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Title and description of data: Self-administered questionnaire for women 3 and 4â months after delivery in Suzaka City (English translation). (PDF 345 kb)

Title and description of data: Self-administered questionnaire for pregnant women when they turned in their pregnancy notification form in Suzaka City (English translation). (PDF 392 kb)

Title and description of data: The results of the psychosocial questionnaire of the intervention groupâ s participants at T1. (PDF 276 kb)

Background Proton pump inhibitors (PPI) are widely used for the treatment of gastric acid-related disease, but they are not approved for use in children in Japan. To assess the safety, pharmacokinetics, pharmacodynamics, and efficacy (gastrointestinal symptom improvement) of PPI in Japanese pediatric patients with gastric acid-related disease, we conducted an 8 week, open-label, parallel-group, multicenter, phase I/III study of once-daily oral esomeprazole use. Methods Japanese children, aged 1-14 years with gastric acid-related disease, were stratified by weight and age into five groups (10 patients/group) to receive esomeprazole as granules for suspension (10 mg) or capsules (10 mg or 20 mg) once daily. Results Esomeprazole was absorbed and eliminated rapidly in all groups, with a median time to reach maximum plasma concentration of 1.47-1.75 h, an arithmetic mean terminal elimination half-life of 0.80-1.37 h, and a weight-correlated apparent total body clearance of 0.216-0.343 L/h/kg. Area under the plasma concentration-time curve during a dosage interval and maximum plasma drug concentration were generally higher in groups given a higher dose (20 mg) or with a lower age/weight, but also in patients identified as poor metabolizers on cytochrome P450 2C19 genotype. Most patients who had any upper gastrointestinal symptoms at baseline were asymptomatic at the end of the study. Thirty-three patients (66%) reported ≥1 adverse events, including three patients who reported serious adverse events not judged to be causally related to esomeprazole. Conclusions Oral esomeprazole, at 10 mg or 20 mg once daily, had a similar safety, efficacy, and pharmacokinetic profile in Japanese pediatric patients to that previously seen in adults and Caucasian children.

The International Immune Tolerance Induction (I-ITI) Study in hemophilia A patients with inhibitors included 16 Japanese patients among a total of 115 test subjects. The results within this group of Japanese patients were 11 cases of I-ITI off-study, three cases of I-ITI on-study, and two cases of tolerance on prophylaxis. There was no significant difference in success rate between the low-dose and high-dose groups (Study I). Successively, independent follow-up survey in Japan was conducted in 14 cases, with consent (Study II). Ten cases were off-study at the end of the I-ITI Study. Of these 10 cases, seven of seven successful cases remained clinical successes at the end of the follow-up study, one partial success became a full success while a second relapsed, and one failure was subsequently evaluated as a partial success. Four cases that were on-study at the end of I-ITI Study were classified as three successes and one failure at the end of the follow-up study. As a result, the status at the end of follow-up study was: 11 ITI successes (78.6 %); one partial success; one failure; and one relapse. Thus, the ITI follow-up study was helpful in providing a long-term prognostic determination of inhibitors.

Background The pain associated with bone marrow aspiration and biopsy (BMAB) has an enormous impact on pediatric cancer patients and their families, but no specific reference standards for sedation and analgesia have been developed in Japan. To determine the problems associated with pain management during BMAB, a cross-sectional investigation was conducted. Methods A survey was sent in October 2011 to data managers in institutions belonging to the Tokyo Children's Cancer Study Group, addressing the non-pharmacological and pharmacological pain management for BMAB performed on pediatric cancer inpatients between January 2010 and December 2010. Results The eligible response rate was 41 of 57 institutions (71.9%). Non-pharmacological pain intervention was provided in 68% of surveyed institutions. All institutions provided pharmacological pain management. In most institutions, sedation/analgesia was performed by pediatric oncologists in a treatment room in the ward. Standards for pain management were developed and utilized in only four institutions. Other means of pain management were provided in various settings. Twelve institutions reported insufficient sedation/analgesia. In total, 80% of institutions reported some adverse events. Two serious adverse events were reported in cases of underlying or complicated conditions. No serious long-term consequences were reported. Conclusions Significant issues were identified regarding the efficacy and safety of pain management. Adverse events can occur in any institution. Children with underlying or complicated conditions are at high risk for serious adverse events. Therefore, adequate and systematic assessment, patient monitoring, preparation and treatment for adverse events, and cooperation with skilled specialists of pediatric oncology, anesthesiology, and intensive care are essential.

Summary 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 Children's 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.

PURPOSE Three familial cases of each of severe congenital neutropenia (SCN) and cyclic neutropenia (CN) in addition to 3 sporadic cases of SCN were analyzed for neutrophil elastase (Ela2) gene mutation. The contents of the neutrophil-specific granule proteins cathelicidin antimicrobial peptide and neutrophil gelatinase-associated lipocalin were also analyzed in SCN. METHODS Genomic DNA was extracted from the patients' peripheral blood or bone marrow, and the coding sequence of the Ela2 gene was amplified by polymerase chain reaction and subjected to direct sequencing. The contents of antimicrobial peptides were analyzed by flow cytometry. RESULTS Three cases of familial SCN (P13L, R52P, and S97L), 2 of familial CN (W212stop and P110L), and 1 of sporadic SCN (V72M) were shown to have heterozygous mutations in the Ela2 gene. W212stop found in a familial CN case was a novel mutation of Ela2. Prophylactic treatment for growth factors or antibiotic prophylaxis against bacterial infection was useful for lowering the frequency of infectious episodes. Adult patients tended to have less frequent infections compared with minors in the same family. The contents of both cathelicidin antimicrobial peptide and neutrophil gelatinase-associated lipocalin were significantly reduced in SCN compared with healthy controls. CONCLUSIONS Prophylaxis by growth factor or antibiotics is useful for decreasing risks of bacterial infections in SCN and CN. Adults were likely to have less frequent infections than children in familial cases of SCN and CN with the same mutation of Ela2.

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 patient's 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.

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.

A 10-year-old boy who presented with hypopituitarism was diagnosed with supraseller germinoma based on the findings of brain magnetic resonance imaging (MRI). Dissemination of the tumor to the spinal cord was also evident. The patient was first treated with irradiation to both the brain and spinal cord, followed by chemotherapy with cisplatin (CDDP) and etoposide (VP-16). Peripheral blood stem cell transplantation (PBSCT) with extensive chemotherapy was performed when he was 14 years old. During and after the treatment for the brain tumor, he was placed on hormone replacement with corticosteroid hormone, l-thyroxine, and 1-deamino-8-D-arginine-vasopressin (DDAVP). Growth hormone was also replaced after 2 years from PBSCT. The patient was noted to have gait disturbance, osteoporosis and spondylolisthesis of the lumbar vertebrae (L) at 18 years old. His mean bone mineral density (BMD) of the 2nd to 4th lumbar vertebrae was 0.59 g/m2. There was no evidence of recurrence of the tumor. It is possible that a high dose of local irradiation, long-term corticosteroid hormone replacement, and a hypogonadal state are related to the onset and progression of the skeletal abnormalities.

The X-linked lymphoproliferative disease (XLP) is an inherited immunodeficiency characterized by an abnormal responses to infection with Epstein-Barr virus (EBV), resulting in fatal infectious mononucleosis, hypogammaglobulinemia, virus-associated hemophagocytic syndrome, and malignant lymphoma. Mutations in the gene coding for a T cell-specific SLAM-associated protein (SAP) have been recently identified in XLP patients. We report on a 1-year-old boy representing fulminant hemophagocytic syndrome. He developed high fever, lymphadenopathy, hepatosplenomegaly with liver dysfunction, and pancytopenia with marrow hemophagocytosis. EBV DNA was abnormally increased in the blood. Polymerase chain reaction failed to amplify SAP mRNA and genomic DNA products from the patient' As peripheral blood. A large deletion of the SAP gene was confirmed by fluorescence in situ hybridization (FISH). FISH analysis also disclosed that the patient's mother was a carrier. We conclude that FISH can be useful in the diagnosis of XLP with large deletions of the SAP gene and its carrier state.

The contribution of the thymus-dependent pathway and thymus-independent pathways for T cell regeneration after BMT in children is still unclear. We analyzed the kinetics of T cell regenerative pathways after allogeneic BMT. The number of CD4+CD45RA+ T cells, a thymus-dependent population, was very low until 3 months after BMT. The numbers of CD28- T cells and CD8+ T cells expressing CD8alpha/alpha homodimer (CD8alpha/alpha+ T cells), a thymus-independent population, increased shortly after BMT, beyond the levels of healthy children in some patients. The numbers of Vgamma9+Vdelta2+ and Valpha24+ T cells, which represent populations of extrathymic development, were less than 200/microl during the 6 months after BMT. There was a significant inverse correlation between the percentages of CD4+CD45RA+ and CD28-T cells at 1 month, and a positive correlation between the percentages of CD28- and CD8alpha/alpha+ T cells at 2 and 3 months after BMT. The mean age at BMT was higher in patients with a high level of CD8alpha/alpha+ T cells than in those without an increase in these cells, suggesting the influence of thymic function on the regenerative pathways. These results suggest that the thymus-independent pathway is the dominant source of T cells even in children shortly after allogeneic BMT.

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/microl 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.

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.

In normal hematopoiesis, stem cell factor (SCF) stimulates survival, proliferation and differentiation of hematopoietic progenitors. Although SCF acts synergistically with a variety of cytokines, the mechanism of growth factor-cooperation remains to be determined. To analyze the synergism between SCF and granulocyte-macrophage colony-stimulating factor (GM-CSF), we established a new megakaryoblastic cell line, HML-2, by culture in the presence of both SCF and GM-CSF. While SCF alone or GM-CSF alone supported modest cell growth, SCF and GM-CSF together induced substantial growth of this cell line. SCF alone tyrosine-phosphorylated several bands including the 145 kDa subunit of c-kit. GM-CSF alone did not cause the tyrosine phosphorylation of the 145 kDa subunit, but markedly up-regulated the expression of the 145 kDa subunit of c-kit. The combination of SCF and GM-CSF resulted in a synergistic increase in tyrosine phosphorylation of the 145 kDa subunit of c-kit. Several proliferation inhibitors which removed the two-factor interaction on the growth of the HML-2 cells down-regulated the 145 kDa subunit of c-kit. Thus, a synergistic increase in tyrosine phosphorylation of the 145 kDa subunit of c-kit may be one possible mechanism underlying the cooperation of SCF and GM-CSF on the HML-2 cell growth.

The pain associated with bone marrow aspiration and biopsy (BMAB) has an enormous impact on pediatric cancer patients and their families, but no specific reference standards for sedation and analgesia have been developed in Japan. To determine the problems associated with pain management during BMAB, a cross-sectional investigation was conducted.A survey was sent in October 2011 to data managers in institutions belonging to the Tokyo Children's Cancer Study Group, addressing the non-pharmacological and pharmacological pain management for BMAB performed on pediatric cancer inpatients between January 2010 and December 2010.The eligible response rate was 41 of 57 institutions (71.9%). Non-pharmacological pain intervention was provided in 68% of surveyed institutions. All institutions provided pharmacological pain management. In most institutions, sedation/analgesia was performed by pediatric oncologists in a treatment room in the ward. Standards for pain management were developed and utilized in only four institutions. Other means of pain management were provided in various settings. Twelve institutions reported insufficient sedation/analgesia. In total, 80% of institutions reported some adverse events. Two serious adverse events were reported in cases of underlying or complicated conditions. No serious long-term consequences were reported.Significant issues were identified regarding the efficacy and safety of pain management. Adverse events can occur in any institution. Children with underlying or complicated conditions are at high risk for serious adverse events. Therefore, adequate and systematic assessment, patient monitoring, preparation and treatment for adverse events, and cooperation with skilled specialists of pediatric oncology, anesthesiology, and intensive care are essential.

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.

Background: 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. Methods: Between 2005 and 2008, we recruited 23 male patients with suspected XLP, including mainly EBV-associated hemophagocytic lymphohistiocytosis (HLH), and attempted to evaluate SAP expression in fresh lymphoid cells using Alexa Fluor 488-conjugated secondary antibody instead of an FITC-conjugated one. Results: The method demonstrated that SAP was intensely expressed in CD8+ T cells and NK cells in normal fresh blood samples, thus suggesting the possible rapid identification of individuals with SAP deficiency. SH2D1A mutations were identified in six patients with SAP deficiency, but not in patients with normal SAP expression. Conclusion: The outcomes from this trial were verified by a flow cytometric assay using KST-3 and Alexa Fluor 488 secondary antibody. Based on the demonstration SAP deficiency in patients with suspected XLP, including mainly EBV-associated HLH, this approach could serve as a method for the early and rapid detection of patients with XLP-1. © 2010 International Clinical Cytometry Society

Three in vitro human germ cell tumor cell lines with different cell lineage and variable capability of differentiation were established from testicular germ cell tumors. NCR-G1 corresponding to yolk sac tumor were induced α-fetoprotein (AFP) production with retinoic acid (RA) treatment. NCR-G2 and -G3 were human embryonal carcinoma (EC) cell lines. They have cytological and immuno-phenotypic characteristics common to human EC cells as has been described in other EC cells. NCR-G2 grew as floating cell aggregates in the culture medium, whereas NCR-G3 grew as floating cell aggregate and flattered cells attached to the surface of culture dish. These flattened cells of NCR-G3 immunohistochemically expressed a variety of differentiation antigens including myogenic markers, extraembryonic trophoblastic cell marker (human chorionic gonadotropin (hCG)), and extracellular proteins. Differentiation capabilities of NCR-G3 cells became more evident when they were treated with RA. By flowcytometric analysis, human EC cell surface markers 2D7, 2H2 and 5D4 which were mouse monoclonal antibodies we obtained from immunization by NCR-G2 cells, disappeared from cell surface of RA-treated NCR-G3 cells. Consistent with these findings, the flattened cells of NCR-G3 that attached to the culture dish were negative for these antibodies. Moreover, longer exposure to RA enhanced hCG production in exposure time dependent fashion. These observations clearly indicate that NCR-G3 possess multipotent differentiation capabilities and they differentiated into the trophoblastic cell lineage other than somatic cells. In contrast to NCR-G3, NCR-G2 did not show any differentiation capabilities with RA treatment. All three germ cell tumor lines produced tumors in athymic mice with 100% efficiency. High AFP content was observed in the sera of NCR-G1 tumor-bearing mice. High hCG and AFP contents were detected in the sera of the NCR-G3 tumor-bearing mice. Thus, our newly established human germ cell tumor cell lines add new insights of molecular mechanism of human embryogenesis and differentiation of EC cells.

To study murine major histocompatibility complex (MHC)-haploidentical bone-marrow transplantation (BMT), B6C3F1 mice (H-2b/k) underwent BMT using syngeneic [B6C3F1 (H-2b/k)], haploidentical [CB6F1 (H-2d/b)], or fully allogeneic [DBA/2 (H-2d)] donor mice. As pretreatment, dimethyl myleran (DMM), an alkylating agent that produces effective myeloablation but little immunosuppression, was used with total body irradiation (TBI). Four conditioning regimens were studied: TBI 800 rads (1 rad = 0.01 Gy), TBI 950 rads, TBI 800 rads plus DMM (0.2 mg per mouse), and TBI 950 rads plus DMM. Survival rates, chimerism, proliferative responses in mixed-lymphocyte culture, specific cell-mediated lympholysis, and in vivo plaque-forming cell responses to several antigens were compared. TBI 800 rads plus DMM was maximally effective. Haploidentical BMT was as successful in inducing long-term survival and immune and hematologic reconstitution as was syngeneic BMT. This regimen plus haploidentical BMT of T-cell-purged marrow yielded survivors tolerant of donor and recipient major histocompatibility complex. Such myeloablation and immunosuppression prevented graft rejection, immunodeficiency due to histoincompatibility, and damage to a radiosensitive cell population. A microenvironmental influence crucial to some antibody responses was thus revealed. Delayed recovery of antibody production after BMT in humans may be due partly to suboptimal myeloablation or excess irradiation.

We report the case of an infant with acute myeloblastic leukemia who had the abnormal karyotype 46,XX,t(2;11;9)(q31;p15;q22),t(6;11;15)(q21;q23;q22),t(8;10)(q13;q22). At relapse, a different three-way translocation emerged. Fluorescence in situ hybridization and a reverse transcription-polymerase chain reaction assay detected the NUP98-HOXD13 fusion gene in bone marrow cells of the patient at diagnosis and at relapse. Sequence analysis showed that exon 12 of NUP98 was fused in-frame with exon 2 of HOXD13. The patient had neither a rearrangement of the MLL gene nor aberrations for FLT3, KIT, NRAS, KRAS, or PTPN11. The NUP98-HOXD13 fusion transcript created by t(2;11;9)(q31;p15;q22) may play an important role in the leukemogenesis in this case.

The clinical utility of minimal residual disease (MRD) measurements following allogeneic stem cell transplantation (SCT) in childhood ALL is controversial. We therefore performed a multi-institutional study of MRD in bone marrow samples taken before SCT and at 1, 3, 6 and 12 months after SCT. Case-specific clonal rearrangements of IgH and TCR genes and expression levels of Wilms' tumor 1 (WT1) mRNA were determined by PCR or RT-PCR methods. In total, 95 cases met all criteria for analysis of informative IgH/TCR markers and quantitative WT1 mRNA expression levels. During the 2-year (median 414 days) study period, 20 patients relapsed. Although the proportion of patients with a positive IgH/TCR result before SCT was significantly reduced at 1 month after treatment (P

A rare beta-thalassemia mutation at the splicing junction [namely, G-->C in intervening sequence (IVS) I-1] was found in a Japanese family. The proband and his mother were heterozygous for the mutation. Analysis of mRNA extracted from the reticulocyte-rich fraction obtained from the proband's mother revealed that the mutant beta-globin gene did not produce any detectable, stable mRNA including exon 1 and exon 2, since the polymorphism in exon 1 on her mutant gene was not detected in the RT-PCR products.

Synergism between stem cell factor (SCF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to be essential for hematopoietic cell proliferation. Since HML-2 cells proliferate exponentially in the presence of SCF and GM-CSF together, we analyzed the molecular mechanism of the interaction between these two factors in the cells. An immediate-early gene product, c-myc, was additively upregulated in HML-2 cells by addition of a combination of SCF and GM-CSF. c-myc antisense oligonucleotides effectively suppressed cell proliferation and downregulated the induction of D3, E, A, and B cyclins in HML-2 cells stimulated with the two-factor combination. HML-2 cells arrested at the G0/G1 phase with SCF alone and expressed modest amounts of c-myc and cyclin D3, but not cyclin E. With GM-CSF treatment alone, the cells could not progress to the G2/M phase and expressed c-myc, cyclin D3 and cyclin E but not cyclins A or B. The addition of the counterpart cytokine resulted in cell cycle completion by induction of the deficient cyclins. Taken together, it appears that the induction of c-myc is an indispensable event in the proliferation of HML-2 cells and that the cytokines SCF and GM-CSF interact reciprocally for expression of all cyclins required for cell cycle progression.

HML/SE is a cytokine-dependent cell line established from childhood acute megakaryoblastic leukemia. Granulocyte-macrophage colony-stimulating factor or stem cell factor (SCF) alone could stimulate proliferation of HML/SE cells, however interleukin-3, interleukin-6, granulocyte colony-stimulating factor and thrombopoietin could not. Although erythropoietin (EPO) alone stimulated neither proliferation nor differentiation of HML/SE cells, it did stimulate proliferation of HML/SE cells and production of hemoglobin in the presence of SCF. SCF activated the human EPO receptor promoter and induced EPO receptor gene expression. Given these results, we speculate that HML/SE cells acquired responsiveness to EPO via the EPO receptor induced by SCF. Mutation analysis of putative transcription factor binding sites in the human EPO receptor promoter suggested that Sp1, rather than the GATA-1 binding site, contributed to the induction of the hEPOR gene. Although it is well documented that hematopoietic stem cells and primitive progenitors require both an early-acting cytokine and a lineage-specific cytokine to differentiate to a certain lineage, related mechanisms are not well understood. HML/SE may serve as an excellent model system to analyze functions of early-acting cytokine SCF and lineage-specific cytokine EPO related to proliferation and differentiation of hematopoietic stem cells.

Development of prophylactic cranial irradiation (CRT) has greatly contributed to the improvement of outcome of childhood ALL. However, late complications, including secondary malignant neoplasms, arising from CRT are becoming big problems. Furthermore, the prognosis of the patients with relapse in central nervous system (CNS) after CRT is extremely poor. To eliminate CRT in the treatment for majority of the patients with B-cell precursor (BCP) ALL, we had attempted to reduce the indication of CRT by conducting randomizations in 3 studies. We present here the long-term results of 4 consecutive clinical trials, to assess the effect of reducing the proportion of patients who received CRT on the treatment outcome. Of the 2,116 children with newly diagnosed ALL with 1–18 years of age who were enrolled to the 4 consecutive TCCSG ALL studies between 1989 and 2003, 1845 BCP ALL patients were evaluable: 354 Pts in L89-12 protocol (1989–1992), 291 Pts in L-92-13 (1992–1995), 536 Pts in L95-14 (1995–1999) and 664 Pts in L99-15 (1999–2003). The probability of event-free survival (EFS) and overall survival (OS) were constructed using the Kaplan-Meier method. Events in the analysis of EFS included induction failure, death, relapse and secondary malignant neoplasm. The initial status of CNS disease was examined after 7 or 10 days of pre-phase treatment consisted of steroid and/or other drugs. The definition of the CNS status was as follows: CNS positive = CNS-1 with symptomatic CNS disease, CNS-2, CNS -3 and traumatic lumbar puncture (TLP) with leukemic blasts; CNS negative = CNS-1 and TLP without leukemic blasts. In L89-12 study, all but a half of the standard risk group (SR) patients had received CRT. After this study, no CRT was given for the patients in SR. Patients in the intermediate risk group (IR) were randomly assigned into either high-dose methotrexate (HD-MTX) arm or CRT arm in L92-13 study. In L95-14 study, patients with initial leukocyte count exceeding 50,000/ul received CRT, whereas the others were treated with HD-MTX in IR. A part of patients in the high risk group (HR) in L95-14 study proceeded to stem cell transplantation during the first remission. In L99-15 study, only patients with high WBC, more than 100,000/ul, at diagnosis received CRT. Overall, no increase in CNS relapse was observed. The EFS rate and the OS rate at 8 years in L99-15 study were significantly improved compared to those in L89-12 study. Details of the results are shown in the table. By intensifying systemic chemotherapy and intrathecal injections, the proportion of the patients who received CRT in L99-15, which was the last study, was reduced to as small as 4% without increase in CNS relapse. These remaining 4% of the patients who had received CRT included those with CNS-3 disease (= 0.3 %), and they are the ultimate target who needs innovative treatment. In conclusion, we have succeeded to reduce the proportion of the patients with BCP ALL receiving CRT from 79% to 4% in the past 20 years, without compromising the treatment outcome. Table. Treatment and outcome in each study L89-12 L92-13 L95-14 L99-15 LP: lumbar puncture, IT: intrathecal injection, *randomization, **Pts with WBC higher than 50,000/ul at diagnosis received CRT. ***Pts with WBC higher than 100,000/ul at diagnosis received CRT. Rate of CNS positive 2.0% 3.4% 1.3% 2.1% Date of 1st LP/IT Day8 Day8 Day11 Day8 Rate of Pts receiving CRT 79% 36% 23% 4% Dose of CRT 18Gy 1y:12Gy, 2-18y: 18Gy 1y:12Gy, 2–18y: 18Gy 1–6y:12Gy, 7–18y: 18Gy CNS directed therapy SR*: A; HD-MTX 9g B; CRT IR: CRT HR: CRT SR: HD-MTX 6g IR*: A; HDMTX 6g B; CRT HR: CRT SR: HD-MTX 9g IR**:A;MTX9g B;CRT HR: CRT SR: HD-MTX 9g IR***: HD-MTX 9g (*HD-or LD-Ara-C) HR***: HD-MTX 6g Rate of Pts receiving SCT 0% 0% 15% 8% 8y-EFS 65+/−3% 55+/−3% 76+/−2% 74+/−2% 8y-OS 76+/−2% 79+/−2% 84+/−2% 86+/−2% Rate of BM relapse 25.6% 31.1% 17.6% 18.7% Rate of Isolated CNS relapse 2.6% 0.72% 1.2% 1.5% Rate of all CNS relapse 3.2% 1.4% 1.7% 3.2%

Immunophenotypic features of human sarcomatous Wilms’tumor (SWT) were studied using a newly established mouse monoclonal antibody (5H10, IgG). 5H10 was produced against CR SW2, one of several transplanted SWT lines in nude mice, and defines a 200-kDa cell surface protein. The antibody was found to react equally with all subtypes of SWT; clear cell sarcoma of the kidney, malignant rhabdoid tumor of the kidney, and unclassified sarcoma. Furthermore, it reacted equivalents with surgically resected tumors, transplanted tumors in nude mice, and cell lines in vitro. On the other hand, 5H10 was entirely negative for any of the components of nephroblastic Wilms’tumor (NBW). Considering these results, 5H10 appears to recognize the antigen expressed preferentially on SWT, and therefore the subtypes of SWT may be closely related to one another immunophenotypically. In normal human tissues, however, 5H10 only reacted with fetal kidneys, its reactivity being restricted to the lower limbs of S bodies in both the metanephros and mesonephros. No reactivity was identified in any other tissues including adult kidney. These results indicate that 5H10 detects an oncofetal antigen expressed preferentially in both SWTs and fetal kidney and that the histogenesis of SWT should be considered in connection with nephrogenesis. Acta Pathol Jpn 39: 656-663, 1989.

The common acute lymphoblastic leukemia antigen(CALLA) on acute lymphoblastic leukemia(ALL) cells and granulocytes were compared by newly developed and other anti-CALLA monoclonal antibodies(anti-CALLA). New anti-CALLA(IF-3 through IF-7) were effectively selected by immunostaining on kidney sections. By competitive binding three antigenic determinants were separated on ALL cells by IF and other anti-CALLA. All three determinants existed on granulocytes although the reactivity of each anti-CALLA was variable. Such a variability was partly due to the heterogeneous terminal sialic acid compositions. Although CALLA from granulocytes and ALL cells differed in molecular weight they showed identical peptide mapping patterns. These results strongly suggest that CALLA on ALL cells and granulocytes are structurally related although they are different in post-transulational modification.

Establishment of monoclonal antibodies against human germ cell tumors which detected cell surface antigens, 2A2 and 2G10 (subclass; IgM) are described. Distributions of these antigens in human tumors were studied immunohisto-chemically. They were expressed on immature endodermal components often seen in yolk sac tumors but not on the other solid tumors in childhood. Regarding their specificities on human germ cell tumor, we have established an ELISA system to detect these antigens from patient's sera. In normal and fetal tissue, on the other hand, they showed quite different distributions. In the kidney, for example, 2A2 and 2G10 were present on collecting tubules and proximal/distal tubules respectively which were already observed in fetal kidney, gestational ages 10 weeks. These results indicate the usefulness of these antibodies as the markers for human germ cell tumors and also for somatic cell differentiation.

Two cases of Ki-1 lymphomas in childhood were analyzed immunohistochemically and immunoelectron microscopically. They expressed Hodgkin's disease associated antigen, Ki-1, interleukin-2 receptor (IL2R), OKT9, and HLA-DR. Histologically, the tumour cells were large in size with abundant cytoplasm and atypical nuclei. Lymph node involvement was characterized by parafollicular and marginal sinus infiltration. These features were identical to those reported in Ki-1 lymphomas. Electron microscopically tumour cells had abundant cytoplasmic organelles with pleomorphic nuclei but had no specific granules. Some tumour cells had marked interdigitation of cell membrane. Immunoelectron microscopically Ki-1 was positive on cell membrane. Tumour cells had no T-cell or B-cell antigens except for Leu-3 (T4). Unexpectedly they expressed epithelial membrane antigen (EMA) strongly. EMA was positive on cell membrane and in the cytoplasm. EMA was detected effectively in paraffin-embedded sections. Among the malignant lymphomas in childhood tested, two cases were EMA-positive. The pattern of EMA-reactivity and the histology were very similar to Ki-1 lymphomas. These results strongly suggest that Ki-1 lymphomas in childhood may arise from non-lymphoid haematopoietic cells and that EMA can be used as a new marker to distinguish certain type of Ki-1 lymphomas in childhood.

Adult and fetal human kidneys were investigated for the reactivities of monoclonal antibodies, BA-1, BA-2 and BA-3 against human leukocytes. In developing metanephros, their reactivities changed reflecting the stage of nephrogenesis. Thus BA-1 stained both metanephric blastema and ureteric bud. Glomerular and proximal tubular development was characterized by the disappearance of BA-1 reactive antigen and the appearance of CALLA defined by BA-3. Immuno-electron-microscopically CALLA was solely located on the glomerular epithelial membrane and on the microvilli of the proximal tubules. BA-2 constantly stained ureteric bud-derived tissues. These observations were applied for the analysis of histogenesis of Wilms' tumour. Tumour blastema of the classical type reacted only with BA-1. Epithelial components in the classical as well as the epithelial type reacted both with BA-1 and BA-2. CALLA was only detectable in glomeruloid and the connecting tubular structures, while these were unstained by BA-1. Stromal components in the classical and the sarcomatous type did not express any of these antigens. Staining patterns were identical in surgically removed and xenotransplanted tumours. These studies establish that each component of Wilms' tumour can clearly be interpreted regarding its histogenesis and that epithelial components corresponding to proximal tubules are identified in Wilms' tumour.