Eizaburo Ishii

Treatment of intracranial nongerminomatous germ-cell tumor by high-dose chemotherapy and autologous stem-cell rescue.

Nongerminomatous germ-cell tumor (NGGCT) in the central nervous system (CNS) is still highly lethal. The present study evaluated the outcome of high-dose chemotherapy followed by autologous stem-cell rescue (ASCR). The patients included three cases of choriocarcinoma, two cases of embryonal carcinoma and one case of yolk sac carcinoma. High-dose cisplatin (200 mg/m2), etoposide (1250 mg/m2) and ACNU (150 mg/m2) were administrated in combination with ASCR to patients at complete remission as a result of surgical removal, irradiation, and from four to seven courses of induction chemotherapy. All the patients treated with this therapy were alive from one to seven years after the diagnosis, living with good performance status. The patients have not required any additional treatments after ASCR. The myelosuppression period, characterized by fewer than 500/µl peripheral neutrophils, ranged from 8 to 15 days (median, 11.5 days). Within seven days of ASCR, high fever was found in four patients. Although mild liver dysfunction was found in all patients, renal dysfunction was not observed. Hearing disturbance was found in 50% of the patients. This treatment regime will improve long-term survival for patients with NGGCT.

Establishment of a GM‐CSF‐dependent megakaryoblastic cell line with the potential to differentiate into an eosinophilic lineage in response to retinoic acids

We recently established a human granulocyte‐macrophage colony‐stimulating factor (GM‐CSF)‐dependent cell line (HML) from colony‐constituent cells grown by peripheral blood cells of a patient with acute megakaryoblastic leukaemia. The HML cells possessed megakaryocytic features, as determined by cytochemical, electron microscopic and flow cytometric analysis. In the present study we examined the effects of retinoic acid (RA) on the development of HML cells. All‐trans‐RA, 13‐cis‐RA and 9‐cis‐RA at 10−8 mol/l to 10−5 mol/l inhibited the GM‐CSF‐dependent cell growth. Some of the RA‐treated cells contained prominent azurophilic granules and were positive for peroxidase. They also reacted with Biebrich scarlet, Luxol fast blue and a monoclonal antibody against eosinophil peroxidase. In addition, exposure to RA increased the frequency and the intensity of major basic protein‐positive cells. However, eosinophil‐derived neurotoxin and eosinophil cationic protein were not detected or were only detected at a low level in the lysates of the HML cells treated with RA. Although IL‐5 alone could not stimulate cell growth, the addition of IL‐5 to the cultures containing stem cell factor + all‐trans‐RA was required for the expression of the eosinophilic phenotype. These results suggest that the HML cell line is a megakaryoblastic cell line with the potential to differentiate into the eosinophilic lineage. HML cells may be a useful model for elucidating the eosinophilic differentiation programme.

Secondary cancers among children with acute lymphoblastic leukaemia treated by the Tokyo Children's Cancer Study Group protocols: a retrospective cohort study

With improvement in survival, it is important to evaluate the impact of treatment on secondary cancers in acute lymphoblastic leukaemia (ALL) survivors. A retrospective cohort study comprising 2918 children diagnosed with ALL and enrolled on Tokyo Childrens Cancer Study Group (TCCSG) protocols between 1984 and 2005 was conducted to evaluate the incidence of secondary cancers and associated factors including treatment protocol, cranial irradiation and other characteristics of the primary ALL. Thirty‐seven patients developed secondary cancers, including acute myeloid leukaemia (n = 11), myelodysplastic syndrome (n = 5), non‐Hodgkin lymphoma (n = 2), brain tumours (n = 13) and other solid carcinomas (n = 6) within a median follow‐up duration of 9·5 years. The cumulative incidence of any secondary cancers was 1·0% (95% confidence interval (CI), 0·7–1·4%) at 10 years and 2·4% (95% CI, 1·5–3·7%) at 20 years, respectively. Standardized incidence rate ratio of secondary cancers was 9·3 (95% CI, 6·5–12·8). Multivariate analyses showed an increased risk of secondary cancers associated with the recent treatment protocol and cranial irradiation. There was no evidence of a reduction in secondary cancer incidence despite marked decreases in cranial irradiation use in the recent protocols.

Early and rapid detection of X-linked lymphoproliferative syndrome with SH2D1A mutations by flow cytometry

X‐linked lymphoproliferative syndrome (XLP) is a rare immunodeficiency with extreme vulnerability to Epstein‐Barr virus (EBV) infection. It presents with fatal infectious mononucleosis, lymphoproliferative disorder, or dysgammaglobulinemia. The majority of affected males have mutations in the SH2D1A/SLAM‐associated protein (SAP) gene. We previously generated an antihuman SAP monoclonal antibody (KST‐3) for a flow cytometric assay and described the activation of T cells to be necessary for the flow cytometric assessment of the SAP expression using an FITC‐conjugated secondary antibody.

Retrospective analysis of risk factors for development of liver dysfunction in transient leukemia of Down syndrome

About 20% of patients with transient leukemia (TL), which is a disease noted in Down syndrome, are reported to develop hepatic fibrosis, which has a poor prognosis. The clinical factors related to the poor prognosis of TL were retrospectively analyzed in 25 patients, and criteria for starting chemotherapy were established. The initiation of chemotherapy was recommended when two or more of the following categories were fulfilled in the process of the disease: (1) a reduced hepatic functional reserve estimated by direct bilirubin, prothrombin time, and the presence of ascites, (2) an elevated level of hyaluronic acid (>500 U/mL), (3) respiratory failure or poor sucking associated with hepatosplenomegaly, and (4) demonstration of fibrosis by liver biopsy. When these criteria were applied to our cases, all patients who received chemotherapy remained alive. Our criteria are useful for selecting patients with TL at high risk of developing hepatic fibrosis and for starting chemotherapy.

In vitro expression of β-thalassaemia gene (IVS1-1G>C) reveals complete inactivation of the normal 5' splice site and alternative aberrant RNA splicing

We previously reported a case of heterozygous β-thalassaemia with IVS1-1G > C substitution in the β-globin gene and a non-detectable level of mutant mRNA in the patients reticulocytes. The purpose of this study was to determine whether the transcription and RNA splicing and processing of the mutant gene occurred. We analysed the expression of the mRNA encoded by the cloned mutant gene in COS-1 cells by reverse transcription-polymerase chain reaction followed by agarose gel electrophoresis and nucleotide sequencing. The G > C mutation completely inactivated the normal 5- splice site and resulted in the activation of two cryptic 5- splice sites, located 16 and 38 nt upstream of the normal site. The usage of these two cryptic sites accords with the findings of reports on IVS1-1G > A or IVS1-1G > C substitution of exon 1 of the β-globin gene. Additional experiments that involved transfection of equal amounts of both normal and mutant vectors into COS-1 cells indicated the presence of mutant mRNAs. In conclusion, the β-thalassaemia gene (IVS1-1G > C) was expressed in transfected cells, but showed aberrant RNA splicing. Further studies will be required to clarify the molecular mechanism that results in severe reduction in the mutant mRNA level in vivo.

Pretransplant-corrected QT dispersion as a predictor of pericardial effusion after pediatric hematopoietic stem cell transplantation

Pericardial effusion is a potentially fatal complication following hematopoietic stem cell transplantation (HSCT). Therefore, the identification of risk factors could improve the outcome. Prolonged QT dispersion (QTD) and corrected QTD (QTcD) are associated with serious arrhythmias and sudden death in many forms of heart disease. However, no study has evaluated the efficacy of QTD and QTcD to predict pericardial effusion post‐HSCT. We studied 89 pediatric HSCT patients to identify preoperative risk factors for pericardial effusion with particular focus on QTD and QTcD. Pericardial effusion occurred in 15 patients (cumulative onset rate: 17.4%) within one year post‐HSCT, of which 8 (9.2%) were symptomatic. Patients with pericardial effusion following allogeneic HSCT showed significantly lower overall survival; however, pericardial effusion was not the direct cause of death in any patient. Univariate and multivariate analyses revealed that transplantation‐associated thrombotic microangiopathy (TA‐TMA) was an independent risk factor for post‐HSCT pericardial effusion. In addition, pretransplant QTcD was significantly prolonged in the pericardial effusion group. These results suggest that pediatric patients with abnormally prolonged QTcD before the preparative regimen for HSCT should be regularly followed‐up by echocardiography to detect pericardial effusion, particularly when accompanied by complications including TA‐TMA.

Transfusion-related acute lung injury in an infant

Ryu Yanagisawa, Kouichi Takeuchi, Takashi Kurata, Kazuo Sakashita, Shigetaka Shimodaira and Eizaburo Ishii Departments of Hematology/Oncology and General Pediatrics, Nagano Children’s Hospital, Azumino and Department of Pediatrics, Shinshu University School of Medicine, and Division of Blood Transfusion and Center for Advanced Cell Therapy, Shinshu University Hospital, Matsumoto and Department of Pediatrics, Nagano Prefectural Suzaka Hospital, Suzaka, Nagano, Japan

Wilms tumor accompanied by premature chromatid separation

Cancer-prone syndrome of premature chromatid separation (PCS) with mosaic variegated aneuploidy syndrome (PCS [MVA] syndrome) is a rare autosomal recessive disorder characterized by constitutional aneuploidy with PCS in more than 50%metaphase cells and a high risk of pediatric cancers.1 Central nervous systemanomalies found in these patients include microcephaly, Dandy–Walker malformation characterized by brain hypoplasia, and bilateral cataracts.1 Wilms tumor is thought to be the most developed of all neoplasms.2 Intergenic mutation upstream of BUB1B or mutation of BUB1B has been reported to cause thePCS (MVA) syndrome.1,3 However, the significanceof detecting a PCS in Wilms tumor accompanied with normal karyotype in the germline is unknown. A 4-month-old male was admitted to our hospital due to abdominal swelling. His individual and family histories were unremarkable. Ultrasonography and computed tomography revealed a unilateral right renal neoplasm that could be removed completely with surgery, and

Myelomonocytic differentiation associated with NPM1-RARA rearrangement

A 5-year-old boy presented with a left femoral bone lesion. Laboratory tests showed: WBC count 4 98 9 10/l; haemoglobin concentration 71 g/l; platelet count 181 9 10/l; International Normalised Ratio 1 08; activated partial thromboplastin time 28 8 s; fibrinogen 3 7 g/l; fibrin degradation products 392 ng/ml; and D-dimer 8 5 lg/ml. Of the bone marrow cells, >95% showed myeloperoxidase activity with heterogeneous morphologies: 28% were promyelocyte-like cells that lacked Auer rods while others had similar features but lacked granules (left). Most cells were chloroacetate esterase positive and 38% were strongly positive for nonspecific esterase (right). Immunophenotyping showed CD13 positivity; some cells showed CD14, CD11b and lysozyme positivity. Although circulating mature monocytes were not increased, we diagnosed acute myelomonocytic leukaemia. Chemotherapy was discontinued because of severe disseminated intravascular coagulation. G-banding analysis showed 46,XY, t(2;9)(q31;p13),t(5;17)(q35;q12). Reverse transcription polymerase chain reaction detected the NPM1-RARA rearrangement. With all-trans-retinoic acid (ATRA) and combination chemotherapy, the patient achieved remission with no NPM1-RARA mRNA being detectable. The NPM1-RARA fusion gene, created by the chromosomal translocation t(5;17)(q35;q12-21), is associated with a rare acute promyelocytic leukaemia (APL) variant. Typical APL harbouring a PML-RARA rearrangement shows an increase of either hypergranular or variant promyelocytes and is sensitive to ATRA. Acute leukaemia with an NPM1RARA rearrangement differs cytologically but its recognition is important because it is also ATRA sensitive.