7-Deoxyloganin 7-hydroxylase in Lonicera japonica cell cultures.

The activity of 7-deoxyloganin 7-hydroxylase, an enzyme catalyzing the conversion of 7-deoxyloganin into loganin, was detected in a microsomal preparation from the cell suspension cultures of Lonicera japonica. It was dependent on NADPH and molecular oxygen. The enzymatic reaction was inhibited by carbon monoxide as well as by several cytochrome P450 inhibitors, especially ketoconazole, indicating that the reaction was mediated by cytochrome P450. The enzyme showed substrate specificity for 7-deoxyloganin. The K(m) values for 7-deoxyloganin and NADPH were estimated as 170 and 18 microM, respectively, from Lineweaver-Burk plots.

[Treatment and prognosis of children with relapsed non-Hodgkin's lymphoma--a report from CCLSG-NHL 890 Study. Children's Cancer and Leukemia Study Group (CCLSG)].

To address the issue of salvageability in relapsed children with NHL who had all received the same frontline therapy, we retrospectively studied the treatment response and the outcome of 27 children who relapsed following the CCLSG-NHL890 protocol. The reinduction rates and 3-year survival rates (mean +/- SD) were as follows: lymphoblastic lymphoma (LB, n = 9), 44% & 17 +/- 14%; leukemia lymphoma syndrome (LLS, n = 8), 25% & 0%; large cell lymphoma (LC, n = 3) 100% & 67 +/- 27%; Burkitt's lymphoma (B, n = 7) 0% & 0%. Thus, the salvageability of LC lymphoma was good, but the outcome of Burkitt's lymphoma was very poor. CCLSG-NHL960 protocol for LB lymphomas and intensive multiagent regimens for LC lymphomas produced favorable response rates, but the effect of the high-dose Ara-C regimen for Burkitt's lymphoma was not determined. The initial stages of the disease seemed to be associated with the patient outcome: the outcome of the patients in stage IV was inferior to that of patients in stages II or III. Other clinical variables, such as relapse sites, relapse time and BM rescue did not affect the patients' outcome.

[Prognostic significance of chromosome analysis in childhood acute lymphoblastic leukemia. Children's Cancer and Leukemia Study Group (CCLSG)].

We analyzed the prognostic significance of chromosomal findings in children with acute lymphoblastic leukemia (ALL), treated according to the Children's Cancer and Leukemia Study Group protocols between 1987 and 1993. Patients were classified into 5 groups according to chromosome number. The patients with a hyperdiploid(> 50) karyotype(13%) had the best prognosis [4-year event-free survival (EFS): 83 +/- 6%], while those with a pseudodiploid karyotype (24%) had the worst prognosis(4-year EFS: 52 +/- 6%) (log-rank, p = 0.03). However, multivariate analysis revealed that the ploidy classification had no prognostic significance in terms of EFS. When patients were classified according to chromosome abnormalities, those with any type of translocation had a worse outcome (4-year EFS: 33 +/- 9%) than those with hyperdiploidy(> 50), normal diploidy, and other abnormalities(log-rank, p < 0.0001). Multivariate analysis revealed that chromosome abnormalities were an independent prognostic factor (relative risk 3.98; p < 0.0001). Patients with t(1; 19) had an EFS similar to that of patients with chromosome abnormalities other than translocations or normal diploidy. We conclude that chromosomal findings have prognostic significance, although some chromosome abnormalities lost their statistical significance after modern intensified chemotherapy. Childhood ALL should be further stratified according to chromosome classification.

[A newly developed method for taking pictures of tooth crown cracks for clinical records].

It is very important to examine fine cracks on the crown surface of traumatized tooth in the dental clinic because the presence of these cracks may cause discomfort or hypersensitivity of the tooth. By the emission of routinely used light, it is difficult to illuminate cracks, and the usual intraoral photography using strobe light emission is not useful to make records of cracks in most cases. In the present study, a method for taking pictures to record tooth crown cracks was newly developed using LED as a light source, and applied to patients with traumatized teeth in the dental clinic. The results were as follows: 1. Cracks on the tooth surface could be confirmed with the LEDs of all the colors used in this study. However, the blue LED enabled cracks in the incisal edge part to be more easily confirmed, and the white LED enabled cracks in the cervical part to be more easily confirmed. 2. Cracks of multiple modalities on the tooth surface was illuminated more often when the LED light was emitted at 45 degrees to the axis of the tooth than 90 degrees. 3. The light exposure field became wider by the use of the LED of diameter 5 mm than that of diameter 3 mm. However, the LED of diameter 3 mm was more favorable than that of diameter 5 mm for the observation of the cracks because the former could more easily change the direction of light emission.

Secologanin synthase which catalyzes the oxidative cleavage of loganin into secologanin is a cytochrome P450.

Secologanin synthase, an enzyme catalyzing the oxidative cleavage of the cyclopentane ring in loganin to form secologanin, was detected in microsomal preparations from cell suspension cultures of Lonicera japonica. The reaction required NADPH and molecular oxygen, and was blocked by carbon monoxide as well as by several other cytochrome P450 inhibitors, indicating that the reaction was mediated by cytochrome P450. Of the substrates examined, only specificity for loganin was demonstrated. A possible reaction mechanism is described.

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/MLL rearrangements 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) ort(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). 11q231MLL rearrangements 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.

Improvement in CNS protective treatment in non-high-risk childhood acute lymphoblastic leukemia: report from the Japanese Children's Cancer and Leukemia Study Group.

BACKGROUND: Prevention of central nervous system (CNS) leukemia by early introduction of therapy to this sanctuary site is an essential component of modern treatment strategy for acute lymphoblastic leukemia (ALL). However, the optimal form of preventive CNS therapy remains debatable. PROCEDURE: To address this issue, we evaluated the efficacy of CNS preventive therapy for 572 children with ALL who achieved complete remission in the Children's Cancer and Leukemia Study Group (CCLSG) ALL874 (1987-1990) and ALL911 (1991-1993) studies. They received risk-directed therapy based on age and leukocyte count. In the ALL 874 study, the non-high-risk (low-risk [LR] + intermediate risk [IR]) patients were randomly assigned to the conventional cranial irradiation (CRT) regimen (L874A and I874A) and the high-dose methotrexate (HDMTX) regimen without CRT (L874B and I874B). The former patients received 18-Gy CRT plus 3 doses of intrathecal (i.t.) MTX and the latter patients received 3 courses of HDMTX at 2 g/m2 plus 13 doses of ITMTX (L874B) or 4 courses of HDMTX at 4.5 g/m2 plus 1 dose of ITMTX (I874B). RESULTS: The 7-year probabilities (+/- SE) of CNS relapse-free survival were 97.3% +/- 2.6% (L874A, n = 41) vs. 90.3% +/- 5.3% (L874B, n = 39) (P = 0.25) in the LR patients, and 100% (I874A, n = 55) vs. 78.5% +/- 6.5% (I874B, n = 54) (P = 0.002) in the IR patients. The corresponding disease-free survival (DFS) rates were 79.4% +/- 6.5% vs. 74.4% +/- 7.3% (P = 0.62) in the LR group and 63.3% +/- 6.8% vs. 58.3% +/- 7.2% (P = 0.66) in the IR group. Thus, the HDMTX regimen could not provide better protection of CNS relapse as compared with the CRT regimen, although their overall efficacy was not significantly different. In the ALL 911 study, intensive systemic chemotherapy with extended i,t, injections of MTX plus cytarabine achieved a high CNS relapse-free survival (98% +/- 1.9% at 7 years) and a favorable DFS (85.5% +/- 5% at 7 years) in the IR patients. The patients in the high-risk (HR) group in both ALL874 and ALL911 studies received the 18-Gy or 24-Gy CRT with intensive systemic chemotherapy. Their 7-year probabilities of CNS relapse-free survival ranged from 88% to 95%, among which the T-ALL patients had a risk of CNS leukemia, which was 3-4 times higher compared with B-precursor ALL patients. CONCLUSIONS: These results indicate that long-term intrathecal CNS prophylaxis as well as appropriate systemic therapy for the non-high-risk patients can provide protection against CNS relapse equivalent to that provided by cranial irradiation.

[Studies of childhood non-Hodgkin's lymphoma--treatment results with the CCLSG NHL 960 protocol. Children's Cancer and Leukemia Study Group (CCLSG)].

We report here on the preliminary treatment findings of a CCLSG NHL 960 study that was initiated in March 1996. In this study, 37 patients with non-Hodgkin's lymphoma were assigned to 4 different treatment groups according to disease stage and histology: (1) localized disease; (2) advanced disease, lymphoblastic type; (3) advanced disease, large cell type; and (4) advanced disease, Burkitt type. The first three groups received the modified protocols of the NHL 890 study. Groups 1 and 3 received COPADM induction therapy (CPM, VCR, PRD, ADR, and MTX). After achieving remission, Group 1 received only maintenance therapy consisting of alternate administration of 7 drugs, while Group 3 received additional intensification therapy with combination chemotherapy consisting of MTX and Ara-C, followed by a maintenance phase involving the administration of 9 drugs. Group 2 received COPADL induction therapy (CPM, VCR, PRD, ADR, and LASP) and consolidation/intensification therapies followed by a maintenance phase. Group 4 received short-term intensive COPADM polychemotherapy. Twelve patients with localized with localized disease (stage I-II) and 25 patients with advanced disease (stage III-IV) were enrolled in this study. Except for 2 patients in the advanced disease stages who died earlier in the course of the study, all patients remained in remission.

[Bone marrow relapse in high-risk pediatric patients with acute lymphoblastic leukemia: a comparison of relapse times and initial clinical features of patients on different protocols. Children's Cancer and Leukemia Study group (CCLSG)].

To clarify the efficacy of modern intensive chemotherapy for ALL patients with unfavorable features, we compared the time to failure and initial clinical features of children who relapsed in the bone marrow or combined sites, as documented by early CCLSG studies (H811 and H851; 1981-1987) and later studies (H874 and H/HH911; 1987-1993) concerning high-risk ALL patients. In the later studies patients outcomes with new intensive regimens employing early intensification and reinduction therapy were apparently better than those of patients in the early studies with conventional regimens. When we compared the number of relapsed patients based on duration of first remission, we found that the improved outcomes for patients in the later studies were due to a decrease in the number who relapsed 7-36 months after the start of treatment (intermediate relapse), and that the percentage of those who relapsed within the first 6 months of therapy (early relapse) was higher. Patients with high initial WBC counts tended to relapse much earlier than those with low initial WBC counts. However, in the later studies, patients with high WBC counts often relapsed after the termination of therapy (late relapse). These results suggest that the intensive chemotherapy regimens used in the later studies can prevent the development of drug resistant leukemic clones, except in extremely high-risk patients likely to relapse within the first 6 months of therapy.

[Treatment of children with non-Hodgkin's lymphoma with CCLSG NHL 855/890 protocols long-term outcome and incidence of secondary malignancies].

We report here on treatment results of consecutive CCLSG NHL studies (NHL855, 1985-1989; NHL890, 1989-1996). The NHL855 protocol consisted of an induction phase of five drugs (VCR, PRD, CPM, DXR, and high-dose MTX) and a maintenance phase of 7 drugs. The probabilities of EFS at 7 years were 78% (SE, 10%) for the patients with localized disease, and 38% (SE, 7%) for those with advanced disease. In the NHL 890 protocol, the patients were assigned to two different treatment groups according to their histology and received different consolidation therapy; non-lymphoblastic subtype was treated almost identically to NHL855 while LASP and VP-16 were newly added for the lymphoblastic subtype. The 7-year EFS improved to 91% (SE, 6%) for localized disease, and 61% (SE, 6%) for advanced disease. A remarkable improvement was particularly evident for lymphoblastic type with mediastinal mass. Optional trial of high-dose sequential chemotherapy and peripheral blood progenitor cell auto grafting resulted in an unfavorable outcome. The 7-year EFS according to main histological subgroups were as follows: 84% (10%) for large cell type, 67% (11%) for Burkitt's-type, 58% (10%) for lymphoblastic type. Secondary cancer occurred in two of the 163 patients studied. Both patients were AML (M0/M4) and MLL rearrangement was detected in the M4 case.

Bcl-2 expression and prognosis in childhood acute leukemia. Children's Cancer and Leukemia Study Group.

Bcl-2 expression and its prognostic value were evaluated in 42 children with acute leukemia. The Bcl-2 expression of the leukemic blast cells was measured quantitatively by flow cytometry and was further analyzed by the simultaneous immunostaining of Bcl-2 with the surface membrane antigens, DNA, Ki-67 antigen. All of the cases showed a consistent expression of Bcl-2 protein; virtually all leukemic lymphoblasts were Bcl-2 positive. Although the expression of Bcl-2 varied widely from 7 to 80 x 10(3) MESF units, no significant difference was found in the mean value between the patients with acute lymphoblastic leukemia and those with acute myeloblastic leukemia. In more than half of the patients with AML, intraclonal heterogeneity of Bcl-2 expression was observed. The expression of Bcl-2 showed no apparent fluctuations during the different phases of the cell cycle. However, the proportion of Bcl-2-positive and -negative cells during the cell cycle was different between ALL and AML patients. In the ALL patients, few Bcl-2-negative cells were detected only in the GI phase, whereas in the AML patients Bcl-2-negative cells were detected in the S and G2/M phases, as well as in the G1 phase. No apparent difference was found in Bcl-2 expression between the Ki-67-negative noncycling population and the Ki-67-positive cycling population. Of the clinical features of these patients, only CD34 expression in the ALL patients was associated with high levels of Bcl-2 expression. In the 28 untreated cases of ALL, high expression of Bcl-2 was not an unfavorable factor for the outcome of this disease.

[Pilot study of relapsed osteosarcoma and brain tumor with ifosfamide, carboplatin and etoposide (ICE therapy)].

Ifosfamide, Carboplatin and Etoposide (ICE) therapy was used to treat 4 patients, 2 with refractory osteosarcoma, and one each with relapsed brain tumor and newly diagnosed brain tumor. ICE therapy was administered in doses of Ifosfamide 1,800 mg/m2 x 5, Carboplatin 400 mg/m2 x 2 and Etoposide 100 mg/m2 x 5. A total of 30 courses were administered. Two cases of osteosarcoma had a stable disease (range, 3-9 months) and 2 cases of brain tumor had a complete response by magnetic resonance imaging. Moderate or severe toxicity evaluated on a per course basis included: neutropenia 83%, thrombocytopenia 93%, fever 30%, hepatotoxicity 3%, and hemorrhagic cystitis 3%. The median time to hematologic recovery was 20 days. ICE therapy is highly effective for the treatment of refractory or recurrent solid tumors with acceptable toxicity.

[Cytogenetic abnormality and prognosis in childhood acute myeloblastic leukemia. Children's Cancer and Leukemia Study Group (CCLSG)].

We report on the leukemic cell karyotype of 180 children with acute myeloblastic leukemia (AML). They were treated by the protocols of chemotherapy for the Children's Cancer and Leukemia Study Group (CCLSG) in the last decade. Of 132 cases with adequate banding analysis, 24.2% had normal karyotype, 21.2% had miscellaneous clonal abnormalities and 54.6% were classified into known cytogenetic subgroups: t(8;21) (n = 35), t(15;17) (n = 23), inv (16) (n = 6), t(11q23;V) (n = 6), -7/7q-(n = 2). Each karyotype was closely correlated with a particular FAB subtype such as t(8;21) in M2, t(15;17) in M3, inv (16) in M4, t(11q23;V) in M5. In the M1+M2 group, although patients with t(8;21) had favorable clinical features such as low WBC counts and less frequent lymphadenopathy, their treatment outcome was not significantly better than those of patients with a normal karyotype (3-year EFS: 58 +/- 11% vs. 47 +/- 12%). Patients with miscellaneous chromosomal abnormalities had a significantly shorter EFS (22% +/- 10%) (p < 0.05) than those with t(8;21) or normal karyotype. In M4+M5 group, 2-year EFS of patients with inv (16) (40 + 30%) was longer than that of patients with normal karyotype (25 +/- 19%), and t(11q23;V) or miscellaneous chromosomal abnormalities (0 +/- 25%). These results suggest that cytogenetic data may be useful for risk-based treatment assignments for children with AML.

Treatment outcome and prognostic factors in childhood acute myeloblastic leukemia: a report from the Japanese Children's Cancer and Leukemia Study Group (CCLSG)

Treatment outcome and prognostic factors were evaluated in 152 children with acute myeloblastic leukemia (AML) treated on three consecutive protocols (ANLL 861, 8912, 9205) of the Children's Cancer Leukemia Study Group (CCLSG, Japan). In the ANLL 9205 protocol, anthracycline was used with a continuous infusion of cytosine arabinoside, followed by an intensive sequential post remission chemotherapy of short duration. Forty-two of these 46 patients (91.3%) achieved complete remission, and 58.8% of these patients projected a 3-year disease-free survival. These results were apparently superior to those obtained with the ANLL 861 and 8912 protocols, which used conventional doses of multiple drugs followed by a moderate post remission chemotherapy of long duration. This favorable response with the ANLL 9205 protocol was attributed mainly to the high induction rate of patients with the M4 and M5 FAB subtypes, as compared to those in the previous two protocols (93.3% in ANLL 9205 vs. 57.9% in ANLL 861 + 8912; P < 0.05). The ANLL 861 and 8912 protocols, an older age (> or = 8 years), higher WBC counts (> or = 10 x 10(9)/1) and all predicted an increased risk of relapse and decreased the survival following univariate analysis (P < 0.05). An older age and high WBC count continued to predict an increased risk of relapse in multivariate analyses: patients with an age > 8 years and WBC counts > 10 x 10(9)/1 had a 4.5 times higher risk of relapse than patients without these adverse features.

[Prognostic implication of DNA contents on long-term outcome of childhood acute lymphoblastic leukemia].

Prognostic value of cellular DNA content was evaluated in 189 children with acute lymphoblastic leukemia. Treatment outcome of the three different DNA index (DI) groups (Group A, DI = 1.0 vs. Group B, DI 1.01-1.15 vs. Group C, DI > or = 1.16) was compared between the two treatment risk groups (standard-risk and high-risk groups) stratified by the initial leukocyte count and age. In the standard-risk group, these groups had 10-year event free survival (EFS) rate (SE) of 62% (6%), 40% (21%) and 87% (6%), respectively (p < 0.05). In the high risk group, they had 10-year EFS rate of 30% (5%), 33% (27%) and 60% (19%), respectively (p < 0.01). Use of the DI, leukocyte count and age may be sufficient to distinguish the patients with an extremely low risk of failing to the standard ALL therapy from the patients with a relatively high-risk of treatment failure.

[Treatment results in childhood acute myeloblastic leukemia--a report of clinical trials of a past decade from the Japanese children's Cancer and Leukemia Study Group (CCLSG)].

Treatment results were evaluated in 167 children with acute myeloblastic leukemia (AML) treated on four protocols (ANLL 861, 8912, 9205, APL-ATRA) of the Children's Cancer Leukemia Study Group. In the ANLL 9205 protocol, anthracycline was used with a continuous infusion of cytosine arabinoside, followed by an intensive sequential post remission chemotherapy of short duration, 42/46 patients (91.3%) achieved complete remission, and 58.8% of these patients projected a 3-year disease free survival. These results were apparently superior to those obtained with the ANLL 861 & 8912 protocols, which used conventional doses of multi drugs followed by a moderate post remission chemotherapy of long duration. This favorable response with the ANLL 9205 protocol was attributed mainly to the high induction rate of patients with the M4 and M5 FAB subtypes, as compared to those in the previous two protocols (91.3% in ANLL 9205 vs 57.9% in ANLL 861 + 8912; p < 0.05). No significant difference in the patients outcome was found between the chemotherapy group and allogenic bone marrow transplantation group in the ANLL 9205 study. The patients with the M3 FAB subtype treated with the APL-ATRA protocol which consisted of an alternative use of all-trans retinoic acid and chemotherapy significantly prolonged event free survival as compared with the patients treated with ANLL 861/8912 protocols without all-trans retinoic acid.

[Pharmacokinetic studies of all-trans retinoic acid (ATRA) and pilot study of intermittent schedule of ATRA and chemotherapy in childhood acute promyelocytic leukemia. Children's Cancer and Leukemia Study Group].

A pharmacokinetic study of all-trans retinoic acid (ATRA) was performed in 8 patients with various types of leukemia and MDS. After oral administration at a dose of 30 mg/m2, the mean peak plasma concentration was 430 ng/ml and was reached at 150 min. In one patient who failed to respond a very low plasma ATRA level was seen. Though the plasma ATRA exposure decreased significantly with daily drug administration, an intermittent schedule of ATRA administration would yield higher plasma drug concentrations. We treated 2 patients with refractory acute promyelocytic leukemia (APL) in a pilot study of ATRA followed by intensive chemotherapy (APL-ATRA protocol). Two patients successfully achieved complete remission with ATRA after failing under conventional chemotherapy. Based on the pharmacokinetic study of ATRA, an intermittent schedule of ATRA in addition to chemotherapy suggests an effective regimen for children with APL. Phase II trials to evaluate the role of intermittent schedules of ATRA are planned in Children's Cancer and Leukemia Study Group.

[Treatment results of intermittent and cyclic regimen with ATRA and chemotherapy in childhood acute promyelocytic leukemia. Children's Cancer and Leukemia Study Group].

An intermittent and cyclic regimen with All-Trans Retinoic Acid (ATRA) and intensive chemotherapy was conducted due to pharmacokinetic studies on ATRA for acute promyelocytic leukemia (APL) in children. We have treated 17 children with APL using ATRA for remission induction followed by an intermittent schedule of ATRA plus intensive chemotherapy (APL-ATRA protocol). There were 10 males and 7 females. The median age was 9.0 years old. The median baseline white blood cell count was 12.1 x 10(3)/microliter, hemoglobin 7.8 g/dl, platelet 4.5 x 10(4) microliters at diagnosis. Sixteen patients showed t(15; 17) translocation. RT-PCR analysis was available in 15 patients and showed PML/RAR alpha rearrangement in all patients. Overall, 13 or 17 newly diagnosed patients (88%) achieved complete remission and EFS was 67%. Compared to the control (same chemotherapy without ATRA regimen), remission induction and EFS were significantly increased. The toxicity of ATRA consisted of retinoic acid syndrome in 1 and pseudotumor cerebli in another. Other toxicities included headache, chelitis, gastrointestinal trouble and bone pain. These results suggest that intermittent and cyclic regimen with ATRA and intensive chemotherapy (APL-ATRA protocol) is highly effective for APL patients.

[Antiemetic efficacy of granisetron in pediatric cancer treatment--(2). Comparison of granisetron and granisetron plus methylprednisolone as antiemetic prophylaxis].

A crossover clinical trial was carried out to compare the effectiveness and safety of granisetron alone (40 micrograms/kg) with that from a combination of granisetron plus methylprednisolone (MPL, 10 mg/kg) for control of emesis and vomiting induced by anticancer drugs in children with cancer. Complete control of emesis and vomiting were achieved in 95% (19/20 cases) of patients receiving the combination compared to 85% (17/20 cases) of patients receiving granisetron alone. There were no clinical toxicities or side effects in either treatment group. These data indicated that the combination of granisetron plus MPL was superior for control of emesis and vomiting in children receiving cytostatic anticancer drugs.