Shinya Osone

Fenretinide induces sustained‐activation of JNK/p38 MAPK and apoptosis in a reactive oxygen species‐dependent manner in neuroblastoma cells

Fenretinide, which mediates apoptosis in neuroblastoma cells, is being considered as a novel therapeutic for neuroblastoma. The cytotoxic mechanisms of fenretinide, however, have not been fully elucidated. Sustained‐activation of JNK and p38 MAPK signaling has been shown recently to have a pivotal role in stress‐induced apoptosis. Whether fenretinide activates the signaling in neuroblastoma cells is not known. In the present study, fenretinide induced sustained‐activation of both JNK and p38 MAPK in neuroblastoma cells. Pretreatment with the antioxidant L‐ascorbic acid almost completely inhibited the accumulation of fenretinide‐induced intracellular reactive oxygen species (ROS), activation of JNK and p38 MAPK and apoptosis. Intracellular ROS production and activation of stress signaling was not altered by fenretinide in resistant neuroblastoma cells. Our study demonstrates that in neuroblastoma cells, fenretinide induces sustained‐activation of JNK and p38 MAPK in an ROS‐dependent manner and indicates that JNK and p38 MAPK signaling might mediate fenretinide‐induced apoptosis. Our results also indicate that suppression of the fenretinide‐induced ROS productive system and the downstream JNK and p38 MAPK signaling pathways causes neuroblastoma cells to become resistant to fenretinide.

Prediction of MYCN Amplification in Neuroblastoma Using Serum DNA and Real-Time Quantitative Polymerase Chain Reaction

PURPOSE MYCN amplification (MNA) indicates a poor prognosis in neuroblastoma (NB) and is routinely assayed for therapy stratification. We aimed to develop a diagnostic tool to predict MYCN status using serum DNA, which, in cancer patients, predominantly originates from tumor-released DNA. PATIENTS AND METHODS Using DNA-based real-time quantitative polymerase chain reaction, we simultaneously quantified MYCN (2p24) and a reference gene, NAGK (2p12), and evaluated MYCN copy number as an MYCN/NAGK (M/N) ratio in 87 NB patients whose MYCN status had been determined by Southern blotting. Of these patients, 17 had MYCN-amplified NB, and 70 had nonamplified NB. RESULTS The serum M/N ratio in the MNA group (median, 199.32; range, 17.1 to 901.6; 99% CI, 107.0 to 528.7) was significantly (P < .001) higher than the ratio in the non-MNA group (median, 0.87; range, 0.25 to 4.6; 99% CI, 0.82 to 1.26; Mann-Whitney U test). The sensitivity and specificity of the serum M/N ratio as a diagnostic test were both 100% when the serum M/N ratio cutoff was set at 10.0. Among six MNA patients whose clinical courses were followed, the serum ratios decreased to the normal range in the patients in remission (n = 3), whereas the ratios increased to high levels in the patients who relapsed (n = 2) or failed to achieve remission (n = 1). CONCLUSION Measurement of the serum M/N ratio seems to be a promising method for accurately assessing MYCN status in NB, although a larger set of patients needs to be examined to confirm this result.

Potential use of procalcitonin concentrations as a diagnostic marker of the PFAPA syndrome

PFAPA syndrome is a clinical entity of unknown etiology characterized by periodic episodes of high fever accompanied by aphthous stomatitis, pharyngitis/tonsillitis, and cervical adenitis [3, 5]. Since specific laboratory abnormalities for the PFAPA syndrome are inexistent, it is usually diagnosed clinically after excluding other probable causes of the fever, such as infection [1]. In PFAPA patients, discriminating between a fever attack due to bacterial infection and a fever attack due to noninfectious inflammation constitutes a major difficulty. Because procalcitonin, a propeptide of calcitonin, is reported to be a sensitive marker of systemic bacterial infection [2, 4], we followed peripheral leukocyte counts, CRP values and procalcitonin concentrations during the fever attacks associated with PFAPA syndrome in the hope of defining reliable criteria for its diagnosis. We determined serum procalcitonin concentrations in six PFAPA syndrome patients (two males and four females) with a median age of 7.5 (range 3–10) years and in 32 controls (bacterial, n=10 and non-bacterial, n=22). Sampling was performed on the third to fifth day of fever. In the PFAPA syndrome patients, febrile episodes started at the median age of 2.5 (range 1–7) years with each episode lasting 5–7 days and recurring every 3–4 weeks. The ethical committees of our institutes approved the study protocol and the guardians of all the patients gave their informed consent. Serum procalcitonin concentrations were measured by using the fully automated enzyme immunoluminescent assay (Wako Pure Chemical Industries, Ltd.), which employs katacalcin monoclonal antibody and calcitonin polyclonal antibody labeled with peroxidase for SphereLight 180 (Olympus Corporation). The detection limit was 0.1 ng/ml and the normal reference was set at <0.5 ng/ml. In PFAPA patients, the correlations between procalcitonin, CRP values and leukocyte counts were examined over 13 febrile episodes. Serum procalcitonin values ranged from 0.20 to 11.36 (median value 1.05) ng/ml in positive control subjects (Table 1), while all the negative controls had undetectable levels. During febrile episodes in PFAPA patients, which were confirmed not to be due to adenoviral or group A streptococcal infections, leukocyte counts and serum concentrations of CRP were invariably and significantly Eur J Pediatr (2007) 166:621–622 DOI 10.1007/s00431-006-0281-2

Antitumor Activity of Gefitinib in Malignant Rhabdoid Tumor Cells In vitro and In vivo

Purpose: Malignant rhabdoid tumor (MRT) is a rare and highly aggressive neoplasm of young children. Current treatments have had only limited success. Epidermal growth factor receptor (EGFR) was found recently to be expressed on MRT cell lines. Gefitinib (trade name Iressa) is an oral and selective EGFR-tyrosine kinase inhibitor and has been demonstrated to be effective in inhibiting the proliferation of cancer cells in vivo as well as in clinical trials. This encouraged us to examine the antitumor effects of gefitinib on MRT cells in vitro and in vivo. Experimental Design: The expression of EGFR in two MRT tumors and two MRT cell lines (MP-MRT-AN and KP-MRT-NS), established from these two tumor tissues, was examined by immunohistochemistry, immunofluorescence, and immunoblot. The effect of gefitinib on EGFR phosphorylation was examined by immunoblot. The effects of gefitinib on cell growth and apoptosis were examined by cell growth assay and terminal deoxynucleotidyl transferase-mediated nick end labeling assay. The in vivo effect of gefitinib was assessed in athymic mice that had been xenografted with MRT cells. Results: The expression of EGFR was detected in both tumor tissues and cell lines. Gefitinib inhibited EGFR-phosphorylation (IC50 < 0.1 μmol/L) and in vitro cell growth (IC50 = approximately 10–12 μmol/L), and a high concentration of gefitinib (20 μmol/L) induced apoptosis in vitro (MP-MRT-AN, 42.9% and KP-MRT-NS, 47.2%). Furthermore, gefitinib at 150 mg/kg had a cytostatic effect on established MRT xenografts (MP-MRT-AN, P = 0.039 and 0.0014; and KP-MRT-NS, P = 0.048 and 0.0086). Conclusions: Our results demonstrate that gefitinib has antitumor effects in MRT cells in vitro and in vivo and, thus, has promise as a novel and therapeutic strategy for MRT.

Successful treatment of idiopathic colitis related to XIAP deficiency with allo-HSCT using reduced-intensity conditioning

Recently, it has been reported that Crohns‐like intractable colitis occurred in approximately 20% of the patients with XIAP deficiency, also known as X‐linked lymphoproliferative disease type 2. Because treatment used for Crohns disease is not always effective for Crohns‐like colitis related to XIAP deficiency, more effective treatment should be established. Although several studies reported allo‐HSCT might be promising even for Crohns‐like colitis related to XIAP deficiency, the outcome of allo‐HSCT using MAC for XIAP deficiency is extremely poor due to frequent TRM. In addition, there is little information about the outcome of allo‐HSCT for intractable colitis related to XIAP deficiency. Herein, we describe a patient with intractable colitis related to XIAP deficiency who was successfully treated with allo‐HSCT using a reduced‐intensity conditioning regimen. Although allo‐HSCT using the RIC regimen might be a curative therapeutic option for intractable colitis with XIAP deficiency, the prognostic factors that will determine the success of allo‐HSCT require further clinical information of more patients.

Allo-SCT in a patient with CRMCC with aplastic anemia using a reduced intensity conditioning regimen

Allo-SCT in a patient with CRMCC with aplastic anemia using a reduced intensity conditioning regimen

ISOLATED RELAPSE OF ACUTE LYMPHOBLASTIC LEUKEMIA IN THE BREAST OF A YOUNG FEMALE

A 20-year-old female developed a relapse of B-precursor acute lymphoblastic leukemia (ALL) as a mass in her left breast after 6 years of maintained continuous complete remission. No leukemic lesions were identified in other sites such as the bone marrow or cerebrospinal fluid. The relapsed leukemic cells in the breast revealed the same immunophenotypes (CD10+, CD19+, CD20+, HLA-DR+, CD34+) as those of the onset ALL cells in the bone marrow. A literature survey found 10 other cases of ALL relapse in the breast without bone marrow involvement, mostly consisting of adolescent girls. Including the present report, a total of 11 cases were analyzed; the onset ages of ALL were a median of 16.5 (range 5–50) years old and the ages of relapse in the breast a median of 20 (range 12–51) years old. Data suggest that, although rare, the breast could become one of the extramedullary relapse sites of ALL developed in adolescent girls.

Hematopoietic stem cell transplantation- associated thrombotic microangiopathy accompanied by renal arteriolar C4d deposition

HSCT‐associated thrombotic microangiopathy (TA‐TMA) is a severe complication with a poor prognosis. Recently, it has been reported that complement system dysregulation, such as CFH autoantibodies and deletions CFH‐related genes 3 and 1, induced TA‐TMA. In addition, C4d‐positive renal arterioles are both a good marker of complement system activation and a useful diagnostic tool for TA‐TMA. Because dysregulation of the complement system is associated with TA‐TMA, the complement system might be a therapeutic target, such as eculizumab, a terminal complement inhibitor. Herein, we describe an eight‐yr‐old boy who developed TA‐TMA accompanied by severe renal dysfunction. His renal specimen showed diffuse C4d deposition in the renal arterioles, which is consistent with TA‐TMA. Although the patient gradually improved without eculizumab, renal arteriolar C4d staining in sample with TA‐TMA shows the complement system activation and may guide the target therapy using the eculizumab.

BCL2 Inhibitor (ABT-737): A Restorer of Prednisolone Sensitivity in Early T-Cell Precursor-Acute Lymphoblastic Leukemia with High MEF2C Expression?

Early T-cell precursor-acute lymphoblastic leukemia (ETP-ALL) has been identified as a high-risk subtype of pediatric T-cell acute lymphoblastic leukemia (T-ALL). Conventional chemotherapy is not fully effective for this subtype of leukemia; therefore, potential therapeutic targets need to be explored. Analysis of the gene expression patterns of the transcription factors in pediatric T-ALL revealed that MEF2C and FLT3 were expressed at higher levels in ETP-ALL than typical T-ALL. Using human T-ALL and BaF3 cell lines with high expression levels of MEF2C, the present study tested whether the BCL2 inhibitor (ABT-737) restores the sensitivity to prednisolone (PSL), because MEF2C causes PSL resistance, possibly by augmenting the anti-apoptotic activity of BCL2. Treatment with PSL and ABT-737 caused a significant reduction in the IC50 of PSL in the MEF2C-expressing LOUCY cells, in addition to the MEF2C-transduced BaF3 cells, but not in the non-MEF2C-expressing Jurkat cells. The combination treatment significantly accelerated the killing of primary leukemic blast cells of ETP-ALL with high expression levels of MEF2C, which were co-cultured with murine stromal cells. These findings suggest that BCL2 inhibitors may be a therapeutic candidate in vivo for patients with ETP-ALL with high expression levels of MEF2C.

Case Reports of Severe Congenital Neutropenia Treated With Unrelated Cord Blood Transplantation With Reduced-intensity Conditioning.

Allogeneic stem cell transplantation is a curative treatment for severe congenital neutropenia (SCN). However, a standard conditioning regimen and donor source have not been established. We report 3 consecutive cases of SCN who were successfully treated by cord blood transplantation (CBT) with reduced-intensity conditioning consisting of fludarabine, melphalan, and low-dose total body irradiation. All cases achieved complete donor chimerism without severe infectious complications and have maintained normal neutrophil counts for between 3 and 9 years after CBT. These results suggest that CBT with reduced-intensity conditioning can be an alternative therapy for SCN when human leukocyte antigen-matched bone marrow donor is unavailable.