamide (50u2009mg/kg once daily i.v. on days −9, −8, −7 and −6), fludarabine (30u2009mg/m2 once daily i.v. on days −5, −4, −3 and −2) and thymoglobulin (2.5u2009mg/kg once daily i.v. on days −3, −2 and −1) from HLA-matched unrelated donors. Complete donor type hematologic recovery was achieved in all patients. No serious complication occurred during BMT. Only one patient developed grade I acute GVHD resolved spontaneously. Except for one who had rupture of hepatic adenoma 78 days after BMT, all the other four patients are still alive with median 566 days. Fludarabine, cyclophosphamide plus thymoglobulin conditioning allows for the promising results of good engraftment, tolerable toxicity and minimal GVHD.

Early engraftment kinetics of two units cord blood transplantation.

Cord blood transplantation (CBT) is a promising alternative means of allogeneic stem cell transplantation. However, limited cell doses may compromise outcome. To enhance engraftment, CBT has been conducted using two units with promising results. However, little is known about the mechanism of engraftment. Here, we analyzed the early engraftment kinetics of eight patients given two unit umbilical CBT. Early engraftment kinetics revealed dominancy of one of two units from the day of engraftment (absolute neutrophil count >0.5 × 109/l). The median value of percentage of the predominant unit by chimerism analysis at the time of engraftment was 88% (60–100%). Two units CBT was found to be a safe, effective and promising alternative treatment option with good engraftment potential. Dominancy occurred early after CBT and is probably influenced by multiple factors.

Apoptosis and megakaryocytic differentiation during ex vivo expansion of human cord blood CD34+ cells using thrombopoietin

Thrombopoietin (TPO), the primary regulator of megakaryocytopoiesis, plays important roles in early haematopoiesis. Previously, we have demonstrated that TPO induces a characteristic pattern of apoptosis during ex vivo expansion of cord blood (CB) CD34+ cells. In this study, we have demonstrated that the TPO‐induced apoptotic cells belong to the megakaryocytic (MK) lineage and that initially expanding MK progenitors declined along with the appearance of TPO‐induced apoptosis. Human CB CD34+ cells were expanded in serum‐free conditions with TPO. Multidimensional flow cytometry using simultaneous measurement of apoptosis and immunophenotyping showed that the TPO‐induced apoptotic cells appeared in CD61+ fractions. Immunocytochemical analysis of the fluorescent activated cell‐sorted fractions showed that the apoptosis‐associated CD44low fraction expressed CD61. Clonogenic assay revealed 7·4u2003±u20030·50‐fold increase of total megakaryocyte colony‐forming units (CFU‐MKs) during the initial 9u2003d. Thereafter, the number of CFU‐MKs decreased in parallel with the increase of apoptosis. When the MK colonies were subdivided according to size, the proportion of large colonies progressively decreased, while that of medium and small colonies increased. In particular, from du20036 small colonies became predominant. These results suggested that the MK progenitors matured as they expanded during ex vivo expansion with TPO and then proceeded to apoptosis.

ARSENIC TRIOXIDE INHIBITS CELL GROWTH IN SH-SY5Y AND SK-N-AS NEUROBLASTOMA CELL LINES BY A DIFFERENT MECHANISM

Neuroblastoma, characterized by heterogeneous cell population, is a common solid tumor in childhood and some malignant neuroblastomas are refractory to conventional chemotherapy. Recently, treatment with arsenic trioxide (As2O3) was found effective in the treatment of acute promyelocytic leukemia as well as neuroblastoma cells by inducing apoptosis. To define the mechanism contributing to cell death in those heterogenous cell populations, the authors used two different types of neuroblastoma cells, SH-SY5Y and SK-N-AS, to compare the pathways that mediate death response to arsenic trioxide. With arsenic trioxide exposure, both cell lines were arrested at the S-G2/M phase with the increase of cyclin B expression and CDK1 activity. Although caspase 3 was activated in both cell lines, the NF-κB activity and the expression of cyclin D1, cyclin E, and p27 were different. Therefore, arsenic trioxide could be an effective cytotoxic drug for the treatment of heterogeneous cellular population of neuroblastoma.

Peri-engraftment syndrome in allogeneic hematopoietic SCT.

Engraftment syndrome (ES) and pre-engraftment syndrome (pre-ES) are both inflammatory conditions that occur after hematopoietic SCT (HSCT) and are characterized by non-infectious fever and skin rash. Although the pathogenesis is not fully understood, both syndromes are similar, and could be defined as a new clinical syndrome, named as peri-engraftment syndrome (peri-ES). We retrospectively analyzed the clinical records in 176 pediatric patients, following allogeneic HSCT. We utilized the definition of ES by Spitzer as the diagnostic criteria, excluding ‘within 96u2009h of engraftment’ criteria. Thirty cases developed peri-ES with a cumulative incidence of 17.0%. High cumulative incidence (50%) was seen in patients who underwent a double-unit cord blood transplantation (DUCBT; P<0.01). Clinical findings of peri-ES are similar, regardless of the onset day, and encephalopathy was the most severe complication. In the DUCBT cohort, the use of TBI and early complete chimerism (⩽day 21) were identified as risk factors that predispose the development of peri-ES. We determined that both, ES and pre-ES, might have similar causes, which could be included in peri-ES. Particularly, it occurred more in DUCBT patients, which means that not only neutrophil engraftment but also immune reactions within the two units might contribute to peri-ES.

Founder effects in two predominant intronic mutations of UNC13D, c.118-308C>T and c.754-1G>C underlie the unusual predominance of type 3 familial hemophagocytic lymphohistiocytosis (FHL3) in Korea

Familial hemophagocytic lymphohistiocytosis (familial HLH or FHL) is a potentially fatal autosomal recessive disorder. Our previous study demonstrated that UNC13D mutations (FHL3) account for ∼90xa0% of FHL in Korea with recurrent splicing mutation c.754-1G>C (IVS9-1G>C). Notably, half of the FHL3 patients had a monoallelic mutation of UNC13D. Deep intronic mutations in UNC13D were recently reported in patients of European descent. In this study, we performed targeted mutation analyses for deep intronic mutations and investigated on the founder effect in FHL3 in Korean patients. The study patients were 72 children with HLH including those with FHL3 previously reported to have a monoallelic UNC13D mutation. All patients were recruited from the Korean Registry of Hemophagocytic Lymphohistiocytosis. In addition to conventional sequencing of FHL2-4, targeted tests for c.118-308C>T and large intronic rearrangement mutations of UNC13D were performed. Haplotype analysis was performed for founder effects using polymorphic markers in the FHL3 locus. FHL mutations were detected in 20 patients (28xa0%). Seventeen patients had UNC13D mutations (FHL3, 85xa0%) and three had PRF1 mutations (FHL2, 15xa0%). UNC13D:c.118-308C>T was detected in ten patients, accounting for 38xa0% of all mutant alleles of UNC13D, followed by c.754-1G>C (26xa0%). Haplotype analyses revealed significantly shared haplotypes in both c.118-308C>T and c.754-1G>C, indicating the presence of founder effects. The deep intronic mutation UNC13D:c.118-308C>T accounts for the majority of previously missing mutations and is the most frequent mutation in FHL3 in Korea. Founder effects of two recurrent intronic mutations of UNC13D explain the unusual predominance of FHL3 in Korea.

Autologous peripheral blood stem cell transplantation with BCVAC conditioning in childhood acute myeloid leukemia.

Summary:Autologous peripheral blood stem cell transplantation (APBSCT) after intensifying conditioning is one of the post-remission therapeutic options in childhood acute myeloid leukemia (AML) patients without a matched family donor, but the optimal conditioning regimen has not been defined. This study was performed to evaluate the efficacy of a novel conditioning regimen without busulfan or total body irradiation. In total, 28 children with AML underwent APBSCT with BCVAC (BCNU, etoposide, cytosine arabinoside and cyclophosphamide) conditioning regimen during first remission. The event-free survival rate was 71.43% for all patients and the only cause of treatment failure was relapse. Eight male patients recurred at 1–11 months (median 5 months) after APBSCT. One patient remains alive with salvage therapy after relapse. With the exception of fever, mucositis and diarrhea, no serious complications occurred during APBSCT, including veno-occlusive disease (VOD), and there was no transplantation-related mortality. One patient developed secondary MDS after APBSCT but recovered hematologically on medication. APBSCT with BCVAC conditioning was found to be a safe and effective alternative option for patients with childhood AML in first remission, without a matched family donor.