Interaction between NUDT15 and ABCC4 variants enhances intolerability of 6-mercaptopurine in Japanese patients with childhood acute lymphoblastic leukemia.

6-Mercaptopurine (6-MP) is a main component of childhood acute lymphoblastic leukemia (ALL) treatment. Some candidate gene variants are associated with its toxicities, but the major variants and effects of combined variants remain unclear. We used Cox regression analysis to evaluate the time-dependent association between candidate variants and the cumulative incidence of 6-MP intolerability in 95 Japanese patients. The major risk factors for severe leukopenia were ABCC4 rs3765534, NUDT15 rs116855232 and rs186364861 in multi-covariate analysis (P<0.05). NUDT15 intermediate activity variant, that is, heterozygous rs116855232 or rs186364861 variant, and the ABCC4 rs3765534 variant showed leukopenia more frequently than either variant alone. All patients with both the intermediate activity NUDT15 variant and the ABCC4 rs3765534 variant suffered from leukopenia, and 57.1% patients required 50% protocol dose by day 168. These data indicate that NUDT15 and ABCC4 are major factors for 6-MP intolerability and that the interaction between these variants enhances intolerability to 6-MP.

Multidrug resistance protein 4 (MRP4) polymorphisms impact the 6-mercaptopurine dose tolerance during maintenance therapy in Japanese childhood acute lymphoblastic leukemia.

Multidrug resistance protein 4 (MRP4) is involved in the efflux of nucleoside derivatives and has a role in the determination of drug sensitivity. We investigated the relationship between MRP4 genetic polymorphisms and doses of the 6-mercaptopurine (6-MP) and methotrexate. Further, we evaluated the frequency of therapeutic interruption during maintenance therapy in Japanese children with acute lymphoblastic leukemia (ALL). Ninety-four patients received an initial 6-MP dose in the range of 30-50 mg m(-2) in this analysis. Patients with homozygous variant allele in any of MRP4 G2269A, C912A and G559T required high frequency of 6-MP dose reduction compared with non-homozygous individuals. Average 6-MP dose for patients with homozygous variant allele on either MRP4 or inosine triphosphate pyrophosphatase was significantly lower than that for patients with non-homozygous variant allele during maintenance therapy (30.5 versus 40.0 mg m(-2), P=0.024). Therefore, MRP4 genotyping may be useful for personalizing the therapeutic dose of 6-MP during the ALL maintenance therapy in Japanese.

Genetic and epigenetic alterations on the short arm of chromosome 11 are involved in a majority of sporadic Wilms' tumours.

Wilms' tumour is one of the most common solid tumours of childhood. 11p13 (WT1 locus) and 11p15.5 (WT2 locus) are known to have genetic or epigenetic aberrations in these tumours. In Wilms' tumours, mutation of the Wilms tumour 1 (WT1) gene at the WT1 locus has been reported, and the WT2 locus, comprising the two independent imprinted domains IGF2/H19 and KIP2/LIT1, can undergo maternal deletion or alterations associated with imprinting. Although these alterations have been identified in many studies, it is still not clear how frequently combined genetic and epigenetic alterations of these loci are involved in Wilms' tumours or how these alterations occur. To answer both questions, we performed genetic and epigenetic analyses of these loci, together with an additional gene, CTNNB1, in 35 sporadic Wilms' tumours. Loss of heterozygosity of 11p15.5 and loss of imprinting of IGF2 were the most frequent genetic (29%) and epigenetic (40%) alterations in Wilms' tumours, respectively. In total, 83% of the tumours had at least one alteration at 11p15.5 and/or 11p13. One-third of the tumours had alterations at multiple loci. Our results suggest that chromosome 11p is not only genetically but also epigenetically critical for the majority of Wilms' tumours.

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.

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.

Long survivors with Ki-1 lymphoma having t(2;5) (p23;q35). Does the presence or absence of t(2;5) influence the prognosis of patients with Ki-1 lymphoma?

We experienced three patients with CD30+ diffuse large cell lymphoma having chromosomal abnormalities. The first patient was an 8-year-old girl with bilateral cervical lymphadenopathy. A biopsy of a cervical lymph node revealed diffuse large cell lymphoma (stage III), positive for CD30 and a chromosomal abnormality, t(2;5). She attained a remission and is now in complete remission 108 months after diagnosis, despite frequent relapses. The second patient was a 13-year-old boy with right axillar and supraclavicular lymph-node adenopathy. A biopsy of a cervical lymph node revealed diffuse large cell lymphoma (stage III), positive for CD30 and a chromosomal abnormality, t(2;5). He attained remission and was in continuous first remission 112 months after diagnosis. The third patient was an 11-year-old boy with fever and bilateral cervical lymph node revealed diffuse large cell lymphoma (stage III), positive for CD30 and chromosomal abnormality without t(2;5). He showed a very aggressive clinical course. Only the patients with Ki-1 lymphoma having t(2;5) survived over 100 months from the diagnosis, despite the advanced stage of the disease. These findings and a review of the literature showed that the presence or absence of t(2;5) may influence the outcome of Ki-1 lymphoma.

[Childhood acute promyelocytic leukemia treated with all-trans retinoic acid].

We treated two children with acute promyelocytic leukemia (APL) in whom complete remission was successfully induced by oral administration of all-trans retinoic acid (ATRA). We followed these patients with conventional chemotherapy. The first patient has remained in continuous complete remission. However, the other patient relapsed during the maintenance therapy and died of progressive disease in spite of a second treatment with ATRA and chemotherapy. From a clinical point of view, the latter case had a hyperleukocytosis on admission. Also morphologically speaking, this patient had a different M3 variant than the first case. There are two major isoforms of PML/RAR alpha transcripts, so called short and long type transcripts, according to the breakpoints in the PML genes. In the first case the "long type' isoform was detected by reverse transcriptase polymerase chain reaction (RT/PCR) amplification. On the other hand the "short type' isoform was observed in the latter case. Also the second case became PCR positive at relapse, although the detectable isoform was negative during remission. The "short type' isoform may be related to the poor prognosis and RT/PCR analyses may be a powerful to detect early relapse.

Acute lymphoblastic leukemia and non-Hodgkin's lymphoma with mediastinal mass--a study of 23 children; different disorders or different stages?

Mediastinal tumor was found in both acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphoma (NHL). Most cases showed the T-cell phenotype. We query whether these two diseases are in fact different disorders or merely different stages of the same disease. Twelve ALL patients with a mediastinal mass and eleven NHL patients with a mediastinal mass under 15 years of age were studied with respect to cytogenetics, immunophenotype, genotype and clinical features. Clonal chromosome abnormalities were found in 75% (9/12) of the ALL patients and 100% (11/11) of the NHL patients. Of the 20 patients with chromosome abnormalities, 12 (60%) had translocations involving 14q11-13 and 7q35 (8 ALL, 4 NHL). t(9;17)(q34;q23) was found only in 3 patients with NHL. All showed the T-cell phenotype except two, who had none of the chromosomal abnormalities frequently detected in T cell ALL/NHL. In T-cell patients, immunophenotypical staging of ALL showed a predominance of early and common thymocyte phenotypes while that of NHL showed a predominance of common thymocyte phenotypes. All 7 of the T-cell patients examined showed rearrangements of the T-cell receptor beta chain gene. On the other hand, two non-T-cell, non-B-cell patients showed no rearrangement. There were no apparent clinical differences between ALL and NHL patients in age (median 8.6 vs 8.9 years), sex ratio (F/M 9/3 vs 7/4) or in the rate of complete remission (90% vs 100%). Our study demonstrated no relevant clinical, prognostic, or immunophenotypic differences between ALL and NHL with mediastinal mass.(ABSTRACT TRUNCATED AT 250 WORDS)

[Primary myelofibrosis in an infant--a case report and review of the literature].

Primary myelofibrosis (PMF) is regarded as a chronic myeloproliferative disorder. It is characterized by marrow fibrosis, leukoerythroblastosis, tear drop erythrocytes and extramedullary hematopoiesis. Most patients are in their late 50s when first diagnosed. Pediatric PMF is said to be quite rare. Here describe a female infant with PMF. The patient was born on Aug. 7, 1991. The pregnancy and delivery were uneventful. Hepatomegaly was noted soon after birth. Combined blood counts showed polycythemia and leukocytosis. It was thought to be extramedullary hematopoiesis due to intrauterine infection. She was followed up in another hospital, but since her condition was unchanged she was admitted to our hospital for further medical examinations at age 7 months. On the peripheral blood smear, there were tear drop erythrocytes, normoblasts and early myeloid elements. Repeated bone marrow aspirations were dry taps. This case presented the classical findings of fibrosis of the bone marrow on bone marrow biopsy. She is in good health without any therapy until now. A review of 7 cases of PMF, including our case, in Japanese children was made and discussed in comparison to adult cases.

[Clinical characteristics and treatment results of acute promyelocytic leukemia in children (Children's Cancer and Leukemia Study Group)].

The clinical characteristics and treatment outcome in 40 children with acute promyelocytic leukemia (APL) treated at institutions participating in the Children's Cancer and Leukemia Study Group (CCLSG) were studied retrospectively. The median age at diagnosis was 8 years old. Bleeding diathesis was the predominant presenting symptom (90%), associated with laboratory findings of disseminated intravascular coagulation. Hepatomegaly, splenomegaly and lymphadenopathy were observed in 35%, 10%, and 15% of the cases, respectively. The median WBC count was 4.25 x 10(9)/l. Anemia (hemoglobin < 8 g/dl) and thrombocytopenia (< 30 x 10(9)/l) were present in more than half of the patients. Cytogenetic studies demonstrated the characteristic 15; 17 translocation in about 90% of the patients analyzed. Induction therapy consisted of cytosine arabinoside and an anthracycline, with or without other agents. Twenty-nine patients (73%) achieved complete remission (CR) while early fatal hemorrhage was the predominant cause of induction failure. The survival rates continued to decrease (28% at 3 years, 24% at 5 years, and 7.9% at 10 years) due to late marrow relapses. Anthracycline cardiotoxicity was fatal in three patients in remission. These clinical features of childhood APL should be taken into account in the development of new protocols.

[A multi-institutional study on the efficacy and toxicity of imipenem/cilastatin sodium in severe infections complicating hematological diseases and cancers in children. Study Group of Infectious Diseases in Pediatric Hematology/Oncology in Hokkaido].

A multi-institutional study was conducted between September 1990 and April 1992 to evaluate the efficacy and toxicity of imipenem/cilastatin sodium (IPM/CS) in severe infections in cases of granulocytopenia in children with hematological diseases and cancers. A total of 60 episodes of infection were treated with the drug, and an overall efficacy rate of 80% (48/60) was obtained. The efficacy rate in patients who were positive for Endospecy test was 90.0%. A group of patients who had previously received other antibiotics showed an efficacy rate of 79.2%, while the patients who had not received previous antibiotic treatment showed an efficacy rate of 80.6%. The difference between the 2 groups was statistically insignificant, however. Granulocyte counts appeared to have influence on the efficacy of the drug, but the influence was not strong. Three patients had nausea, vomiting and/or diarrhea, and 2 other patients showed abnormal liver function test parameters though they recovered soon after the cessation of the drug treatment. From these results, we have concluded that IPM/CS is an effective antibiotic for treatment of severe infections with hematological diseases and cancers in children.

[Analysis of cytoplasmic antigens in acute leukemia by flow cytometry].

The expression of cytoplasmic antigens in 77 cases of acute leukemia were analyzed by flow cytometry using the following monoclonal antibodies: CD3, CD22, anti-myeloperoxidase (MPO-7) and anti-mu-heavy chain. CD22 antigen was detected in the cytoplasm of all non-T-ALL patients excluding one not-tested patient. In two patients with unclassified ALL, surface CD22 antigen was not expressed but cytoplasmic CD22 antigen was strongly expressed. Three out of 9 patients with common ALL were cytoplasmic mu-heavy chain-positive, so these patients were diagnosed as Pre-B ALL. In four out of 8 patients with T-ALL, CD3 antigen was not expressed on the cell surface membrane. However all of T-ALL patients excluding one non-tested patient were cytoplasmic CD3-positive. The cytoplasmic expression of myeloperoxidase antigen was detected in twenty out of 21 patients with acute non-lymphoblastic leukemia (ANLL). One megakaryocytic leukemia patient was MPO-negative. In two ANLL patients, the percentage of MPO for conventional cytochemical staining was undetectable or low, but MPO antigens were positive (77% and 70%) for flow cytometric analysis. All of 46 non-T ALL patients were cytoplasmic MPO-negative, however 4 out of 10 T-ALL patients were cytoplasmic MPO-positive. The study proved that the analysis of cytoplasmic CD3, CD22, mu-chain and MPO antigens were very useful to define the cell lineage of leukemia and to classify ALL and ANLL. It is necessary to study further whether the expression of MPO in the cytoplasm of T-ALL was non-specific reaction or whether MPO precursors are expressed in the cytoplasm of T-ALL.

Cytogenetic characteristics of childhood non-Hodgkin lymphoma.

Cytogenetic studies were performed successfully on 24 patients with non-Hodgkin lymphoma (NHL) who were younger than 15 years of age. Of these, 22 patients (92%) had abnormal clones. With respect to histologic findings, 3 (25%) of the 12 patients with lymphoblastic lymphoma had 14q11 translocations and 2 (17%) had t(9;17) (q34;q23). Four (80%) of the five patients with small non-cleaved cell lymphoma had t(8;14)(q24;q32). With respect to immunologic findings, four (44%) of the nine patients with T-cell lymphoma had abnormalities consisting of 14q11 and 7q36 translocations, in which the T-cell receptor genes resided. Three (33%) of the patients with T-cell lymphoma had t(9;17)(q34;q23). However, three (43%) of the seven patients with B-cell lymphoma had t(8;14) (q23;q32), and two (29%) of the patients with B-cell lymphoma had an extra i(11q) chromosome with a resultant 11q tetrasomy. Non-T-cell non-B-cell lymphomas, which occurred in 21% of all patients, showed various chromosomal abnormalities. This study demonstrated that, in childhood NHL, karyotype correlates closely with immunophenotype, clinical features, and histologic findings.

Analysis of 13 postmortem examinations with regard to the effect of chemotherapy on neuroblastoma patients.

Thirteen postmortem examinations on neuroblastoma patients were reviewed clinicopathologically. Commonly found metastatic sites were bone, liver and lung. The introduction of massive doses of cyclophosphamide and cisplatinum into chemotherapy protocol markedly reduced the tumor burden: no tumor focus in liver or lung, small tumor burden in bone. Lymph node metastasis remained, however, and was refractory to the chemotherapy. The intensive chemotherapy caused severe side effects: interstitial or bacterial pneumonia and intrathecal bleeding, which sometimes proved fatal. Such side effects must, therefore, be carefully controlled in order to maximize the survival rate.

[Clinical evaluation of severe toxicity in two patients with acute lymphoblastic leukemia receiving outpatient methotrexate therapy].

Methotrexate (MTX) is now widely used for the treatment of acute leukemia and non-Hodgkin lymphoma in the pediatric oncology field and is thought to be one of the key drugs for this treatment. A regimen utilizing high dose MTX (HD-MTX) with leucovorin rescue is being investigated as effective chemotherapy in the patients with these kinds of cancer. Relatively large amounts of MTX (225 mg/m2) are given to such outpatients by intravenous push as a course of maintenance therapy. It is said that those amounts will infuse safely. However, we experienced two serious cases-patients T.H. and M.Y.--which developed into severe side effects after this treatment. Both patients showed acute renal failure, severe myelosuppression, erosion around the oral and anal region, and continuous diarrhea. Judging from the serum concentration of MTX, patient T. H. was exposed to more than the maximum allowance serum MTX level for 9.6 days, patient M. Y. for 6.5 days. This suggests physicians must pay attention to the clinical symptoms even after treatment using MTX without HD-MTX.

[Cytogenetic studies on 53 childhood acute nonlymphocytic leukemia].

Cytogenetic study in 53 children (aged less than 15 years) with acute non-lymphocytic leukemia (ANLL) were studied. The cytogenetic findings were compared with those of ANLL patients (136 aged less than 19 years and 747 aged over 20 years) in the Fourth International Workshop on Chromosomes in Leukemia (IV IWCL) and also with those of childhood acute lymphoblastic leukemia (ALL) cases (previously reported as our 124 ALL case). Of the ANLL patients, 77.4% had acquired chromosomal clonal abnormalities. As abnormalities, t(15;17), all cases which were seen in M3 or M3V cases, t(8;21), which was seen in M1 or M2, and rearrangements of 11q23, which were seen in M5, were more frequently seen than was reported at the IV IWCL (20.8%, 17.0% and 7.5% vs 6.3%, 6.3% and 3.2% respectively). 5q-, monosomy 7, t(6;9) and t(9;22), which have been noted previously in this disease, were not seen. Besides structural abnormalities, some cytogenetic differences in numerical abnormality between ALL and ANLL were observed as follows: 1) Hyperdiploidy of greater than 51 chromosomes noted in ALL was not found in ANLL. 2) Isolated trisomy 8 was frequently found in ANLL, but not in ALL. 3) Loss of a sex chromosome was frequently found in ANLL, but not in ALL. Our study revealed a different frequency of non-random chromosome abnormality in children with ANLL as compared with that of adults, and clarified the differences in numerical abnormalities, as well as structural abnormalities, between ALL and ANLL.

[Significance of the 14q32 translocations in childhood acute lymphoblastic leukemia].

To assess the frequency and significance of 14q32 translocation abnormalities in childhood acute lymphoblastic leukemia (ALL) and the differences between the clinical and cytogenetic features of patients with the 8; 14 translocation and those of patients with other 14q32 translocations, we analyzed our experience with 124 consecutive cases with completely banded karyotype. Eight cases (6.5%) with 14q32 translocation were identified :5 with the 8; 14 translocation and 3 with other 14q32 translocations. As compared with ALL children lacking 14q32 translocations, these 8 cases had a higher serum lactic dehydrogenase (LDH) level, more L3 (FAB classification), and a poorer outcome. On the other hand, in comparison with ALL patients with other 14q32 translocations, patients with the 8:14 translocation were likely to be younger (median age 4.5 years vs 10.4 years), to have a higher serum LDH level (median 5832 IU/l vs 504 IU/l), to have more L3 (3/5 vs 0/3), to have a higher induction failure rate (4/5 vs 1/3), and to have more partial duplication of the long arm of chromosome 1 (4/5 vs 0/3). These results helped clarify the characteristic features of ALL children with 14q32 translocations and showed that ALL children with the 8 ; 14 translocation have different clinical and cytogenetic findings from those of ALL children with other 14q32 translocations.

[Transient abnormal myelopoiesis followed by acute leukemia in children with Down syndrome].

This report describes three cases with Down's syndrome. These cases initially had transient abnormal myelopoiesis (TAM), from which they recovered spontaneously. They finally developed into overt acute leukemia characterized by an increase of blasts, hepatosplenomegaly, and elevated lactic dehydrogenase. Of these three cases, one was thought to have ANLL, which broke out 5 months after spontaneous remission. The other two had ALL, each occurring 8 and 9 years later. Chromosomal abnormality, in addition to trisomy 21, was detected in blast cells from one of the patients with acute leukemia. All three patients with acute leukemia experienced complete remission. However, two of the three patients relapsed and died. It is noted in the literature that remission is permanent in most cases of TAM, and is rarely terminated by leukemic relapse. In view of our observations, the importance of following up on such patients who evidence apparent remission of their leukemia-like disorder is emphasized.