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Background Quality of life (QOL) as a treatment outcome has not yet been evaluated among patients receiving a specific treatment regimen by treatment phase in a consistent manner. This exploratory cross-sectional study evaluated the QOL of children with acute lymphoblastic leukemia (ALL) receiving one of the most popular treatment regimens in Japan (Japan Association of Childhood Leukemia Study ALL-02 revised protocol). Methods Children aged 5–18 years with newly diagnosed B-cell precursor ALL were included. The Pediatric Quality of Life Inventory™ 4.0 Generic Core Scales (PedsQL-J) were completed by children with ALL and their siblings, as well as by age- and sex-matched healthy controls. PedsQL Cancer Module (PedsQL-C) scores were also collected from children with ALL. Results QOL in children with ALL of the consolidation phase group was significantly decreased compared with that of healthy controls, except in the area of emotional functioning. Regarding the maintenance phase group, QOL impairment was noted in the physical and school functioning, but no differences were noted in social functioning. The off-treatment group had a large effect size only for physical functioning, and the social functioning score was even better in children with ALL than in matched controls. QOL of children with ALL differed with treatment phase. Effect size varied with function and treatment phase. Conclusions QOL may change with the progression of treatment, and the timing of these changes varied according to function and problem.

During electron microscopic examination of 156 consecutive human endomyocardial biopsy specimens, we found marked mitochondrial deformity within a single cardiomyocyte in each of 4 specimens. The deformed mitochondria were unevenly distributed, but the deformities were confined to the one cardiomyocyte. Those affected cardiomyocytes were accompanied by nonspecific degenerative changes such as nuclear hypertrophy and/or rarefaction of the myofibrils. Mitochondria in all other cells within the specimens appeared normal. Such an abnormality has never been reported to date. Each of the four cases was diagnosed with a different ailment: post-myocarditis, dilated cardiomyopathy, amyloidosis, and tachycardia-induced heart failure. However, all four cases were accompanied by left ventricular systolic dysfunction at biopsy. The very limited mitochondrial deformation may thus reflect a type of degenerative change that accompanies heart failure. .

Peritonitis remains an important complication of peritoneal dialysis and is mostly caused by aerobic enteric bacteria. Non-tuberculous mycobacteria (NTM)-associated peritonitis is an unusual but serious infection, requiring special culture techniques to avoid delay in diagnosis. We report the case of an 11-year-old girl with aplastic anemia on ambulatory peritoneal dialysis who had Mycobacterium avium complex-associated peritonitis after allogeneic hematopoietic stem cell transplantation (allo-HSCT). This case emphasizes that we should be constantly cautious about NTM infection in allo-HSCT recipients, especially when standard cultures are negative and the infection is refractory to empirical antibiotic therapy.

Background Peripheral blood stem cells (PBSC) may be used as an alternative to bone marrow (BM) for allogeneic transplantation. Since peripheral blood stem cell bank from unrelated volunteer donor has been started in Japan, use of PBSC allografts may be increased. Therefore we surveyed the outcomes of Japanese leukemia children after PBSC and BM transplantation. Procedure This retrospective study compared the outcomes of 661 children (0–18 years) with acute lymphoblastic leukaemia (ALL) or acute myeloid leukaemia (AML) who received their first allogeneic peripheral blood stem cell transplantation (PBSCT; n = 90) or bone marrow transplantation (BMT; n = 571) from HLA-matched siblings between January 1996 and December 2007. Result Neutrophil recovery was faster after PBSCT than after BMT (ALL: P

Anthracyclines are key drugs for the treatment of children with acute lymphoblastic leukemia (ALL). However, anthracyclines are known to induce QT prolongation, and life-threatening complications, such as torsades de pointe may also occur. To date, there have been no reports on the use of anthracyclines in patients with congenital long QT syndrome (LQTS). We report a child with ALL complicated by congenital LQTS who was treated with anthracyclines. The administration of anthracyclines caused QT-interval prolongation, but this was uneventful with the concomitant administration of magnesium sulfate.

The prognosis of high-risk rhabdomyosarcoma (RMS) with metastatic or recurrent disease remains poor. We report a 6-year-old girl who successfully underwent allogeneic hematopoietic stem cell transplantation against recurrent metastatic alveolar RMS. The disease recurred at distant lymph node metastasis with bone marrow involvement. After chemotherapy and radiotherapy for the metastatic site, she underwent allogeneic bone marrow transplantation during complete remission from her 5/8 HLA-matched father. She developed acute graft-versus-host disease after preemptive donor lymphocyte infusion and remains in a disease-free condition for 31 months after transplantation. A graft-versus-tumor effect through allogeneic immune cells might produce a beneficial effect for high-risk RMS.

HSCT is the optimal treatment for patients with SCID. In particular, HSCT from a HLA-identical donor gives rise to successful engraftment with long survival. We report a six-month-old girl with JAK3-deficient SCID who developed hemophagocytosis after BMT without conditioning from her HLA-identical father. She had suffered from pneumonia and hepatitis before BMT. Prophylaxis for GVHD was short-term methotrexate and tacrolimus. On day 18 after BMT, the patient developed hemophagocytosis in bone marrow when donor lymphocytes were increasing in peripheral blood. Analysis of chimerism confirmed host origin of macrophages and donor origin of lymphocytes. Thus, host macrophage activation was presumably induced in response to donor lymphocytes through immunoreaction to infections and/or alloantigens. HSCT for SCID necessitates caution with respect to hemophagocytosis.

Anaplastic large cell lymphoma (ALCL) is chemosensitive, but recurrence is frequently observed. Allogeneic hematopoietic stem cell transplantation (HSCT) has recently been reported to be effective against recurrent disease, suggesting a graft-versus-lymphoma effect. We present a 3-year-old child with recurrent ALCL who underwent HSCT from an HLA-1-locus mismatched cord blood unit following reduced intensity conditioning with fludarabine, melphalan, and low-dose thoraco-abdominal irradiation. Engraftment was uneventful, but grade III acute graft-versus-host disease was observed. He is well and free of disease 25 months after HSCT, which implies that reduced intensity conditioning may allow a sufficient graft-versus-lymphoma effect against ALCL while lessening treatment-related toxicities.

Wilms tumor gene (WT1) can be overexpressed in childhood cancers. We evaluated the efficacy of WT1 vaccination for five children with solid cancer or acute leukemia. WT1 vaccine was administered to HLA-A*2402-positive patients with WT1-overexpressing residual tumor despite prior conventional treatment. One patient showed complete response and one patient showed stable disease according to the Response Evaluation Criteria in Solid Tumors; the remaining three showed progressive disease. Treatment-related adverse effects were limited to local injection site erythema. These results suggest that WT1 vaccination has therapeutic potential, but any beneficial effect may be insufficient in the presence of gross residual disease.

Myeloid/NK cell precursor lymphoma/leukemia has been suggested to be of precursor NK origin. We report a 1-year-old boy with myeloid/NK cell precursor lymphoma/leukemia who presented with a skin nodule. Biopsy of the skin nodule specimen revealed CD45(+), CD56(+), myeloid antigen(+), CD7(-), CD3(-), CD19(-), CD34(-), CD4(-) by flow cytometry and myeloperoxidase (MPO)(+), TdT(+), cytoplasmic CD3(+) by immunohistochemistry. He received acute myeloid leukemia-oriented combination chemotherapy. Although his skin nodule disappeared, regrowth was observed; a biopsy was performed again, showing that the immunophenotype was CD56(+), myeloid antigen(+), HLA-DR(+), MPO(-), CD3(+) and TCRgammadelta(+). Being refractory to chemotherapy, he underwent stem cell transplantation from his father. However, he relapsed again and died from septic shock. Although myeloid/NK cell precursor acute leukemia is characterized by CD7(+), CD56(+), CD3(-), CD34(+) and myeloid antigen(+) phenotype, the blast cells of our patients lacked CD34 and CD7 expression while expressing myeloid antigens. Furthermore, CD94 1A transcripts were predominantly expressed rather than CD94 1B, implying their origin in immature NK cells. After acute myeloid leukemia-oriented combination chemotherapy, his blasts acquired stable CD3 expression and TCRgammadelta rearrangement at recurrence. The blast cells possessed features overlapping both myeloid/NK precursor acute leukemia and blastic NK/precursor acute lymphoma/leukemia. These observations suggest that two subclones originated from common precursors.

X-linked adrenoleukodystrophy (X-ALD) is a genetic disease associated with demyelination of the central nervous system, adrenocortical insufficiency and accumulation of very long chain fatty acids. It is a clinically heterogeneous disorder ranging from a severe childhood cerebral form to an asymptomatic form. The incidence in Japan is estimated to be between 1:30,000 and 1:50,000 boys as determined by a nationwide retrospective survey between 1990 and 1999, which found no cases with Addison's form. We reviewed the medical records of eleven Japanese boys with X-ALD from 1990 to 2010 in our institute. Eight patients were detected by neuropsychological abnormalities, whereas a higher prevalence of unrecognized adrenocortical insufficiency (5/11: 45%) was observed than previously recognized. While no neurological abnormalities were demonstrated in two brothers, the elder brother had moderate Addison's disease at diagnosis and the presymptomatic younger brother progressed to Addison's disease six months after the diagnosis of X-ALD. Early detection of impaired adrenal function as well as early identification of neurologically presymptomatic patients by genetic analysis is essential for better prognosis. Addison's form might be overlooked in Japan; therefore, X-ALD should be suspected in patients with adrenocortical insufficiency.

Post-transplant lymphoproliferative disorder (PTLD) is a well recognized and potentially fatal complication after pediatric cardiac transplantation. PTLD encompasses a wide spectrum, ranging from benign hyperplasia to more aggressive lymphoma. Most cases are Epstein-Barr virus (EBV)-related B-cell tumors resulting from impaired immunity due to immunosuppressive therapy. Pediatric recipients, often seronegative for EBV at transplantation, have a greater risk for PTLD than adults. The clinical presentation of PTLD varies from isolated lymphadenopathy to systemic disease; common sites involved are gastrointestinal tract, lung or airway, and cervical lesions. Timely and accurate diagnosis based on histological examination of biopsy tissue is essential for early intervention. Immunostaining for EBV and evaluation for clonality are needed. For prophylaxis when EBV viral loads are increasing or for initial treatment of early lesions or polymorphic PTLD, a reduction in immunosuppressive treatment is a key component of therapy, but caution is needed for possible rebound allograft rejection. Chemotherapy is indicated for patients with poor response to reduced immunosuppression and for highly aggressive monomorphic PTLD. The use of rituximab in combination with chemotherapy is effective. For the time being, avoiding excessive immunosuppression is the most effective strategy for reducing the incidence of PTLD. Calcineurin inhibitor (CNI) minimization with proliferation signal inhibitors (PSIs) or conversion from a CNI to a PSI might be useful for preventing both development of PTLD and allograft rejection.

Post-transplant lymphoproliferative disorders (PTLDs) occur in 3.5-9% of patients after pediatric cardiac transplantation. Caution is needed when treating patients with PTLD because of the risk of allograft rejection frequently caused by withdrawal of immunosuppression. In this report, we describe a 47-month-old boy who developed PTLD as an ileocecal mass 29 months after cardiac transplantation. Immunosuppressive therapy with cyclosporine A (CyA) had been reduced due to an elevation of Epstein-Barr virus (EBV) titer for 8 months before development of PTLD. Histology of the tumor was diffuse large B cell lymphoma. EBV was detected by in situ hybridization assay. Cytogenetic analysis revealed t(8;14)(q24;q32) and Southern blot analysis detected a c-Myc rearrangement. He was treated with rituximab and combination chemotherapy with excellent response. CyA dose was maintained at reduced levels during chemotherapy and later minimized with introduction of everolimus. The child is free of both PTLD and allograft rejection 41 months after the diagnosis of PTLD.

Immunotherapy using a Wilms tumor (WT1) peptide has been undergoing clinical trials for adulthood leukemia and solid cancer with promising results. In this study, the authors used WT1 peptide vaccination to treat a 6-year-old girl with metastatic alveolar rhabdomyosarcoma. She received weekly intradermal injection with HLA-A*2404-restricted, 9-mer WT1 peptide against residual bone disease. After 3 months her bone disease disappeared, concurrent with an increase in the frequency of WT1-specific cytotoxic T lymphocytes (CTLs). A high proportion of WT1-specific CTLs with effector or effector memory phenotype were detected in peripheral blood of this patient. She is currently still on continued WT1 peptide immunotherapy in a disease-free condition for 22 months. WT1 peptide-based immunotherapy should be a promising option for high-risk rhabdomyosarcoma in childhood.

We evaluated the feasibility of UCBT from unrelated donors and a myeloablative preparative regimen that did not involve anti-thymocyte globulin in five children with lysosomal and peroxisomal diseases. Patients with MPS II (n = 1), adrenoleukodystrophy (n = 1), metachromatic leukodystrophy (n = 2), and Krabbe disease (n = 1) received UCBT between December 2001 and September 2005. All patients received oral Bu (600 mg/m2), CY (200 mg/kg IV), and fludarabine (180 mg/m2 IV). Prophylaxis for GVHD consisted of a combination of tacrolimus and a short methotrexate course. Neutrophil engraftment occurred a median of 24 days (range, 21–25) after transplantation. None had graft rejection. One patient developed grade III acute GVHD and the other four patients had grade I acute GVHD; none had extensive chronic GVHD. One patient developed hemorrhagic cystitis. There were no treatment-related deaths. Although one child with MPS II died of PTLD 10 months after the UCBT, four of the five children are alive 14, 20, 31, and 55 months after transplantation with complete donor chimerism. These results suggest the feasibility of the UCBT with Bu, fludarabine, and CY-preparative regimen for patients with inherited metabolic diseases.

With recent improvements in the diagnosis and treatment of cancer, the number of childhood cancer survivors (CCSs) has been increasing in Japan. The importance of quality of life during the lifetime of CCSs has now been recognized, and the late effects of cancer treatments are essential and important issues. In this study we analyzed the endocrinological abnormalities of CCSs by retrospectively evaluating 122 outpatients (62 males and 60 females) who had been referred from pediatric oncologists to our follow-up clinic among 151 CCSs attending our hospital more than two years after their cancer treatment. Follow-up duration varied from 2 to 30 (median 8.0) years. Their average age was 17.3 (range 4-36, median 17.0) years, and 38 patients (31.1%) reached adulthood. Endocrinological abnormalities were found in 82 (67%) of 122 survivors. Gonadal dysfunction was observed in 60 patients (49%). Thirty-nine patients (32%) were short or grew at a slower rate. Twenty-six patients (21%) showed thyroid dysfunction. Other abnormalities were as follows: obesity in 20 patients (16%), leanness in 10 (8%), central diabetes insipidus in 11 (9%) and adrenocortical dysfunction in 9 (7%). Low bone mineral density was observed in 41 (42%) of 98 patients evaluated. These endocrinological abnormalities were caused by the combined effects of cancer itself and various treatments (chemotherapy, radiation therapy, surgery, and hematopoietic stem cell transplantation). Lifetime medical surveillance and continuous follow-up are necessary for CCSs, because treatment-related complications may occur during childhood and many years after the therapy as well. Endocrinologists should participate in long-term follow-up of these survivors in collaboration with pediatric oncologists.

The hypolipidemic effect of dietary methanol fraction (BMMF) extracted from bitter melon (Koimidori variety), at the levels of 0.5% and 1.0%, was examined in male golden Syrian hamsters fed diets supplemented with and without cholesterol. The feeding of BMMF at 0.5% and 1.0% levels in the diets for 4 wk tended to reduce food intake and growth, although there was no difference in food efficiency (weight gain/food intake). An effect of dietary BMMF on serum triglyceride was not seen in hamsters fed diets free of cholesterol, while hypertriglyceridemia induced by dietary cholesterol was significantly lowered in a dose-dependent manner in those fed diets containing the BMMF Serum total cholesterol concentration also tended to decrease in a dose-dependent manner following feeding of increasing amounts of BMMF in the presence and absence of cholesterol in the diet. The effects of dietary BMMF on liver triglyceride and total cholesterol levels were marginal, although dietary cholesterol caused a marked accumulation of these lipid molecules in the liver. These results suggest that the BMMF contains some components that could ameliorate lipid disorders such as hyperlipidemia.

Background: Thromboembolic and bleeding events are serious complications associated with the administration of L-asparaginase (ASP) during the induction phase in children with acute lymphoblastic leukemia (ALL). Prophylactic supplementation of plasma-derived coagulation products remains controversial. The purposes of this study were to examine the plasma levels of hemostatic proteins during the induction phase and the efficacy of prophylactic replacement of plasma-derived products. Methods: A multicenter retrospective survey on the incidence of hemostatic complications and the frequency of prophylactic replacement of plasma-derived products among children with ALL who underwent ASP therapy was conducted. All patients were treated according to the Japan Association of Childhood Leukemia Study (JACLS) ALL-97 protocol. Results: Thirty-six (67%) of the 54 JACLS-treatment centers responded and provided information on 127 patients. Clinically relevant hemostatic complications occurred in two patients (1.6%; 2/127); one patient suffered intracranial bleeding 5 days after beginning the protocol, and the second patient suffered an ischemic attack during an ASP treatment. The administration of ASP (10 000 u/m2 × six doses) led to significant reductions in the plasma fibrinogen (180 ± 74 to 105 ± 57 mg/dL), antithrombin III (AT III) (126 ± 21 to 73 ± 18%), and plasminogen levels (97 ± 17 to 52 ± 18%). These laboratory parameters returned to normal levels 2 weeks after completion of the ASP treatment. Forty-eight patients (38%) received AT III concentrate (AT III-supplement group). The AT III level of the AT III-supplement group was significantly lower than that of the AT III non-supplement group, not only during the ASP therapy, but also prior to the ASP therapy. A greater percentage of patients in the AT III-supplement group received fresh-frozen plasma and in greater amounts than those in the AT III non-supplement group. Conclusions: Severe hemorrhage and thrombosis were uncommon in the ALL patients who underwent ASP therapy in comparison with the degree of coagulation changes. Prophylactic administration of AT III concentrate or fresh-frozen plasma did not demonstrate clear beneficial effects in the treatment of ALL in preventing thromboembolic events.

Unrelated cord blood transplantation (CBT) has been worldwide for bone marrow reconstitution. CBT is associated with a high frequency of engraftment failure and rejection due to a small dose of graft cells. In cases of engraftment failure or rejection following unrelated CBT, retransplantation from the original donors is impossible. We report a successful transplantation with CD34p+ blood cells selected from a 2-loci HLA-mismatched mother to a child with acute monocytic leukemia after engraftment failure of the primary CBT. Selected CD34p+ blood cell transplantation is a useful approach for retransplantation in the setting of engraftment failure.

All-trans retinoic acid (ATRA) improves the outcome of patients with acute promyelocytic leukemia (APL) [1, 2]. ATRA administration leads to complete remission in APL patients by inducing growth arrest and differentiation of the leukemic clone. Caution is needed during ATRA treatment because of the potential development of differentiation syndrome (DS), formerly known as retinoic acid syndrome. It is a cardiorespiratory syndrome characterized by unexplained fever, weight gain, respiratory distress, interstitial pulmonary infiltrates, pleural and pericardial effusions, acute renal failure, cardiac failure, and hypotension [3, 4]. Although this syndrome commonly develops during the first week after starting ATRA treatment [3, 4], late-onset DS has been observed in a substantial number of patients [4]. We report a child with APL who developed DS after 21 days on ATRA treatment. A 9-year-old boy was diagnosed as having APL with t(15;17)(q22;q11.12) and PML/RARa transcript. He was registered on the JPLSG (Japan Pediatric Leukemia/Lymphoma Study Group) AML-P05 protocol and ATRA was administered at 45 mg/m/day, PO. When his WBC count was over 5,000/lL, he received dexamethasone (DEX: 0.4 mg/kg/day, IV) for DS prevention (Fig. 1). Promyelocytes disappeared in his peripheral blood after starting daunorubicin and cytosine arabinoside. He became febrile on the 16th day and antibiotics were given with successful resolution of fever. On the 17th day, serum C-reactive protein (CRP) level increased to 1.6 mg/dL and he complained of chest pain. Chest X-ray, electrocardiogram, and echocardiography showed normal findings. On the 20th day, he again became febrile at 39 C and antibiotics were changed. On the 21st day, his urine output decreased from 549 to 248 mL in 8 h and he produced foamy sputum with fresh blood. He subsequently became dyspneic. Pulse oxymetry showed oxygen saturation at 85% and BP rapidly fell from 92/56 to 80/50 mmHg. Weight gain or peripheral edema was not observed, but serum creatinine level transiently increased from 0.26 to 0.32 mg/dL. Chest X-ray revealed bilateral patchy confluent opacities. Although echocardiography demonstrated no pericardial fluid, left ventricular ejection fraction (LVEF) decreased to 26%. At this point, serum CRP level was 5.6 mg/dL and brain natriuretic peptide was 757.4 pg/mL. Cultures of blood, urine, and sputum were negative. A probable diagnosis of DS was made. ATRA was discontinued and DEX (0.4 mg/kg/day, IV) was started. He was also treated with dobutamine and milrinone, leading to an increase of LVEF to 55% several hours later. His respiratory status promptly improved with resolution of pulmonary infiltrates. Myelocyte and metamyelocyte counts increased up to 200/lL from the 21st day through to the 24th day (Fig. 1). The pathogenesis of DS is not completely understood, although several mechanisms have been proposed. First, adhesion and aggregation of APL cells may be promoted by increased expression of CD11b, CD11c and CD54 (ICAM-1) during ATRA treatment [5]. Second, differentiated APL cells secrete serine proteases [6] and matrix metalloproteases [7], which can promote coagulopathy and cellular invasion, respectively. Third, ATRA-treated APL cells secrete inflammatory cytokines such as IL-8, IL-1b, G-CSF, and TNF-a [8] as well as CC chemokines such as monocyte chemoattractant protein-1, macrophage inflammatory protein (MIP)-1a, and MIP-1b [9]. H. Yoshida R. Matsumura E. Sato Y. Hashii H. Ohta (&) K. Ozono Department of Pediatrics, Osaka University Graduate School of Medicine, Yamadaoka 2-2, Suita 565-0871, Japan e-mail: ohta@ped.med.osaka-u.ac.jp

Pneumocystis jiroveci pneumonia (PCP) is a serious complication in patients receiving chemotherapy or hematopoietic stem cell transplantation. Current recommendations for trimethoprim-sulfamethoxazole (TMP-SMZ) dosing as PCP prophylaxis in immunocompromised patients are based on either daily dosing or dosing three consecutive days per week. We report our experience of prophylaxis with TMP-SMZ twice daily on two non-consecutive days per week in 145 immunocompromised children with hematologic disorders, cancer, or metabolic disorders following chemotherapy or hematopoietic stem cell transplantation. There were no breakthrough cases of PCP. We therefore conclude our prophylaxis regimen is effective against PCP in immunocompromised children.

LAD is a rare and fatal congenital disorder in which there is a defect of the leukocyte adhesion molecule (CD18) on neutrophils. Severe LAD results in frequent and potentially fatal infections. Although allogeneic HSCT is the only curative treatment for severe LAD, strategies for HSCT remain to be established since a high engraftment failure rate and severe persistent infection are still seen. We report a four-month-old boy with LAD who was successfully treated with allogeneic HSCT from an HLA-complete matched sibling following a conditioning regimen of pharmacokinetically adjusted individual busulfan doses.

Hemophagocytic lymphohistiocytosis (HLH) is caused by the hyperactivation of T cells and macrophages. The clinical characteristics associated with this disease result from overproduction of Th1 cytokines including interferon-γ (IFN-γ), interleukin-2 (IL-2), and tumor necrosis factor-α (TNF-α). In this study, we analyzed the production of IL-12 and IL-4, which determine Th1 and Th2 response, respectively, and IL-10, which antagonizes Th1 cytokines, in 11 patients with HLH. IL-12 was detected in plasma in all patients (mean peak value, 30.0 ± 5.0 pg/mL), while IFN-γ was massively produced in nine patients (mean peak value, 79.2 ± 112.0 U/mL). IL-4 was not detected in any of the patients. Plasma IL10 levels were elevated in all patients (mean peak value, 2,698.0 ± 3,535.0 pg/mL). There was a positive correlation between the levels of IFN-γ and IL-10 (P < .01). The plasma concentrations of these cytokines were initially high, before decreasing after the acute phase. However, the decrease in IL-10 levels was slower than that of IFN-γ. Although the concentration of IL-12 was high at the acute phase, in some patients, a peak in the level was delayed until the chronic phase. Thus, in HLH, production of cytokines that promote development of Th1 cells appears to be predominant over that for Th2 cell development. Overproduction of IL-10 was also observed indicating that a mechanism suppressing hyperactivation of Th1 cells and monocytes/macrophages functions in patients with this disease.

Background Serum β-cryptoxanthin levels are lower in overweight subjects than in normal subjects. Abnormalities of adipocytokine profiles in obesity subjects have been reported. There are several reports that serum β-cryptoxanthin levels in them were relatively lower than normal subjects. Objective We hypothesize that supplementation of highly concentrated β-cryptoxanthin improves serum adipocytokine profiles in obese subjects. This study tested the association between β-cryptoxanthin intake and serum adipocytokine levels. Methods An intervention study consisted of a 3-week long before-and-after controlled trial, where β-cryptoxanthin (4.7 mg/day) was given to 17 moderately obese postmenopausal women. Results The results indicated no significant changes in body weight or body mass index (BMI). Serum β-cryptoxanthin levels increased significantly by 4-fold. Serum high molecular weight (HMW)-adiponectin levels increased significantly, while serum plasminogen activator inhibitor (PAI)-1 levels decreased. Conclusions We concluded that increasing the intake of β-cryptoxanthin to approximately 4 mg per day for 3 weeks may have beneficial effects on the serum adipocytokine status and consequently alleviate progression of metabolic syndrome.

Although the antigen expression patterns of childhood acute lymphoblastic leukemia (ALL) are well known, little attention has been given to standardizing the diagnostic and classification criteria. We retrospectively analyzed the flow cytometric data from a large study of antigen expression in 1,774 children with newly diagnosed ALL in JPLSG. T- and B-lineage ALL accounted for 13 and 87% of childhood ALL cases, respectively. Cytoplasmic CD3 and CD7 antigens were positive in all T-ALL cases. More than 80% of T-ALL cases expressed CD2, CD5 and TdT. In B-lineage ALL, the frequencies of early pre-B, pre-B, transitional pre-B and B-ALL were 81, 15.5, 0.6 and 2.9%, respectively. More than 90% of early pre-B ALL cases expressed CD19, CD79a, CD22, CD10 and TdT. CD34 was expressed in three-fourths of early pre-B ALL cases. The frequencies of TdT and CD34 expression were lower in pre-B ALL than in early pre-B ALL. B-ALL showed less frequent expression of CD22, CD10, CD34 and TdT than other B-lineage ALL cases. Expression of CD13 and CD33, aberrant myeloid antigens, was significantly more frequently associated with B-lineage ALL than with T-ALL. Based on this retrospective study of antigen expression in 1,774 de novo childhood ALL cases in JPLSG, we propose standardized clinical guidelines for the immunophenotypic criteria for diagnosis and classification of pediatric ALL.

The Additional sex combs like 1 (Asxl1) gene is 1 of 3 mammalian homologs of the Additional sex combs (Asx) gene of Drosophila. Asx is unusual because it is required to maintain both activation and silencing of Hox genes in flies and mice. Asxl proteins are characterized by an amino terminal homology domain, by interaction domains for nuclear receptors, and by a C-terminal plant homeodomain protein-protein interaction domain. A recent study of patients with myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML) revealed a high incidence of truncation mutations that would delete the PHD domain of ASXL1. Here, we show that Asxl1 is expressed in all hematopoietic cell fractions analyzed. Asxl1 knockout mice exhibit defects in frequency of differentiation of lymphoid and myeloid progenitors, but not in multipotent progenitors. We do not detect effects on hematopoietic stem cells, or in peripheral blood. Notably, we do not detect severe myelodysplastic phenotypes or leukemia in this loss-of-function model. We conclude that Asxl1 is needed for normal hematopoiesis. The mild phenotypes observed may be because other Asxl genes have redundant function with Asxl1, or alternatively, MDS or oncogenic phenotypes may result from gain-of-function Asxl mutations caused by genomic amplification, gene fusion, or truncation of Asxl1.

We report a 5-year-old boy with juvenile myelomonocytic leukemia (JMML) which relapsed after an allogeneic bone marrow transplant who was successfully treated with interferon-alpha (IFN-alpha). One year after starting the therapy, he remains clinically well and in complete remission while continuing treatment with IFN-alpha and bestatin. Although the precise mechanism by which remission was induced is uncertain, a GVL effect combined with a direct antileukemia effect of IFN-alpha may be responsible. Further assessment of the role of IFN-alpha in relapsed JMLL patients is warranted.

High-risk neuroblastoma has a poor prognosis despite multimodal treatment including high-dose chemotherapy. A 7-year-old male with neuroblastoma received ex vivo-expanded donor CD4+ T lymphocyte infusion (CD4+ DLI) after recurrence in the bone marrow following allogeneic hematopoietic stem cell transplantation from his HLA-identical mother. The disease transiently responded to CD4+ DLI with reduction of tumor cells and a decrease of serum neuron-specific enolase. The response was associated with development of continued high fever and an increase of cytotoxic T lymphocytes in peripheral blood. This case suggests a possibility of a graft-versus-tumor effect against neuroblastoma. Pediatr Blood Cancer 2009;52:895–897. © 2009 Wiley-Liss, Inc.

An 8-year-old boy was referred due to difficulty in walking. T1-weighted MRI detected a well-marginated lesion expanding from the epidural region in the spinal canal to the paravertebral area through the Th9 and Th10 intervertebral foramen. The patient underwent a biopsy under video-assisted thoracoscopy and the tumor was diagnosed as Ewing's sarcoma family of tumor (ESFT). Imaging confirmed that the tumor completely disappeared and his neurologic functions were recovered perfectly at the end of treatment. Very few cases of skeletal ESFT of epidural extension in childhood have been documented. Video-assisted thoracoscopic surgery remains the best option for the diagnosis of endothoracic tumors in children.

Hox genes are clearly implicated in leukemia; however, neither the specificity of the leukemogenic potential among Hox genes of different paralog groups nor the role of the homeodomain is clear. We tested the leukemogenic potential of various NUP98-Hox fusion genes alone and with MEIS1. All genes tested had a significant overlapping effect in bone marrow cells in vitro. However, not all formed strong leukemogenic NUP98 fusion genes; but together with overexpression of MEIS1, all induced myeloid leukemia. This phenomenon was also seen with NUP98 fusions containing only the homeodomain of the corresponding Hox protein. We then exploited the strong transforming potential of NUP98-HOXD13 and NUP98-HOXA10 to establish preleukemic myeloid lines composed of early myeloid progenitors with extensive in vitro self-renewal capacity, short-term myeloid repopulating activity, and low propensity for spontaneous leukemic conversion. We also showed that MEIS1 can efficiently induce their conversion to leukemic stem cells, thus providing a novel model for the study of leukemic progression. In contrast to the leukemogenic effect of most of the Hox genes tested, HOXB4 has the ability to increase the self-renewal of hematopoietic stem cells without disrupting normal differentiation. On the basis of the discovery that the leukemogenic gene HOXA9 can also expand hematopoietic stem cells, we compared the ability of NUP98-Hox fusions to that of HOXB4 to trigger HSC expansion in vitro. Our preliminary results indicate that the expanding potential of HOXB4 is retained and even augmented by fusion to NUP98. Moreover, even greater expansion may be possible using Abd-B-like Hox fusions genes.

Background Obturator hernia is a relatively rare pelvic hernia and usually occurs in elderly, thin, multiparous women. Because symptoms are nonspecific, the diagnosis is often delayed until laparotomy is performed form to treat bowel obstruction. Methods Forty-three patients with obturator hernia undergoing surgery at Tokyo Metropolitan Geriatric Hospital were divided retrospectively into two groups (group A = 23 patients from 1968 to 1986 before computed axial tomography [CAT] was developed; group B = 20 patients from 1987 to 1999 after CAT). Preoperative diagnosis, operative procedures, and postoperative course were reviewed and compared between the 2 groups. Results Preoperative diagnostic accuracy was significantly higher in group B at 80.0% (16 of 20 patients) than in group A at 43.5% (10 of 23 patients) (P = 0.0146). Rate of gut resection and mortality were significantly lower in group B at 25.0% (4 of 20) and 5.0% (1 of 20) than in group A at 52.2% (12 and 23) and 30.4% (7 of 23) (P = 0.0295 and P = 0.0385, respectively). Conclusions The use of pelvic CAT in cases of suspected obturator hernia significantly enhances preoperative diagnostic accuracy and helps to decrease both intestinal resection rate and surgical mortality.

We report three pediatric patients with ganciclovir-resistant cytomegalovirus (CMV) retinitis who were successfully treated with foscarnet. The patients were recipients of hematopoietic stem cell transplantation (SCT) from HLA-mismatched donors. Because these patients had developed or experienced progressive CMV retinitis during ganciclovir therapy, they received foscarnet therapy at 60 mg/kg every 8 h. Their retinitis resolved promptly after initiating foscarnet therapy, suggesting foscarnet's effectiveness in treating ganciclovir-resistant CMV infection. The amount of CMV mRNA was quantitatively measured using an NASBA technique, which amplified the β2.7 transcripts specific for CMV replication. This technique was useful for monitoring disease activity in a more rapid and sensitive manner than the PCR assay for CMV DNA. Bone Marrow Transplantation (2001) 27, 1141–1145.

Human herpesvirus 6 (HHV-6) infection and disease are serious complications of allogeneic hematopoietic stem cell transplantation (allo-SCT). Ganciclovir (GCV) is effective against HHV-6 in vitro but the antiviral susceptibility of HHV-6 has not been well characterized in vivo. We retrospectively compared the HHV-6 reactivation rate in pediatric allo-SCT recipients with and without GCV prophylaxis. The HHV-6 reactivation rate at 3 weeks after allo-SCT in patients without prophylactic GCV administration was significantly higher than that in those receiving prophylactic GCV (11/28 vs 0/13, P < 0.01). Five of 36 patients without prophylactic GCV showed clinical manifestations including skin rash, interstitial pneumonitis, persistent thrombocytopenia, enterocolitis and thrombotic microangiopathy, respectively. HHV-6-associated symptoms were observed in one of the 13 patients receiving prophylactic GCV. This patient showed fever, diarrhea and graft rejection concomitantly with a sudden increase of HHV-6 DNA copy number. Patients who received GCV for treatment of HHV-6 infection showed an improvement in symptoms and/or decrease of HHV-6 copy number. Thus, GCV is effective for treating HHV-6 disease after allo-SCT in vivo.

Bone marrow failure is often associated with skeletal defects, as seen in Fanconi anemia [1], Diamond-Blackfan anemia [2], or thrombocytopenia-absent radius syndrome [3]. Congenital amegakaryocytic thrombocytopenia with radio-ulnar synostosis has been reported to be related with HOXA11 mutation [4]. We present a boy with congenital bone marrow failure with radio-ulnar synostosis, but without HOXA11 mutation. A boy, born to non-consanguineous parents, was delivered via Cesarean section at 31 weeks’ gestation for hydrops fetalis resulting from severe anemia. He had severe anemia (hemoglobin 2.7 g/dl), severe neutropenia (white blood cells 3,220/ll with 1.5% neutrophils), and moderate thrombocytopenia (platelets 89,000/ll) at birth. Neutrophils continued to be nearly absent and platelet counts progressively decreased below 20,000/ll by 2 weeks of age. He had no hepatosplenomegaly, lymphadenopathy, or cafe-au-lait spot, but had hydrocele testicle, sensorineural hearing loss, and overlapping fingers without abnormalities of bone. Supination of his bilateral forearms was restricted and X-rays revealed bilateral proximal radio-ulnar synostosis (Fig. 1). Bone marrow aspiration showed aplasia without dysplasia. Chromosomal analysis revealed a 46, XY, inv(9)(p12q13) karyotype, which is considered to be normal variant without physiologic role. He lacked a family history of hematological or skeletal disorders. No excessive chromosomal breakage was detected by mitomycin C stimulation, and no mutation was found for the Shwachman–Bodian–Diamond syndrome (SBDS) gene. He had repeated bacterial and viral infections after birth, requiring antibiotics and prophylactic antifungal agents. He also needed repeated transfusions of red blood cells and platelets until 8 months of age when he underwent a 5/6 human leukocyte antigen (HLA)-matched unrelated bone marrow transplantation (BMT). Pretransplant conditioning was with fludarabine, cyclophosphamide, and thoracicabdominal irradiation (2 Gy). Prophylaxis for graft-versushost disease (GVHD) was short-term methotrexate and tacrolimus. Engraftment was prompt and grade 1 acute cutaneous GVHD was observed. By 20? days following BMT, he became independent of all transfusions. He has had normal hematological studies for 14 months after BMT. Limb formation is regulated by HOX genes during embryonic development. The most 50 members of the HOXA and HOXD clusters (HOXA9–HOXA13 and HOXD9–HOXD13) are particularly important in limb development [5]. HOX genes are also well known to be associated with hematopoiesis and leukemogenesis [6]. The effects of Hoxa10 and Hoxd11 were found to be on forearm development from studies of mutant mice [7, 8]. Radio-ulnar synostosis with congenital thrombocytopenia has been reported to be associated with HOXA11 mutation [4], which prompted us to examine HOX genes. However, we detected no mutations of HOXA11 and HOXD11. Of the three patients with radio-ulnar synostosis and thrombocytopenia reported by Thompson et al. [9], one H. Yoshida Y. Hashii T. Okuda S. Kusuki E. Sato H. Ohta (&) K. Ozono Department of Pediatrics, Osaka University Graduate School of Medicine, Yamadaoka 2-2, Suita 565-0871, Japan e-mail: ohta@ped.med.osaka-u.ac.jp

To assess the involvement of vascular endothelial cell activation and damage in stem cell transplantation (SCT)-related complications, such as acute and chronic GVHD and thrombotic microangiopathy (TMA), we investigated the changes in serum levels of soluble forms of vascular cell adhesion molecule-1 (sVCAM-1) and E-selectin (sE-selectin) in SCT. The soluble form of intercellular adhesion molecule-1 (sICAM-1) was also analyzed. In patients with acute GVHD (grades II-IV), serum levels of sE-selectin and sICAM-1 increased around onset of GVHD (day 30). While the increase of sE-selectin levels was transient, sICAM-1 levels remained high until day 60. In patients with extensive chronic GVHD, sVCAM-1 as well as sE-selectin levels significantly increased. The appearance of clinical symptoms was preceded by elevations of sVCAM-1 and sE-selectin levels on day 60, and sICAM-1 levels on days 30 and 60. For the analysis of TMA, to exclude the influence of GVHD, serum levels were measured in auto-SCT patients. Around the onset of TMA, sVCAM-1 and sE-selectin levels significantly increased in patients with TMA without an increase of sICAM-1 levels. These findings support the notion that activation and injury of endothelium are commonly involved in the pathogenesis of acute and chronic GVHD and TMA.

Thrombotic microangiopathy (TMA) is a serious complication of BMT. Several factors are important in the etiology of TMA, such as cyclosporin A, GVHD, irradiation, intensive conditioning chemotherapy and infection, which cause damage to vascular endothelial cells leading to activation of these cells. We describe two young children with TMA following high-dose chemotherapy with autologous BMT. Development of TMA was accompanied by reactivation of HHV-6, which was identified by both an increase in the copy number of HHV-6 DNA in the peripheral blood and a significant increase in antibody titers to HHV-6. Thus, it was suggested that reactivation of HHV-6 together with high-dose chemotherapy played an important role in the pathogenesis of TMA in these patients. Since HHV-6 is known to infect vascular endothelial cells, and CMV which is virologically closely related to HHV-6, has been reported to be a pathogen that causes TMA, infection with HHV-6 of vascular endothelial cells may induce TMA via damage and activation of these cells.

The rae28/mph1 gene is the mouse homologue of the Drosophila polyhomeotic gene, which plays a crucial role in the maintenance of the transcriptional repression state of Hox genes. Expression of the rae28/mph1 gene is induced during retinoic acid (RA)-mediated differentiation of embryonal carcinoma F9 cells. By transient-transfection experiments, we identified a pair of inverted differentiation response sequences (DRS(s)) in the 5′ flanking region. Each of the DRS(s) contained the consensus sequence [5′- CCTCCCCXC XGCCC CCTCCXCXC -3′], which is also conserved in the human counterpart of the rae28/mph1 gene. Electrophoretic mobility shift assay and DNase I foot printing with nuclear extracts derived from F9 cells demonstrated the presence of novel DNA-binding factors which specifically interact with DRS(s). Nucleotide substitutions in the 3′ DRS abrogated the factor binding and the transcriptional activation, suggesting that DRS(s) and DRS-binding factors play an important role in the transcriptional regulation of the rae28/mph1 gene.

Morphophenotypic lineage switches occur in a small percentage of those with acute leukemia, and the underlying mechanisms are not clear. In this study, we attempted to induce a lineage switch in acute myelocytic leukemia (AML) with monosomy 7, whose lineage had switched from acute T-lymphocytic leukemia (T-ALL) during chemotherapy, in severe combined immunodeficient (SCID) mice. Although the transplanted myeloid cells were engrafted in SCID mice without cytokine administration, T-ALL developed in SCID mice treated with recombinant human granulocyte-macrophage colony-stimulating factor or recombinant human interleukin 3. Analysis of the nucleotide sequences of the rearranged T-cell receptor γ-chain (TCR-γ) gene revealed that this lineage switch resulted from the selection of the T-lineage subclone in SCID mice, which had expanded at onset. In addition, we found that the T-lineage and myeloid cells belonged to the distinct subclones, which were different in TCR-γ gene rearrangements, but were derived from a common clone with an identical N- ras gene mutation for both subclones. In in vitro cultures, only the myeloid subclone grew; the T-lineage subclone failed to grow even in the presence of recombinant human granulocyte-macrophage colony-stimulating factor or recombinant human interleukin 3. These results suggested that the initial diagnostic T-lymphoid subclone, whose growth was dependent on these cytokines and the hematopoietic microenvironment, emerged from a bipotential T-lymphoid/myeloid leukemic stem cell, and further genetic event(s) induced the myeloid subclone, which grew independently of these cytokines and the microenvironment.

Major dose-limiting factors of high-dose thiotepa (TEPA) and melphalan are life-threatening mucositis and neurotoxicity. To administer a maximum dose of these drugs safely and to obtain a maximum anti-cancer effect, a double-conditioning regimen with a single grafting, two cycles of administration of a combination of TEPA (300-600 mg/m2) plus melphalan (70-150 mg/m2) with a 1-week interval was attempted in 20 patients with pediatric advanced or chemotherapy-resistant solid tumors (seven rhabdomyosarcoma, four hepatoblastoma, three neuroblastoma and four other malignancy). Combinations of TEPA plus melphalan/busulfan (Bu) (8-10 mg/kg) and TEPA plus Bu were given to four and two patients with brain tumors, respectively. In an additional two patients, three cycles of drug administration were performed. According to the results of the dose-escalating study, the maximum tolerable doses of TEPA and melphalan for children aged 2 years old or older were 1000 mg/m2 and 280 mg/m2, respectively. Mucositis was dose-limiting. Renal toxicity was also dose-limiting in young children (

We examined five children who underwent allogeneic peripheral stem cell transplantation (PSCT) using positively selected CD34+ cells from three or two loci-mismatched donors. CD34+ cells mobilized from peripheral blood were separated by immunomagnetic beads. CD34+ cells at 2.2-6.2 x 10(6)/kg were transplanted into three patients with refractory leukemia, a patient with relapsed medulloblastoma and a patient with Fanconi's anemia following a conditioning regimen which included irradiation, alkylating agents and antithymocyte globulin treatment. The number of infused CD3+ cells included in grafts was 2.3-22.7 x 10(4)/kg. Four patients achieved engraftment and hematopoietic reconstitution (> 5 x 10(8)/l of neutrophils on day 10 or 11). Graft rejection was observed in the patient with Fanconi's anemia, but a rapid engraftment was obtained after second PSCT. Although no prophylactic agents other than ATG (included in the conditioning regimen) were used, greater than grade I acute GVHD was not observed, but limited chronic GVHD was observed in two patients. The two patients with leukemia relapsed on days 103 and 210, respectively, and the patient with medulloblastoma died of disease on day 159. The patient with Fanconi's anemia died of fungal infection. CMV and HHV-6 diseases developed in four and two patients, respectively. Thus, although SCT using positively selected peripheral CD34+ cells may be an alternative approach for overcoming graft rejection and GVHD from HLA- mismatched donors, persistent immune deficiency attributing to extremely low numbers of T cells in grafts can potentially lead to reactivation of herpes viruses.

9574 Background: Survival rate of ESFT in Japan was reportedly less than 50% in 1990s. The JESS started in order to improve this condition by conducting clinical trials for ESFT in Japan. Methods: This is a phase II, non-randomized trial to test safety and efficacy of multidisciplinary treatment for non-metastatic ESFT. Age under 30 is eligible. Chemotherapy regimens are alternating vincristine 1.5 mg/m2 on day 1, doxorubicin 37.5 mg/m2 on days 1 and 2, cyclophosphamide 1200 mg/m2 on day 1 (VDC) and ifosfamide 1800 mg/m2 on days 1 through 5 and etoposide 100 mg/m2 on days 1 through 5 (IE) repeating every 21 days for 52 weeks. Local treatment includes surgical resection and/or radiation therapy, of which dosage varies from 0 to 55.8 Gy based on margin of the resection and pathological response. Primary endpoint is 3 year progression free survival (PFS). Statistical design was made to test whether the lower limit of 90% confidence interval (CI) of PFS will exceed the threshold of 60%. Planned sample size is 53 patients including 10% ineligible patients. Results: Fifty-three patients were registered from December 2004 to May 2008. Among the 53 patients, 7 patients were judged ineligible after the registration (metastatic disease 1, changed diagnoses 6). Forty six patients were considered as per protocol set and used for efficacy analyses. Three year PFS is 71.7% (90% CI: 0.59 – 0.81). Estimated 5 year PFS and OS are both 69.6%. Radiographic response rate (RR) in evaluable 38 patients is 71.1% and pathological RR in 25 is 52.0%. In safety data, hematological toxicities are significant such as grade 4 neutropenia observed in 98%. Although there is no previously unknown adverse event reported, there are three patients who experienced secondary malignancies (ALL 1, MDS 1, osteosarcoma 1). Conclusions: A multi-disciplinary treatment comprising standard multi-agent chemotherapy with vincristine, doxorubicin, cyclophosphamide, ifosfamide and etoposide improved outcome of Ewing sarcoma family of tumor in Japan although statistical confirmation of absolute efficacy was not successful.

Abstract 4833 Diagnosis in accordance with the WHO classification provides important information for prognosis and risk stratification in the clinical trials of therapy for AML. However, each of the diagnostic parameter contained in the WHO classification, such as morphologic, cytochemical, immunophenotypic, cytogenetic, and genetic features, requires standardization and accuracy to ensure the validity of such clinical trials. In addition, discrepancies among the results of these parameters are encountered in some patients. Based on these considerations, we attempted to develop a comprehensive and rapid system for the central review of AML diagnosis in a nationwide clinical trial conducted by a multi-institutional cooperative study group. JPLSG AML-05 is a study that was conducted on children with de novo AML, excluding APL and myeloid leukemia associated with Down syndrome. Morphological diagnosis and flowcytometric immunophenotyping were performed through central laboratory systems. Cytogenetic analyses were carried out in regional laboratories, and the reports were reviewed centrally. Expression of chimeric genes including AML1-ETO, CBFB-MYH11, PML-RARA, MLL-AF9, MLL-AF6, MLL-ELL, FUS-ERG, and NUP98-HOXA9, and mutations of FLT3-ITD were also examined for all patients at a single laboratory. In case where cellular bone marrow aspirate smears could not be obtained, we recommended bone marrow biopsy, for which the histology was subjected to central pathologic review. When all the data had been submitted to our data center, a central review of the diagnosis based on the 2001 edition of the WHO classification was performed for each individual case by multidisciplinary conferencing based on electronic mail discussion. Between November 2006 and July 2010, the diagnoses of 383 patients enrolled in AML-05 were reviewed. The diagnosis was undetermined for seven patients because of inadequate submission. Nine patients did not meet the inclusion criteria for AML-05. Among the remaining 367 patients, 184 (48.8%) had AML with recurrent abnormalities, 32 (8.7%) had AML with multilineage dysplasia (AML-MLD), and 151 (41.1%) had AML, not otherwise categorized. Among the 184 patients with AML with recurrent abnormalities, 104 had AML with t(8;21)(q22;q22)/AML1-ETO. A higher prevalence of AML with t(8;21)(q22;q22)/AML1-ETO in our study, compared with those in previous reports from European countries, suggests a difference in genetic background. Two patients with t(8;21)(q22;q22)/AML1-ETO and one patient with inv(16)(p13q22) were assigned to each category through FISH analysis, because their cytogenetic analyses indicated a normal karyotype or suboptimal quality. Among 55 patients who were suspected of having AML with 11q23 (MLL) abnormalities by chromosome or chimeric gene screening tests, 30 had concordant results between chromosomes and chimeric genes. The remaining 25 patients were assigned to AML with 11q23 (MLL) abnormalities by either FISH for MLL or other chimeric gene tests involving MLL (MLL-AF10, MLL-ENL,MLL-AVI1 or MLL-TET1). The prevalence of AML-MLD in this study was higher than that (2.6%) in a previous report for Japanese children with AML, where a central review system of morphology had not been established (Adachi S., et al. Int J Hematol 86;358-63,2007). Patients with AML-MLD had a higher frequency of unfavorable cytogenetics (6/32) and/or FLT3-ITD mutations (7/32) in comparison with other types of AML. One patient with acute megakaryoblastic leukemia was diagnosed on the basis of bone marrow histology in our central pathologic review because the percentage of blasts on the smear did not meet the criteria for AML. We have successfully developed a rapid system for the integrated central review of AML diagnosis based on the WHO classification, which will enable us to evaluate trial data on the basis of standardized diagnosis and risk classification. Disclosures: No relevant conflicts of interest to declare.

Abstract 1161 Poster Board I-183 Introduction The number of cord blood transplantation (CBT) is rapidly increasing. In this setting, development of Epstein-Barr virus (EBV)-associated lymphoproliferative disorder (LPD) appears to be at high risk. To clarify this issue, we retrospectively analyzed EBV serology and clinical course of 57 patients who received CBT during the past 12 years. Patients and methods We underwent 79 CBT until 2008. Fifty-seven patients were available for assessing EBV serology. The mean age of patients was 63 months (range, 6m-28y5m). The underlying disease was hematologic malignancy (n=27), hematologic non-malignancy (n=10), autoimmune disease (n=2), solid tumor (n=7), and EBV-associated T/NK-LPD (n=11). Results Forty-six (80.7%) of the 57 recipients were EBV seropositive before CBT (sero+/). Nineteen (41.3%) of 46 EBV seropositive recipients before CBT became EBV seronegative after CBT (sero+/sero-). Five patients (8.8%) developed EBV-associated post-transplantation LPD. Three of the 5 LPD patients arose from 27 patients of sero+/sero+ group, and the time of onset of LPD was 2-6 months after CBT. Two of them died of LPD. The remaining 2 patients arose from 19 patients of sero+/sero- group, and the time of onset of LPD was 6-9 months. These 2 patients were successfully treated. None of the 11 patients (6 in sero-/sero- group and 5 in sero-/sero+ group) developed LPD. Discussion Among EBV seropositive patients, about 60% of them remained seropositive (endogenous infection group), and 40% of them became seronegative after CBT. The latter group is likely to experience exogenous infection (secondary primary infection). In both instances, incidence of LPD seems to be quite high. Therefore regular monitoring of EBV serology and EBV-DNA load after CBT should be essentially required for all CBT patients. Disclosures No relevant conflicts of interest to declare.

Expanding hematopoietic stem cells (HSCs) ex vivo remains a major challenge due to differentiation. Previous studies have shown that engineered overexpression of HOXB4 increases HSCs >40-fold in short term liquid culture. Most recently we have demonstrated that overexpression of Hox genes of different paralogs fused to the N-terminal region of the nucleoporin98 (NUP98) gene, a common fusion partner of Hox in AML, causes a strong block in differentiation as reflected by marked increases in CFU-S output and lineage negative cell expansion in vitro (Pineault et al, MCB, 2004). NUP98 fusions of Abd-B like HOX genes, HOXA10 and HOXD13 (NA10 and ND13), are more potent in these effects than those of Antennepedia-like-HOX genes, HOXB4 and HOXB3 (NB4 and NB3), prompting us to examine the HSC expanding potential of NUP98 fusions. Following in vitro culture of BM cells transduced with such fusions, we observed that the HSC expanding ability of HOXB4 can be augmented some 10-fold by fusion to NUP98 gene (i.e. NB4) perhaps due to the strong transactivation properties of the NUP98 fragment. Moreover we documented that NA10 has even more potent HSC expansion activity (>1,000-fold net HSC increase in 10 days) (Ohta et al, ISEH 2004 abstract # 24). To further examine NA10’s HSC expansion potency at a clonal level, multiple replicate cultures were initiated with limiting number of 5-FU treated BM cells estimated to contain ~1-2 CRU (5,000 cells per culture). After 2 days of pre-stimulation, individual wells were retrovirally transduced with NA10 for 2 days using an MSCV-based vector and expanded for a further 6 days. After a total 10-day culture, various fractions of individual wells (ranging from 1/2 to 1/250th of a well) were transplanted in limiting dilution assay for lympho-myeloid competitive repopulating cells (CRU). All recipients from individual GFP control wells (initiated with 25,000 cells) were not reconstituted. In marked contrast, all wells assayed for NA10, were positive for lympho-myeloid reconstituting cells at all dilutions tested. At the highest transplant doses (1/2 of a well), 100% of recipients from 4 wells tested were strongly positive for donor cells, averaging 71.5%, 9.0%, 29.0%, 16.4% for myeloid, B-lymphoid, T-lymphoid, RBC for GFP+ donor derived cells respectively. Most strikingly, transplantation of 1/250th of a well yielded 23.4% reconstitution of 5 positive mice (total of 2 wells assayed) and all recipients at this dilution were positive revealing more than a 250-fold increase of HSCs. In support of this, Southern blot analysis showed similar band patterns among different recipients transplanted with cells from the same wells consistent with clonal expansion from 1-2 starting HSC. We further tested the HSC expanding potential of NA10 using highly enriched c-kit+Sca-1+Lin− starting cells, demonstrating >7,000-fold expansion of short-term repopulating cells at 5 weeks post-transplant, and longer term follow-up is in progress. Taken together these results provide strong evidence of the potent ability of NA10 to induce the ex vivo expansion of HSCs at a clonal level. Although the NA10 induced expansion of HSC has not associated with leukemia with observations over 10 months, further development of protein-based delivery systems for NA10 such as TAT-fusion proteins (Krosl et al, Nat Med, 2003) are in progress as a possible novel stem cell expanding agent for safe therapeutic application.