Hiroshi Moritake

Newly established clear cell sarcoma (malignant melanoma of soft parts) cell line expressing melanoma-associated Melan-A antigen and overexpressing C-MYC oncogene

Clear cell sarcoma (CCS), malignant melanoma of soft parts, is a rare malignant tumor with a poor prognosis. In this study, a CCS cell line, designated MP-CCS-SY, was established from a metastatic tumor of a 17-year-old Japanese girl that originated in the left Achilles tendon. A small number of melanosomes were detected in the cytoplasm by electron microscopy. The melanosomes immunoreacted with two melanoma-associated antibodies, HMB45 and Melan-A. A Western blot demonstrated the existence of a Melan-A antigen in this cell line. Although a t(12;22)(q13;q12), which is characteristic of CCS, was not identified by a chromosomal analysis with conventional banding techniques, fluorescence in situ hybridization analysis with painting probes of chromosomes 12 and 22 revealed the insertion of a chromosome 12 fragment into one of the long arms of chromosome 22. The chimeric EWS/ATF1 transcript was detected by the reverse transcriptase polymerase chain reaction. Extra copies and structural abnormalities of chromosome 8 were observed. Overexpression of c-myc mRNA was detected by Northern blot analysis and may have a role in malignant progression of CCS. The availability of this MP-CCS-SY cell line will help to understand the molecular biology of this malignancy and should be useful as a tool for developing an immunotherapy.

Signal Transduction Pathways through TRK-A and TRK-B Receptors in Human Neuroblastoma Cells

Little is known about the signal transduction pathways of TRK family receptors in neuroblastoma (NB) cells. In this study, an NB cell line, designated MP‐N‐TS, was established from an adrenal tumor taken from a 2‐year‐old boy. This cell line expressed both TRK‐A and TRK‐B receptors, which is rare in a single NB cell line. Therefore, the MP‐N‐TS cell line was used to determine whether the signal transduction through these constitutive receptors is functional. Three neurotrophins, nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF) and neurotrophin‐4/ 5 (NT‐4/5), induced tyrosine phosphorylation of panTRK, and BDNF and NT‐4/5 induced tyrosine phosphorylation of TRK‐B. Tyrosine phosphorylation of panTRK and/or TRK‐B by the neurotro‐phins was inhibited in the presence of a tyrosine kinase inhibitor K252a. Tyrosine phosphorylation of Src homologous and collagen (She), extracellular signal‐regulated kinase (ERK)‐l and ERK‐2, and phospholipase C‐γl (PLC‐γl) was increased by the three neurotrophins and the increase was inhibited in the presence of K252a. Activation of Ras, detected as the GTP‐bound form of Ras, was induced by the three neurotrophins. The neurotrophins did not modulate the expressions of TRK‐A or TRK‐B mRNA, but they did induce the expression of c‐fos mRNA. Exogenous NGF induced weak neurite outgrowth, whereas exogenous BDNF and NT‐4/5 induced distinct neurite outgrowth. Exogenous BDNF and NT‐4/5 increased the number of viable cells, while NGF did not. Our results demonstrate that the signal transduction pathways through TRK‐A and TRK‐B in MP‐N‐TS cells are functional and similar, and the main downstream signaling pathways from the three neurotrophins are mitogen‐activated protein kinase (MAPK) cascades through She, activated Ras, ERK‐1 and ERK‐2, and the transduction pathway through PLC‐γl. Further, BDNF and NT‐4/5 increased cell viability. The MP‐N‐TS cell line should be useful for clarifying the TRK‐A and TRK‐B signaling pathways responsible for the different prognoses in patients with NB.

Excess treatment reduction including anthracyclines results in higher incidence of relapse in core binding factor acute myeloid leukemia in children

Excess treatment reduction including anthracyclines results in higher incidence of relapse in core binding factor acute myeloid leukemia in children

Clinical characteristics and outcome of refractory/relapsed myeloid leukemia in children with Down syndrome

Myeloid leukemia in Down syndrome (ML-DS) is associated with good response to chemotherapy and favorable prognosis. Because little research has been focused on refractory/relapsed (R/R) cases, we conducted a retrospective analysis for R/R ML-DS. Among ML-DS patients diagnosed between 2000 and 2010 in Japan, 26 relapsed (25 in the BM and 1 in the skin), and 3 refractory patients were enrolled. The male/female ratio was 18/11. The median age at initial diagnosis of ML-DS was 2 years, and the median time to relapse was 8.6 months. Each patient initially had been treated with ML-DS-specific protocols. Thirteen of the 26 patients achieved complete remission with various kinds of reinduction chemotherapies; 2 of 8 survived without further recurrence after receiving allogeneic hematopoietic stem cell transplantation, and 4 of 5 maintained complete remissions with chemotherapy alone. Treatment failures mostly were associated with disease progression rather than treatment-related toxicities. The 3-year OS rate was 25.9% ± 8.5%. A longer duration from initial diagnosis to relapse was a significant favorable prognostic factor (P < .0001). We conclude that clinical outcome for patients with R/R ML-DS generally are unfavorable, even in those receiving hematopoietic stem cell transplantation. Novel methods to identify poor prognostic factors for ML-DS are necessary.

Paroxysmal nocturnal hemoglobinuria and telomere length predicts response to immunosuppressive therapy in pediatric aplastic anemia

Acquired aplastic anemia is an immune-mediated disease characterized by severe defects in stem cell number resulting in hypocellular marrow and peripheral blood cytopenias. Minor paroxysmal nocturnal hemoglobinuria populations and a short telomere length were identified as predictive biomarkers of immunosuppressive therapy responsiveness in aplastic anemia. We enrolled 113 aplastic anemia patients (63 boys and 50 girls) in this study to evaluate their response to immunosuppressive therapy. The paroxysmal nocturnal hemoglobinuria populations and telomere length were detected by flow cytometry. Forty-seven patients (42%) carried a minor paroxysmal nocturnal hemoglobinuria population. The median telomere length of aplastic anemia patients was −0.99 standard deviation (SD) (range −4.01–+3.01 SD). Overall, 60 patients (53%) responded to immunosuppressive therapy after six months. Multivariate logistic regression analysis identified the absence of a paroxysmal nocturnal hemoglobinuria population and a shorter telomere length as independent unfavorable predictors of immunosuppressive therapy response at six months. The cohort was stratified into a group of poor prognosis (paroxysmal nocturnal hemoglobinuria negative and shorter telomere length; 37 patients) and good prognosis (paroxysmal nocturnal hemoglobinuria positive and/or longer telomere length; 76 patients), respectively. The response rates of the poor prognosis and good prognosis groups at six months were 19% and 70%, respectively (P<0.001). The combined absence of a minor paroxysmal nocturnal hemoglobinuria population and a short telomere length is an efficient predictor of poor immunosuppressive therapy response, which should be considered while deciding treatment options: immunosuppressive therapy or first-line hematopoietic stem cell transplantation. The trial was registered in www.umin.ac.jp with number UMIN000017972.

C-MYC rearrangement may induce an aggressive phenotype in anaplastic lymphoma kinase positive anaplastic large cell lymphoma: Identification of a novel fusion gene ALO17/C-MYC.

Anaplastic lymphoma kinase (ALK) positive anaplastic large cell lymphoma (ALCL) is usually associated with a favorable prognosis. We describe an 11-year-old girl patient with ALK positive ALCL bearing t(2;5)(p23;q35) and t(8;17)(q24;q25) translocations who had an aggressive clinical course despite various combinations of intensive chemotherapy. Southern blot analysis identified C-MYC rearrangement. Immunohistochemistry and Northern and Western blot analyses revealed cmyc overexpression. A new fusion between ALO17 (ALK lymphoma oligomerization partner on chromosome 17) and C-MYC was identified by the 50-rapid amplification of cDNA ends. This new fusion may have possibly provoked the poor prognosis in this patient with ALK positive ALCL, and C-MYC rearrangement may indicate poor prognosis in ALCL.

Cytomegalovirus retinitis as an adverse immunological effect of pulses of vincristine and dexamethasone in maintenance therapy for childhood acute lymphoblastic leukemia

We describe a 5‐year‐old female with acute lymphoblastic leukemia (ALL) who suffered from cytomegalovirus (CMV) retinitis during maintenance therapy consisting of 6‐mercaptopurine (6‐MP) and methotrexate (MTX) with pulses of vincristine (VCR) and dexamethasone (DEX). Administration of anticytomegaloviral drugs led to a complete regression of active retinitis. Her low CD4 positive T cells and serum immunoglobulin G (IgG) recovered when maintenance therapy was resumed without VCR and DEX. The patient has been in complete remission (CR) for more than 5 months after completion of maintenance therapy without recurrence of CMV retinitis. Pediatr Blood Cancer 2013;60:329–331.

Peripheral blood lymphocyte telomere length as a predictor of response to immunosuppressive therapy in childhood aplastic anemia

Predicting the response to immunosuppressive therapy could provide useful information to help the clinician define treatment strategies for patients with aplastic anemia. In our current study, we evaluated the relationship between telomere length of lymphocytes at diagnosis and the response to immunosuppressive therapy in 64 children with aplastic anemia, using flow fluorescence in situ hybridization. Median age of patients was ten years (range 1.5–16.2 years). Severity of the disease was classified as very severe in 23, severe in 21, and moderate in 20 patients. All patients were enrolled in multicenter studies using antithymocyte globulin and cyclosporine. The response rate to immunosuppressive therapy at six months was 52% (33 of 64). The probability of 5-year failure-free survival and overall survival were 56% (95% confidence interval (CI): 41–69%) and 97% (95%CI: 87–99%), respectively. Median telomere length in responders was −0.4 standard deviation (SD) (−2.7 to +3.0 SD) and −1.5 SD (−4.0 to +1.6 (SD)) in non-responders (P<0.001). Multivariate analysis showed that telomere length shorter than −1.0 SD (hazard ratio (HR): 22.0; 95%CI: 4.19–115; P<0.001), platelet count at diagnosis less than 25×109/L (HR: 13.9; 95%CI: 2.00–96.1; P=0.008), and interval from diagnosis to immunosuppressive therapy longer than 25 days (HR: 4.81; 95%CI: 1.15–20.1; P=0.031) were the significant variables for poor response to immunosuppressive therapy. Conversely to what has been found in adult patients, measurement of the telomere length of lymphocytes at diagnosis is a promising assay in predicting the response to immunosuppressive therapy in children with aplastic anemia.

Analysis of PTEN/MMAC1 alteration in neuroblastoma

Neuroblastoma is the most common extracranial solid tumor in children. Although it has been reported that loss of heterozygosity at various loci, including 10q, frequently occurs in neuroblastoma, a bona fide tumor suppressor gene has not been identified. Recently, a gene mapped to chromosome 10q23, PTEN/MMAC1, was identified as a tumor suppressor gene that inhibits cell survival and cell proliferation by catalyzing the dephosphorylation of phosphatidylinositol 3,4,5-triphosphate. To screen for mutations of this gene in neuroblastoma, we analyzed 11 primary neuroblastoma tumors and 16 neuroblastoma cell lines for PTEN/MMAC1 mutations and deletions. All nine exons of the PTEN/MMAC1 gene were examined using the polymerase chain reaction-single strand conformational polymorphism assay and sequencing. Only one of the cell lines showed a mutation, a 1-bp frameshift deletion in exon 7, and an allelic loss in the opposite allele was revealed by a microsatellite analysis. Our results indicate that the disruption of the PTEN/MMAC1 gene is not a frequent event in neuroblastoma, and suggest that this disruption may be responsible for malignant progression in only a limited proportion of cases of neuroblastoma.

Cytomegalovirus infection mimicking juvenile myelomonocytic leukemia showing hypersensitivity to granulocyte–macrophage colony stimulating factor

We describe an infant with cytomegalovirus (CMV) infection presenting as transient myeloproliferation resembling juvenile myelomonocytic leukemia (JMML). The patient fulfilled the international diagnostic criteria of JMML, including hypersensitivity to granulocyte–macrophage colony‐stimulating factor (GM‐CSF). Viral studies using serologic assays and polymerase chain reaction (PCR) were positive for CMV. Clinical symptoms disappeared and laboratory values returned to normal without specific treatment within 1 year. Follow‐up showing a decrease in viral titers suggested CMV infection as an etiologic factor for the development of myeloproliferative features. We conclude that the CMV infection transiently induced abnormal myelopoiesis in this infant. Pediatr Blood Cancer 2009; 53:1324–1326.