E2FBP1/hDril1 modulates cell growth through downregulation of promyelocytic leukemia bodies.

Promyelocytic leukemia nuclear bodies (PML-NBs) comprise multiple regulatory factors and play crucial roles in the maintenance of cellular integrity, while unregulated activation of PML-NBs induces death and premature senescence. Hence, the function of PML-NBs must be directed properly; however, the mechanism that regulates PML-NBs remains unclear. In this paper, we show that PML-NBs are disintegrated by an AT-rich interaction domain family protein E2FBP1/hDril1 through specific desumoylation of promyelocytic leukemia protein (PML) in vivo and in vitro. RNA interference-mediated downregulation of E2FBP1/hDril1 results in hyperplasis of PML-NBs and consequent commitment to PML-dependent premature senescence. Thus, the function of E2FBP1/hDril1 is required for maintenance of survival potential of the cells. Our data suggest a novel mechanism to govern cellular integrity through the modulation of nuclear depots.

Cryptic insertion and translocation or nondividing leukemic cells disclosed by FISH analysis in infant acute leukemia with discrepant molecular and cytogenetic findings.

Of 51 infants with acute leukemia, 13 (25%) had contradictory findings on 11q23/MLL rearrangements that were analyzed by cytogenetic and Southern blot methods: seven had rearranged MLL and normal karyotype, four had rearranged MLL and abnormal karyotype with no 11q23 translocation, and two had germline MLL and 11q23 translocations. Fluorescent in situ hybridization (FISH) analysis using an MLL probe that was performed to elucidate the discrepancy disclosed the presence of normal dividing cells and nondividing leukemic cells in the same bone marrow in five patients, and cryptic insertion or translocation in another five. Subsequent FISH and reverse transcription-polymerase chain reaction analysis identified the MLL-AF10, MLL-AF4, or MLL-AF1q fusions that were produced by the cryptic rearrangements in four of the five patients. In the remaining three patients, the breakpoint of 11q23 translocation was located distal to the MLL locus in one, and the discrepancy was unresolved in two. Thus, FISH should complement cytogenetic analysis when cytogenetic and molecular genetic findings are contradictory in infant leukemia, and when infant leukemia does not show 11q23 translocations or other specific translocations including t(7;12), t(1;22), etc that are recurrently found in infant leukemia.

Mutations/deletions of the WT1 gene, loss of heterozygosity on chromosome arms 11p and 11q, chromosome ploidy and histology in Wilms' tumors in Japan.

Incidence rates of Wilms' tumor (WT) markedly differ in East Asian and Caucasian children. In the present study, we examined WT1 deletions/mutations and loss of heterozygosity (LOH) on 11p and 11q in a large number of WTs and compared our findings with those from 4 series of Caucasian WTs. Incidence rates of the subtle WT1 mutation in 3 of the 5 series of sporadic and unilateral WTs including ours were 4.3-6.2% and similar. However, gross homozygous WT1 deletion was more frequent in our series than in some others. In addition, our series tended to show a higher incidence of LOH limited to 11p13 and a lower incidence of LOH including 11p15 than the Caucasian one. These findings indicate some genetic differences in WT between the 2 regions. One of the 4 Caucasian series reported a correlation of germinal WT1 mutation with the predominantly stromal histology. The present study not only confirms the correlation of germinal WT1 deletion/mutation with predominant stromal histology but also establishes a correlation with somatic WT1 deletion/mutations with predominant stromal histology. While WTs with WT1 abnormalities usually showed pseudodiploidy and predominant stromal histology, those without WT1 abnormalities showed various chromosome numbers and histologic subtypes.

Frequent increase of DNA copy number in the 2q24 chromosomal region and its association with a poor clinical outcome in hepatoblastoma: cytogenetic and comparative genomic hybridization analysis.

In a cytogenetic and comparative genomic hybridization (CGH) study of 38 hepatoblastomas, we found gain of 1q in 17 tumors (44.7%), that of 2 / 2q in 14 (36.8%), that of 20 / 20q in 9 (23.7%) and that of 8 / 8q in 8 (21.0%), loss of 4q in 4 (10.5%) and no DNA copy changes with normal karyotype or no mitotic cells in 11 (28.9%). Eleven tumors with 2 / 2q gain detected by CGH had a total chromosome 2 gain, a partial 2q gain, or a total chromosome 2 gain with an augmented partial 2q region; the common region for DNA copy gain was 2q24. Two-color fluorescence in situ hybridization (FISH) analyses using probes covering the centromere of chromosome 2 or HOXD13 (2q31) confirmed the CGH findings, and showed that the common region for gain in 2q was centromeric to HOXD13. Event-free survival (EFS) +/- standard error (SE) at 5 years was lowest in patients with 2q gain [37 +/- 15%], highest in those with no DNA copy changes [82 +/- 12%], and intermediate in those with DNA copy changes other than 2q gain [74 +/- 13%] (P = 0.0549). Multivariate analysis showed that 2q gain was an independent factor predicting a poor outcome. These findings suggest the presence of a growth-promoting gene or an oncogene in the 2q24 chromosome band, and a tumor suppressor gene in terminal 4q, which have important roles in the development and progression of hepatoblastoma.

Delay of the diagnostic lumbar puncture and intrathecal chemotherapy in children with acute lymphoblastic leukemia who undergo routine corticosteroid testing: Tokyo Children's Cancer Study Group study L89-12.

PURPOSE: To determine the effects of eliminating initial lumbar punctures in 418 consecutively treated children with acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS: Patients were enrolled onto a trial conducted in central Japan between 1989 and 1992. Treatment consisted of standard four-drug induction therapy followed by a risk-based intensification phase, reinduction therapy, late intensification, and remission maintenance therapy (total of 104 weeks). The initial lumbar puncture, with an intrathecal injection of chemotherapy, was performed after 1 week of prednisolone sensitivity testing (day 8). End points included response to prednisolone, CNS status at the time of the day 8 lumbar puncture, subsequent adverse events in CNS and bone marrow, and event-free survival (EFS). RESULTS: The remission induction rate was 93.1% with a 6-year EFS rate (+/- SE) of 68.7% +/- 2.4%, which is similar to historical results for patients who received their diagnostic lumbar puncture and first instillation of intrathecal chemotherapy on day 0. Overall, 84.5% of the patients had good responses to prednisolone, whereas 15.5% had poor responses. Clinical outcome was strikingly better for the good responders (6-year EFS, 74.1% +/- 2.5% compared with 40.1% +/- 6.4% for patients with poor responses), suggesting that omission of intrathecal chemotherapy did not alter the predictive value of drug sensitivity testing. Eighteen patients experienced CNS relapse as their first adverse event (cumulative risk, 5.1%; 95% confidence interval, 2.7% to 7.4%), coincident with reports from groups using conventional strategies of CNS clinical management. Bleeding into the CSF at the time of the day 8 lumbar puncture was apparent in 29 cases (8.1%), but leukemic blasts were identified in only two. CONCLUSION: Delay of the initial lumbar puncture and intrathecal injection of chemotherapy seems to be feasible in children with ALL. Further controlled evaluations are needed to establish the validity of this conclusion.

Differentiation-associated expression and intracellular localization of cyclin-dependent kinase inhibitor p27KIP1 and c-Jun co-activator JAB1 in neuroblastoma.

Neuroblastoma is a unique pediatric cancer, which spontaneously regress in some infants and undergo maturation in older children. The cyclin-dependent kinase inhibitor p27KIP1 negatively control cell cycle progression and its expression is reported to be associated with differentiation and prognosis of some human cancers. To examine whether p27KIP1 is involved in differentiation of neuroblastomas, expression and localization of p27KIP1 in 30 cases of neuroblastic tumors were determined with immunohistochemistry. p27KIP1 was expressed in all cases, but staining intensity and intracellular localization varied in association with tumor differentiation. Primitive small round neuroblasts showed negative or only weak nuclear staining, while differentiating tumor cells displayed a novel, intense cytoplasmic positivity besides the nuclear staining, and mature ganglion cells showed intense positive reaction confined to the nucleus. A neuroblastoma cell line TGW was also immunostained positively for p27KIP1 in the cytoplasm after differentiation induction, and western blot analysis revealed an increase of p27KIP1 in these cells, corroborating the in vivo observations. JAB1, which is thought to bind p27KIP1 and transport it from the nucleus to the cytoplasm for proteasome/ubiquitin-mediated degradation, was found to be localized both in the cytoplasm and the nucleus in undifferentiated and differentiating tumors whereas located predominantly in the nucleus of differentiated tumor cells. These data indicate that the cytoplasmic localization of p27KIP1 in the process of differentiation is due to upregulation of p27KIP1 synthesis and subsequent degradation and suggest a role of p27KIP1 in differentiation of neuroblastoma.

[Li-Fraumeni syndrome].

Germ-line p53 point mutations have been reported for various families with Li-Fraumeni syndrome (LFS) characterized by a dominantly inherited increased susceptibility for the development of early age of onset neoplasms of diverse origin in multiple family members. Recently Bell et al reported that mutations in a known checkpoint gene called Chk2 cause some cases of LFS. This review will present the effective interaction of epidemiologic method and molecular genetics on the identification of cancer predisposition and will discuss about various problems of predictive testing for inherited mutations in cancer susceptibility genes.

Feasibility study of autologous peripheral blood stem cell transplantation for the treatment of childhood acute myelogenous leukemia.

BACKGROUND: The primary object of this study was to identify treatment-related variables that may predict relapse of acute myelogenous leukemia (AML) after autologous peripheral blood stem cell transplantation (PBSCT), which will be critical for the development of a suitable protocol for wider application. METHODS: A total of 28 children (age 0-18 years) with AML underwent PBSCT and have had a minimum follow-up of 25 months; including 24 patients in their first complete remission (CR) and four in their second CR. The patients were divided into two cohorts according to the study phase: 16 patients were treated in an early phase pilot study and 12 patients in their first CR were treated in a prospective trial. Fifteen of the first-CR patients had any of the cited high-risk features of high WBC count (>100x10(9)/l; n = 5), FAB M0/M4/M5/M7 subtypes (n = 11) or delayed achievement of CR (n = 9). Except in one patient, cytoreductive regimens did not include total body irradiation (TBI). RESULTS: After PBSCT, one patient died of veno-occulsive disease (VOD) and another patient relapsed early on day 43, but the remaining patients showed engraftment. Leukemic relapse was observed 1-29 months after PBSCT (median, 8 months); in all of the 4 children treated in their second CR and in 11 of the 24 patients (46%) treated in their first CR. The remaining patients have been disease-free for 24 to 97 months (median, 53 months). Using a multivariate analysis, the timing of apheresis was the most significant prognostic factor for those treated in their first CR (p = 0.03); 12 of the 16 patients whose PBSC were collected beyond 2.5 months of CR continue to remain in CR, while seven of the eight patients whose PBSC were harvested within 2.5 months of CR relapsed. CONCLUSION: Although the small number of patients studied does not allow firm conclusions to be drawn regarding the relative effectiveness of this therapy, the results do suggest the feasibility of further studies of PBSCT for the treatment of childhood AML with high-risk features including the assessment of minimum residual disease.

Hemophagocytosis by leukemic blasts in 7 acute myeloid leukemia cases with t(16;21)(p11;q22): common morphologic characteristics for this type of leukemia.

BACKGROUND: In a previous study of a case of acute megakaryoblastic leukemia with t(16;21)(p11;q22), which displayed hemophagocytosis by leukemic blasts, the authors mentioned that the same type of morphology had been cited in the literature for 4 other cases of acute myeloid leukemia (AML) with the same translocation. This observation prompted the authors to examine more cases of AML with t(16;21)(p11;q22) for this morphology. METHODS: The authors reviewed bone marrow smears for the presence of hemophagocytosis in 7 patients with AML identified as having t(16;21)(p11;q22). RESULTS: The leukemias belonged to the FAB-M1/M7 (n = 5), M5b (n = 2), and contained phagocytic blasts in various percentages (< 0.2-36.7%). The blasts contained either single or multiple cytoplasmic vacuoles, in some of which the phagosomes were visible. The engulfed hemopoietic cells (red cells, erythroblasts, lymphocytes, and thrombocytes) were also noted in their cytoplasm. These observations confirmed that hemophagocytosis by leukemic blasts is a common and characteristic feature of this type of leukemia. CONCLUSIONS: The study of 12 cases (the 7 cases described here and the previous 5 cases) strongly supports the hypothesis that hemophagocytosis by leukemic blasts is common and characteristic in this type of leukemia, which may be related to the specific chromosome aberration of t(16;21)(p11;q22).

Six months of maintenance chemotherapy after intensified treatment for acute lymphoblastic leukemia of childhood.

PURPOSE: We postulated that intensification of chemotherapy immediately after remission induction might reduce the leukemic cell burden sufficiently to allow an abbreviated period of antimetabolite therapy. PATIENTS AND METHODS: Three hundred forty-seven children (ages 1 to 15 years) with previously untreated acute lymphoblastic leukemia (ALL) were enrolled onto the Tokyo L92-13 study, which excluded patients with mature B-cell ALL and patients less than 1 year old. One hundred twenty-four patients were classified as standard risk, 122 as high risk, and 101 as extremely high risk, according to age, peripheral-blood leukocyte count, selected genetic abnormalities, and immunophenotype. All subjects received four drugs for remission induction, followed by a risk-directed multidrug intensification phase and therapy for presymptomatic leukemia in the CNS. Maintenance chemotherapy with oral mercaptopurine and methotrexate was administered for 6 months, with all treatment stopped by 1 year after diagnosis. RESULTS: The mean (+/- SD) event-free survival (EFS) and overall survival rates for all patients were 59.5% +/- 3.4% and 81.5% +/- 2.2%, respectively, at 5. 5 years after diagnosis. EFS rates by risk category were similar (60. 2% +/- 6.0% for standard risk, 57.7% +/- 5.6% for high risk, and 62. 5% +/- 5.7% for extremely high risk), whereas overall survival rates differed significantly (91.2% +/- 2.7%, 80.0% +/- 4.1%, and 72.1% +/- 4.5%, respectively, P <.0001 by the log-rank test). There were 107 relapses. Eighty-five (79.4%) of these 107 patients achieved second complete remissions, with subsequent EFS rates of 61.5% +/- 7. 9% (standard risk), 42.6% +/- 8.1% (high risk), and 9.6% +/- 6.4% (extremely high risk). Of the five risk factors analyzed, only the response to prednisolone monotherapy among extremely high-risk patients proved important. CONCLUSION: Early treatment intensification did not compensate for a truncated phase of maintenance chemotherapy in children with standard- or high-risk ALL. However, 6 months of antimetabolite treatment seemed adequate for extremely high-risk patients who were good responders to prednisolone and received intensified chemotherapy that included high-dose cytarabine early in the clinical course.

[Evidence based chemotherapy for pediatric cancers].

The curability of pediatric cancer has been improved to nearly seventy percent. This change has been achieved by refinements in treatment strategy and supportive care. More than seventy percent of patients with ALL can be cured by modern chemo-radiotherapy with reduced late effects. The stratification of the patients by risk factor, introduction of CNS prophylaxis, shortening of the duration of chemotherapy and intensification of the chemotherapy with agents such as HD-MTX have contributed to this remarkable success. Burkitt's lymphoma is a tumor for which the curability has improved from almost zero to ninety percent. With Wilms' tumor, clinical trials have been used to optimally refine the treatment strategy. The NWTS first compared the efficacy of combined VCR and Act-D with the single use of each drug. The difference was significant. The results of the systematic trials were then used to improve the survival rate of patients with Wilms' tumor from twenty to ninety percent and shorten the duration of chemotherapy to six months. On the other hand, tumors remain with which less than half of patients can survive for long. Advanced neuroblastoma and AML are typical such tumors. With these diseases, refinements in the treatment based on evidence derived from clinical trials have been insufficient. Further intensification of the treatment or novel approaches to control tumor growth are warranted for these diseases. In this article, I would like to describe the "standard" therapy for each tumor and the evidence on which improvements in those strategies have been based.

Long-term follow-up of childhood acute lymphoblastic leukemia in Tokyo Children's Cancer Study Group 1981-1995.

The objectives were as follows: Firstly, to estimate the overall probability of event-free survival (EFS) and isolated CNS relapse in the studies for children with acute lymphoblastic leukemia (ALL) during the 1980s and 1990s. Secondly, to report the EFS according to presenting features and lineage. Thirdly, to evaluate the treatment results re-classified by the risks of NCI criteria. Four consecutive protocol studies were performed in the Tokyo Children's Cancer Study Group: L81-10 protocol (1981-1984, 189 patients), L84-11 (1984-1989, 484 patents), L89-12 (1989-1992, 418 patients) and L92-13 (1992-1995, 347 patients). Overall EFS at 5 years in each protocol was 56.5 +/- 3.8(1 s.e.)%, 71.0 +/- 2.1%, 67.8 +/- 2.3%, and 63.4 +/- 2.7%, respectively. The cumulative isolated CNS relapse rate at 5 years was 8.1 +/- 2.1%, 3.5 +/- 0.9%, 3.6 +/- 1.0%, 1.0 +/- 0.6. The EFS in SR/HR (standard risk/high risk) according to the NCI criteria in B-precursor ALL at 5 years was 61.9 +/- 4.3%/41.4 +/- 7.4% (lineage was not confirmed.), 72.5 +/- 2.6%/63.4 +/- 5.0%, 77.4 +/- 2.7%/56.3 +/- 4.7%, and 67.8 +/- 3.4%/56.7 +/- 5.4% in each protocol. Also EFSs according to NCI SR/HR at 5 years of T-ALL in protocols L84-11, L89-12 and L92-13 were 55.6 +/- 16.6%/60.9 +/- 10.1%, 72.7 +/- 13.4%/51.6 +/- 9.1%, and 77.1 +/- 14.4%/53.6/10.1%, respectively. The truncation of maintenance therapy to 6 months resulted in a decreased EFS in L92-13, particularly due to an increase of bone marrow relapse after cessation of therapy in SR and HR. The NCI risk criteria work properly even in the patients treated by different intensities, so that it makes the comparison possible among the patients in various groups. The overall EFSs in childhood ALL improved in 1980s, but it seemed stable or decreased in 1990s. The short maintenance therapy resulted in poor outcome in SR on the L92-13 protocol. Many of these late relapsers were effectively rescued and overall survival remained at a high level. The proportion of patients who received cranial irradiation reduced without any increase of the CNS events.

Abnormalities of the p53 gene in juvenile myelomonocytic leukaemia.

Juvenile chronic myelomonocytic leukaemia (JMML) is a rare myeloproliferative disorder of childhood. Fewer than 30% of cases of JMML terminate in a blast crisis; however, its molecular mechanism is unknown. Since mutation and/or deletion of the p53 gene has been reported to be associated with disease progression in a wide variety of human cancers, including adult-type chronic myelogenous leukaemia, we studied the p53 gene in 20 patients with JMML (16 samples in chronic phase and seven at blast crisis). Exons 4-8 of the p53 gene, which cover all the hot spots of point mutations, were amplified by the polymerase chain reaction (PCR) method and subjected to mutation screening by single-strand conformation polymorphism analysis. No mobility shift of single-strand DNA of PCR products in polyacrylamide gel electrophoresis, indicating point mutations, was found in 19/20 patients. DNA of the remaining patient in the chronic phase failed to be amplified by PCR and Southern blot analysis with XbaI-digested genomic DNA revealed a gross rearrangement (presumed deletion) of the p53 gene. These data indicate that abnormalities of the p53 gene are rare in JMML and not responsible for acute transformation, but could be involved in the pathogenesis of some cases of JMML.

11p15 translocations involving the NUP98 gene in childhood therapy-related acute myeloid leukemia/myelodysplastic syndrome.

In a survey of childhood therapy-related acute myeloid leukemia/myelodysplastic syndrome (t-AML/MDS) in Japan, we found 11p15 translocations in 5 (6%) of 81 children with t-AML/MDS. t(11;17)(p15;q21), t(11;12)(p15;q13), t(7;11)(p15;p15), inv(11)(p15q22), and add(11)(p15) were each found in one patient. Southern blotting and/or RT-PCR analyses revealed rearrangements of the NUP98 gene in tumor samples of all five patients. Rearrangements of DDX10 were detected in t-AML/MDS cells with inv(11), and rearrangements of HOXA9 were detected in t-AML cells with t(7;11). The 17q21 breakpoint of t(11;17) and the 12q13 breakpoint of t(11;12)(p15;q13) coincided with the loci of the HOXB and HOXC gene families, respectively. Therefore, it is reasonable to speculate that one of the HOXB genes and one of the HOXC genes were fused to NUP98 by t(11;17) and t(11;12), respectively, in t-AML/MDS cells. We propose that NUP98 may be a target gene for t-AML/MDS, and that t-AML/MDS with a fusion of NUP98 and HOX or DDX10 genes may be more frequent in children than in patients of other age groups.

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 (> or =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 (> or =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 (> or =50 chromosomes) Wilms tumors may be characterized by the absence of WT1 abnormalities and possibly also by a favorable prognosis.

The FANCA gene in Japanese Fanconi anemia: reports of eight novel mutations and analysis of sequence variability.

Fanconi anemia (FA), an autosomal recessive disorder characterized by a progressive pancytopenia associated with congenital anomalies and high predisposition to malignancies, is a genetically and clinically heterogeneous disease. At least eight complementation groups (FA-A to FA-H) have been identified with their relative prevalence varying among the ethnical backgrounds. Recently, responsible genes, FANCA and FANCC, have been cloned. This report describes mutations of the FANCA gene, which we studied by direct sequencing of cDNA with confirmation on genomic DNA in 15 unclassified Japanese FA patients. A total of 19 sequence alterations were identified, of which 10 (six missense and four silent alterations) were likely to be nonpathogenic polymorphism. The remaining nine alterations, of which eight were novel mutations, were assumed to be pathogenic and consisted of two missense mutations and seven mutations resulting in truncation of gene product, demonstrating a wide allelic heterogeneity. The pathogenic mutations were found in 12 patients (80%); they were either homozygous or compound heterozygous in 10 patients, apparently heterozygous in two patients and none in three patients. We conclude that the sequence variability is intrinsic to the FANCA gene and that the relative prevalence of the FA-A subtype is unusually high in Japanese FA patients.

Trends of survival in neuroblastoma and independent risk factors for survival at a single institution.

To assess the progress of survival in neuroblastoma which varies with many risk factors and to evaluate the influence of these factors on survival as independent risk factors. The study subjects were 159 neuroblastoma patients seen from 1965-1994 at the oldest and largest children's hospital in Japan. Trends of survival in three treatment eras-1965-81, 1982-86, 1987-94-were assessed by the Kaplan-Meier method for different sex, age at diagnosis, the clinical stage, the site of onset, and the histological type. Then the influence on survival of these factors as independent prognostic variables was evaluated by the Cox proportional hazards regression analysis. Age at diagnosis, the clinical stage, the site of onset, the histological type, and the treatment era were independent risk factors in the order of their influence on survival. Unfavorable survival outcomes were obtained for patients with age at diagnosis above 1 year, the clinical stage of VI by the Evans classification, adrenal onset, and neuroblastoma rather than ganglioneuroblastoma. Survival improved from the first to the second and from the second to the third treatment era. Improvement of survival in neuroblastoma took place during the past 3 decades. Age at diagnosis, the clinical stage, and the histological type have still remained overwhelming prognostic factors over the progress in treatment.