Munenori Miyake

Chromosome abnormalities and MLL rearrangements in acute myeloid leukemia of infants.

Of 29 infants with acute myeloid leukemia (AML), 14 (48%) had various 11q23 translocations. MLL rearrangements were examined in 21 of the 29 patients, and 11 (52%) showed the rearrangements. 11q23 translocations and/or MLL rearrangements were found in 17 (58%) of the 29 patients. While all but one of the 17 patients with 11q23/MLLrearrangements had M4 or M5 type of the FAB classification, the 12 patients without such rearrangements had various FAB types, including M2, M4, M4EO, M6 and M7. Of the 12 patients with other chromosome abnormalities or normal karyotypes, two had inv(16) or t(16;16), one had t(1;22)(p13;q13), and two had a novel translocation, t(7;12)(q32;p13). The breakpoint on 12p of the t(7;12) was assigned to intron 1 or the region just upstream of exon 1 of the TEL/ETV6 gene by fluorescence in situ hybridization. The event-free survival at 5 years for the 17 patients with 11q23/MLL rearrangements was 42.2%, and that for the 12 patients without such rearrangements was 31.3% (P = 0.5544). 11q23/MLLrearrangements have been frequently reported and a poor prognosis in infant acute lymphoblastic leukemia implied. Our study showed that while 11q23/MLL rearrangements were also common in infant AML, AML infants with such rearrangements had a clinical outcome similar to that of AML infants without such rearrangements.

Clonal and non-clonal karyotypically abnormal cells in haemophagocytic lymphohistiocytosis

We studied chromosomes in bone marrow (BM) or peripheral blood cells of nine patients with haemophagocytic lymphohistiocytosis (HLH); three of them had a family history of HLH and four others underwent concurrent Epstein‐Barr virus (EBV) infection. In addition to a large population of normal mitotic cells, karyotypically abnormal clonal cells were found in two patients, abnormal clonal cells and a nonclonal (single) abnormal cell in one, and nonclonal abnormal cells in three. All the six patients with chromosome abnormalities died of progressive disease; one of them also had EBV infection and EBV‐associated clonal proliferation. Two of three patients with EBV infection and only normal mitotic cells in BM completely recovered from the disease.

Transcriptional profiling in hepatoblastomas using high-density oligonucleotide DNA array.

Hepatoblastoma is a common hepatic tumor in children. Although evidence regarding cytogenetic and molecular genetic alterations in hepatoblastomas has been reported, the molecular events affecting the biologic characteristics of this tumor, including alterations of the gene expression profile, are largely unknown. To identify genes differentially expressed between nondiseased liver (NDL) and hepatoblastoma tumor (HBT), we analyzed the gene expression profile in 14 NDL and 16 HBT samples using a high-density oligonucleotide DNA array. Using Mann-Whitney U test followed by the k-nearest neighbor algorithm, we identified 26 genes (predictor genes) that were able to assign unknown samples derived from NDL and HBT to either the NDL group or HBT group with 100% accuracy. Using a cross-validation approach, we confirmed that the k-nearest neighbor algorithm assigned the particular samples derived from NDL and HBT to either the NDL or HBT group with 93.3% (28/30 samples) accuracy. In the 26 predictor genes, we found alteration of the expression of genes regulating cell division (NAP1L1, STMN1, CCNG2, and CDC7L1) and tumor cell growth (IGF2 and IGFBP4) in HBT. Four predictor genes (ETV3, TPR, CD34, and NR1I3) were also found to be mapped to the chromosomal region 1q21 approximately q32, which has been reported to be frequently involved in the development of hepatoblastoma. The findings obtained in this study suggest that alteration of the expression of some genes regulating cell division and tumor cell growth may be characteristics of the gene expression profile in HBT, and that alteration of the expression of the four predictor genes mapped to chromosomal region 1q21 approximately q32 may also contribute to the differences in gene expression profile between NDL and HBT.

High levels of Mn-superoxide dismutase in serum of patients with neuroblastoma and in human neuroblastoma cell lines

Levels of serum manganese superoxide dismutase (Mn-SOD) in normal children aged from 1 to 14 years and children with various hematological and malignant diseases were determined by enzyme-linked immunosorbent assay (ELISA). In the normal children, the serum Mn-SOD levels gradually increased in proportion to age. By 8 years of age, the Mn-SOD level was nearly at the adult level. The normal values of serum Mn-SOD (mean +/- SD) of children below 4 and above 8 years old were 48 +/- 10.2 ng/ml and 84 +/- 22.5 ng/ml, respectively. Assuming the upper limit of normal Mn-SOD level in serum to be the mean value +/- 2 SD of children at each age, high serum levels of Mn-SOD were found for 8 of 12 patients with neuroblastoma, three of four patients with Wilms tumor, and four of five patients with acute myeloid leukemia. The patients with neuroblastoma exhibited a transient increase in Mn-SOD following chemotherapy, but after 1 week the levels decreased markedly to the control levels. The changes in serum Mn-SOD levels in the patients with neuroblastoma correlated well with the levels of neuron-specific enolase. Mn-SOD was intensely stained in bone marrow cells of patients whose cancer cells had moved into the bone marrow. High levels of Mn-SOD were also found in cultured human neuroblastoma cells. These data indicate that Mn-SOD is expressed in neuroblastoma cells, may serve as one of the diagnostic and prognostic markers for the neuroblastoma, and may be useful to predict the effectiveness of chemotherapy for neuroblastoma and the recurrence of this disease.

VITAMIN E AND THE PEROXIDIZABILITY OF ERYTHROCYTE MEMBRANES IN NEONATES

We showed the increased susceptibility of neonatal biomembranes to oxidation by a kinetic analysis using an azo compound as a free-radical initiator and red blood cell (RBC) ghosts as a model membrane. When the RBC ghosts were oxidized, oxygen consumption was suppressed during the induction period in which membrane tocopherol was consumed at a constant rate, while increased oxygen uptake was observed after the tocopherol was exhausted. The total tocopherol content was similar in cord, maternal, and adult RBC ghosts, and there were no differences in the induction period (tinh) among the three types of ghosts. While the oxygen uptake rate during the induction period (Rinh) was similar in cord and adult ghosts, the rate in the subsequent phase (Rp) was considerably faster in the cord ghosts. Fatty acid analysis in the membrane lipids showed that the active bisallylic hydrogen (active H) content was greater in cord ghosts than in adult ghosts. The active H content closely correlated with the Rp, but did not with the Rinh. The kinetic chain length (KCL), i.e., the ratio of the rate of propagation to that of initiation, was calculated from Rp and tocopherol consumption rate and KCL values were higher in cord ghosts than in adult ghosts. The faster Rp and the higher KCL of the cord ghosts were attributable to a greater active H content rather than to the tocopherol content.

Correlation of chromosome abnormalities with presence or absence of WT1 deletions/mutations in Wilms tumor.

Of 40 Wilms tumors with chromosome abnormalities, 6 were hypodiploid, 10 were pseudodiploid, 7 were hyperdiploid with 47 to 49 chromosomes, and 17 were hyperdiploid with 50 or more chromosomes, mostly including +12. WT1 deletions/mutations were found in one hypodiploid, eight pseudodiploid, and one hyperdiploid (47–49 chromosomes) tumor, but in none of the hyperdiploid (≥50 chromosomes) tumors. Of the 10 tumors with WT1 abnormalities, 6 had a homozygous WT1 deletion, 1 had a nonsense WT1 mutation and loss of heterozygosity at 11p, 1 had an intragenic hemizygous WT1 deletion without detectable WT1 mutation, and 2, which occurred in Wilms tumor–aniridia–genitourinary abnormalities–mental retardation syndrome patients, had a hemizygous deletion and a missense or frameshift mutation of WT1. Six of the nine tumors with homozygous or hemizygous WT1 deletions had chromosome aberrations involving chromosome band 11p13 in one of the two chromosomes 11. While one hypodiploid and one pseudodiploid patient died of the disease, and one hyperdiploid (47–49 chromosomes) patient was alive in nonremission, all hyperdiploid (≥50 chromosomes) patients had no evidence of disease at the last follow‐up. Our data show that chromosome aberrations are closely correlated to WT1 abnormalities and suggest that hyperdiploid (≥50 chromosomes) Wilms tumors may be characterized by the absence of WT1 abnormalities and possibly also by a favorable prognosis. Genes Chromosomes Cancer 25:26–32, 1999.

Pharmacokinetics of All-trans Retinoic Acid in Pediatric Patients with Leukemia

Since all‐trans retinoic acid (ATRA) induces complete remission in a high proportion of patients with acute promyelocytic leukemia (APL), and its effectiveness appears to be related to the plasma or serum level, a pharmacokinetic study of ATRA was undertaken in nine patients with various leukemias. After oral administration at a dose of 30 mg/m2, the time required to reach the peak plasma level of ATRA (20–1198 ng/ml) was between 120 and 240 min and the apparent plasma elimination half life was 21–51 min. In addition, 13‐cis retinoic acid was detected in the plasma of seven patients, indicating the occurrence of ATRA isomerization in vivo. ATRA therapy did not induce complete remission in all patients, even when high plasma levels were achieved. Among the six APL patients given ATRA therapy, one who failed to respond had a very low plasma ATRA level. These findings suggest that it may be useful to monitor plasma levels during oral ATRA therapy in order to achieve an appropriate treatment regimen.

Prognostic Impact of CD45 Antigen Expression in High-Risk, Childhood B-Cell Precursor Acute Lymphoblastic Leukemia: Children's Cancer and Leukemia Study Group (CCLSG)

To evaluate the clinical implications of CD45 expression in acute childhood lymphoblastic leukemia (ALL), we measured the CD45 expression of blast cells from 133 untreated patients with childhood B-precursor ALL (n=118) or T-ALL (n=15). CD45 expression (≥20%) was detected in all 15 cases (100%) of T-ALL, and 101 cases (86%) of B-precursor ALL. In 122 cases, the fluorescence intensity of the CD45 expression was measured as a relative value; the ratio of average linear values (RALV) of CD45 on the blasts to that on CD3-positive T-lymphocytes from the same specimen. The expression was more intense in the T-ALL cases than in the B-precursor ALL cases (RALV, mean ± SE: T-ALL 0.230 ± 0.04 vs. pro-B ALL 0.150 ± 0.012/pre-B ALL 0.153 ± 0.019, p < 0.05). However, the intensity of the CD10, CD19, CD20 and CD34 antigen immunoreactivity did not correlate with the CD45 expression. Patients with hyperdiploidy (chromosome number >50) showed significantly lower levels of CD45 expression than patients with t(1;19) or normal karyotypes (RALV, mean ± SE: 0.081 ± 0.022 vs. 0.133 ± 0.03/0.143 ± 0.019, p < 0.05). Other clinical features such as age, gender and WBC count did not correlate with CD45 expression. The prognostic implications of CD45 expression were studied in non-high-risk (low-risk + intermediate-risk) (n=60) and high-risk patients (n=52) with B-precursor ALL who had been treated with the risk-directed protocol of ALL-941 trial. Although CD45 expression did not correlate with the event-free survival (EFS) of the non-high-risk patients, there was a significant correlation between the expression levels and the EFS of the high-risk patients: the 3-year EFS rate of the CD45lowgroup (n=26, RALV=0.017 - 0.132) was 88 ± 7% versus the CD45highgroup (n=26, RALV=0.133 - 0.450) at 34 ± 24% (p < 0.05). These results show that the levels of expression of the CD45 antigen on leukemic lymphoblasts are significantly correlated with the clinical features and prognosis of childhood ALL.

Overview of Clinical Studies of Childhood Acute Lymphoblastic Leukemia for More Than Ten Years by the Japanese Children's Cancer and Leukemia Study Group

Since 1981, the Childrens Cancer and Leukemia Study Group (CCLSG) has developed a series of protocols for treatment of acute lymphoblastic leukemia (ALL) in childhood. In the first randomized controlled study of the 811 protocol (1981-1983) a comparison of conventional daily 6-mercaptopurine and methotrexate with a pulsed regimen of the two drugs was performed. The superiority of the pulsed regimen was shown. In the next 841 protocol (1984-1987) a comparison of two drugs and three drugs during induction therapy was conducted. The three-drug regimen resulted in a significantly higher event-free survival (EFS) rate. In the 874 protocol (1987-1990) two regimens with or without cranial irradiation were randomly compared, and there was no significant difference between the two regimens for the standard-risk group. To further improve the EFS rate a risk group-directed protocol 911 was conducted starting in January 1991. Life-table analysis of serial CCLSG protocols revealed that the outcome of overall ALL has gradually improved with an increase of the EFS rate; 41.4% +/- 3.6% at 14 years for the 811 protocol, 51.3% +/- 3.5% at 11 years for the 841 protocol, 56.7% +/- 3.1% at 8 years for the 874 protocol, and 78.2% +/- 3.1% at 4 years for the more recent 911 protocol.

Kawasaki Disease in a Patient with Wiskott-Aldrich Syndrome: An Increase in the Platelet Count

Figure 1. The clinical course of the patient with Kawasaki Disease. PLT indicates platelet; IVIG, intravenous immunoglobulin; CRP, C-reactive protein; Day, the clinical day after the patient exhibited fever during the course of Kawasaki Disease. We report a unique case of Wiskott-Aldrich syndrome (WAS) associated with Kawasaki Disease (KD). A male Japanese patient had eczema, purpura, and recurrent infections beginning in early infancy and was found to have thrombocytopenia (platelet count, 20-50 109/L). The platelet size was also small (mean platelet volume, 4.0-5.0 fL). The patient was medicated with immunoglobulin and oral prednisolone to increase the platelet count, but counts remained low. The patient developed KD at the age of 6 months (high fever over 5 days, systemic rash, swelling of fingers and toes, conjunctival swelling, strawberrylike tongue, and cervical lymphadenopathy were seen), and the blood platelet count increased transiently in the acute phase (Figure 1) and decreased immediately after administration of high doses of immunoglobulin (1 g/kg 2 days) for the prophylaxis of coronary aneurysm. He was medicated with immunoglobulin (0.5 g/kg 3 days) again to increase platelets after he was cured of KD, but the platelets did not increase. WAS was diagnosed because results of analysis of the WAS protein (WASP) gene showed a missense mutation (in intron 6, 597G→A) and Western blot analysis results showed less progression of WASP than occurs in healthy controls. Although there are a few reports of WAS with vasculitis syndromes (ie, Takayasu arteritis [1] and HenochSchonlein purpura [2]), no report to our knowledge has ever described the combination of WAS and KD. There are some reports that interleukin-6, one of the inflammatory cytokines, is increased in KD patients [3]. Interleukin-6 has been reported to be a potent thrombopoietic Kawasaki Disease in a Patient with Wiskott-Aldrich Syndrome: An Increase in the Platelet Count