Accumulation of methotrexate and methotrexate polyglutamates in lymphoblasts and treatment outcome in children with B-progenitor-cell acute lymphoblastic leukemia: a Pediatric Oncology Group study.

We reported that children with B-progenitor-cell acute lymphoblastic leukemia (BpALL) treated in the early 1980s whose lymphoblasts accumulated high levels of methotrexate (MTX) and of methotrexate polyglutamates (MTXPGs) in vitro had an improved 5-year event-free survival (EFS) (65% (standard error (s.e.) 12%) vs 22% (s.e. 9%)). We repeated this study in children with BpALL treated in the early 1990s. The major change in treatment was the addition of 12 24-h infusions of 1 g/M2 MTX with leucovorin rescue (IDMTX). In 87 children treated on Pediatric Oncology Group (POG) study 9005 and POG 9006, the 5-year EFS for those whose lymphoblasts accumulated high levels of MTX and MTXPGs (79.2%, s.e. 8.3%) was not significantly different from that of patients with lesser accumulation of MTX and MTXPGs (77.7%, s.e. 5.4%). These findings support the notion that higher dose MTX therapy has contributed to increased cure, particularly for patients whose lymphoblasts accumulate the drug less well.

The association of the TEL-AML1 chromosomal translocation with the accumulation of methotrexate polyglutamates in lymphoblasts and with ploidy in childhood B-progenitor cell acute lymphoblastic leukemia: a Pediatric Oncology Group study.

Lymphoblasts from children with B-progenitor cell acute lymphoblastic leukemia (BpALL) with chromosomal hyperdiploidy and with translocations affecting chromosome 12p11-13, accumulate high and low levels of methotrexate polyglutamates (MTXPGs), respectively. Recently a cryptic translocation, t(12;21) (p13;q22), has been demonstrated by molecular and fluorescence in situ hybridization techniques in this disease. The chimeric TEL-AML1 transcript, which has been associated with this translocation, can be detected in up to 25% of children with BpALL. We detected the TEL-AML1 and/or the AML1-TEL transcript in 30 (33%) of 91 patients studied. Levels of lymphoblast MTXPGs were lower in those with than in those without the TEL-AML1 translocation (P = 0.004). Hyperdiploidy was rare in lymphoblasts with the TEL-AML1 translocation (P = 0.047). Both ploidy (P= 0.0015) and TEL-AML1 status (P= 0.0043) were independently and significantly correlated with the log of the lymphoblast MTXPG level. However, the presence of TEL-AML1 or of hyperdiploidy accounted for only 22% of the variation of this value. Our results imply that each of 1.16 > or = DI and the presence of the TEL-AML1 translocation confers a 50% decrease in lymphoblast MTXPG level. When planning reduction of therapy for either of the two excellent outcome categories of hyperdiploid or TEL-AML1 BpALL, one should consider the difference between these two subgroups in the ability of lymphoblasts to accumulate MTXPGs.

Cobalamin and folate deficiency: acquired and hereditary disorders in children.

This review highlights the features of cobalamin and folate deficiency and insufficiency that are particular to children. Maternal deficiency of cobalamin and insufficiency or deficiency of folate are the principal causes of deficiencies of these vitamins in the newborn. Maternal cobalamin deficiency can be caused by pernicious anemia or postgastrectomy, but most often results from a diet lacking in animal protein. The mothers are usually not anemic and failure to thrive and neurological deficits are more common in their infants than is megaloblastic anemia. Inborn errors of cobalamin transport and metabolism present with homocystinuria and methylmalonic aciduria, either alone or in combination. They share many of the clinical features of nutritional cobalamin deficiency. Maternal folate insufficiency results in neural tube defects, fetal loss, prematurity, and fetal growth retardation. Inborn errors of folate metabolism are rare, but polymorphisms affecting the gene for methylenetetrahydrofolate reductase (MTHFR) are common and may have significant health implications. Elevation of plasma methylmalonic acid (MMA) levels reflects a functional lack of cobalamin, whereas elevated total homocysteine levels are associated with a lack of either folate or cobalamin. The determination of these should be part of the investigation of failure to thrive, neurological disorders, and unexplained anemia or cytopenias in children.

Translocations involving chromosome 12p11-13, methotrexate metabolism, and outcome in childhood B-progenitor cell acute lymphoblastic leukemia: a Pediatric Oncology Group study.

Children with B-progenitor cell acute lymphoblastic leukemia whose lymphoblasts at diagnosis accumulate high levels of methotrexate (MTX) and MTX polyglutamates (MTXPGs) appear to have a good prognosis. This has been attributed to increased sensitivity of their blast cells to MTX. However, the proportion of children who are cured of B-progenitor cell acute lymphoblastic leukemia exceeds the number whose lymphoblasts accumulate high MTXPG levels. We report that lymphoblasts from patients with < 50 chromosomes who have translocations that involve the short arm of chromosome 12 accumulate low levels of MTXPGs. These patients appear to have an excellent survival because none of 14 patients with translocations affecting 12p has relapsed, 26-79 months following diagnosis.

Accumulation of methotrexate polyglutamates, ploidy and trisomies of both chromosomes 4 and 10 in lymphoblasts from children with B-progenitor cell acute lymphoblastic leukemia: a Pediatric Oncology Group Study.

Levels of accumulation of methotrexate polyglutamates were measured in vitro in lymphoblasts obtained at diagnosis from children with B-progenitor cell acute lymphoblastic leukemia (pro-B ALL). They were compared to numerical and structural chromosomal abnormalities present in these leukemic cells. In a series of 95 patients, the percent with high lymphoblast methotrexate polyglutamate levels increased with the increase in modal number of total chromosomes (p<0.001). Thus, lymphoblast methotrexate polyglutamate accumulation appeared to be closely linked to the extent of hyperdiploidy in childhood pro-B ALL. Lymphoblasts from 35 (88%) of the 40 children with hyperdiploid (>50 chromosomes) and 23 (88%) of 26 with hyperdiploid (DNA Index >1.16) pro-B ALL accumulated high levels of methotrexate polyglutamate, suggesting that they were more sensitive to methotrexate cytotoxicity. While children with hyperdiploid (DNA Index >1.16) pro-B ALL have a good prognosis, those with trisomies of both chromosomes 4 and 10, almost all of whom are hyperdiploid, have an even better outcome. There was no significant difference in methotrexate polyglutamate levels in lymphoblasts from 19 children with and 21 without trisomies of both chromosomes 4 and 10 (p = 0.25). The improved response to multi-agent chemotherapy conferred by the presence of trisomies of both chromosomes 4 and 10 in such patients may be due to increased sensitivity of their lymphoblasts to one or more anti-leukemic agents in addition to methotrexate.

Changes in red blood cell methotrexate pharmacology and their impact on outcome when cytarabine is infused with methotrexate in the treatment of acute lymphocytic leukemia in children: a pediatric oncology group study.

Since it is unclear whether methotrexate and cytarabine are synergistic or antagonistic in the treatment of acute lymphoblastic leukemia, the Pediatric Oncology Group studied the prognostic significance of a potential interaction between these agents. RBC methotrexate concentrations were compared from 140 patients at lower risk of relapse randomized to two treatment groups: one receiving six methotrexate infusions with overlapping cytarabine; the other, six methotrexate infusions alone. Samples from 248 patients from all risk groups were studied to determine whether patients with extremely low RBC methotrexate concentrations had inferior outcomes. Among low-risk patients studied 3 weeks after the sixth infusion, median RBC methotrexate concentrations were 0.13 nmol/ml RBCs (n = 71) for the methotrexate-only group and 0.02 nmol/ml RBCs (n = 69) for the methotrexate/cytarabine-treated low-risk patients, P < 0.001 by the two-sided Wilcoxon test. For low- and high-risk patients receiving methotrexate/cytarabine infusions, event-free survival at 1 and 3 years after RBC sampling was 97 +/- 2% and 90 +/- 3% for patients with concentrations greater than the median, and 88 +/- 3% and 78 +/- 4% for those with concentrations at or below the median. Log rank comparisons of event-free survival in the first year and overall yielded P = 0.005 and P = 0.04, respectively. Cytarabine altered methotrexate pharmacology when the drugs were infused together. Patients whose levels were extremely low had an adverse prognosis. Although this study could not assess efficacy of the methotrexate/cytarabine combination, it appears that concurrent administration is not optimal.

A phase I study of acivicin in refractory pediatric solid tumors. A Pediatric Oncology Group study.

Forty-two patients with progressive solid tumors and brain tumors were entered in this Phase I study of the glutamine antagonist acivicin given intravenously over thirty minutes daily for five days. The major toxicities encountered were myelosuppression and central nervous system toxicity (nightmares and somnolence). The maximum tolerated dosage on this schedule was 26 mg/M2 daily for five days. Six patients including three patients with brain tumor had stable disease.

A phase I trial of fazarabine in refractory pediatric solid tumors. A Pediatric Oncology Group study.

Fazarabine is a synthetic analog of cytosine arabinoside and 5-azacytidine that incorporates structural features of both compounds. Xenograft studies showed good activity against a variety of transplanted tumors. Initial studies in adults employed both a continuous infusion schedule and a daily bolus x 5 schedule. Myelotoxicity, especially neutropenia, was dose-limiting, with excessive myelotoxicity seen on the daily bolus x 5 at 72 mg/M2/day. Since short infusions may be administered in Ringer's lactate rather than either dimethylsulfoxide or dimethylacetamide required for continuous infusion, this study examined a daily x 5 schedule in children with refractory solid tumors. The initial dosage was 30 mg/M2/day, 80% of the maximum tolerated dosage in adults, with subsequent 30% dosage escalations. A total of 18 patients were enrolled, with a wide spectrum of pediatric solid tumors. Myelosuppression was the only significant toxicity, and was excessive at 78 mg/M2/day. Therefore, on this bolus regimen, 65 mg/M2/day for 5 days was the maximum tolerated dosage. One patient with medulloblastoma had stable disease for 65 days. No other responses were seen.

High glucocorticoid receptor content of leukemic blasts is a favorable prognostic factor in childhood acute lymphoblastic leukemia.

We have previously shown that the number of glucocorticoid receptors (GR) per cell in malignant lymphoblasts from children with newly diagnosed pre-B- and early pre-B-cell acute lymphoblastic leukemia (ALL) has a positive correlation with the probability of successful remission induction (Quddus et al, Cancer Res, 45:6482, 1985). We report now on the long-term outcome for these patients treated on a single protocol with 3 different treatment arms, all of which included glucocorticoid pulses during maintenance therapy. GR were quantitated in leukemic cells from 546 children with ALL at the time of diagnosis. Immunophenotyping studies were performed on all specimens. Prior studies showed that in pre-B- and early pre-B-cell ALL, successful remission induction was associated with a median GR number of 9,900 sites/cell, whereas induction failure was associated with a median receptor number of 4,800 sites/cell. Long-term follow-up of these patients shows an association between higher GR number and improved prognosis. The 5-year event-free survival of 61.0% (SE 2.8%) for patients whose leukemic cells had greater than 8,000 receptors/cell and 47.3% (SE 3.3%) for those with less than 8,000 receptors/cell is significantly different (P < .001). This difference remains significant when adjusted multivariately for blast immunophenotype and clinical risk factors (P < .001) or for treatment type (P < .001). We conclude that GR number greater than 8,000 sites/leukemic cell is a favorable prognostic marker for children with acute lymphocytic leukemia. This finding offers deeper insights into molecular mechanisms of anti-leukemia therapy and suggests that manipulation of steroid receptor number might augment the antitumor response, thus opening new avenues for basic and clinical research.

Ifosfamide with mesna uroprotection and etoposide in recurrent, refractory acute leukemia in childhood. A Pediatric Oncology Group Study.

BACKGROUND: Ifosfamide has previously been shown to be active as a single agent and in combination with doxorubicin, etoposide, and teniposide in pediatric solid tumors and adult acute leukemia. The authors performed a dose-escalation trial of ifosfamide with a fixed dosage of etoposide, with mesna uroprotection, in children with multiply recurrent acute leukemia. METHODS: Chemotherapy was administered daily for 5 days. Etoposide 100 mg/m2 was followed by ifosfamide at an initial dosage of 1.6 g/m2. The ifosfamide was escalated in 20% increments to the maximum tolerated dosage in cohorts of three patients. Mesna 400 mg/m2 was given immediately before the ifosfamide and then at 3 and 6 hours after ifosfamide in the initial patients. Subsequent patients were treated with mesna 400 mg/m2 just before ifosfamide, and then every 2 hours to a total dosage equal to the ifosfamide dosage. RESULTS: Forty-four heavily pretreated patients were entered on study. Forty were evaluable for toxicity and 36 for response as well. The maximum tolerated dosage of ifosfamide was 4.0 g/m2/d for 5 days (20 g/m2/course). Overall, 10 patients achieved complete remission, and 3 achieved partial remission. Remissions were brief, although four patients went on to bone marrow transplant while in remission. One patient is still alive. CONCLUSIONS: The combination of etoposide and ifosfamide with mesna uroprotection showed promising activity in children with multiply recurrent acute leukemia.

Cloning of human cDNAs encoding mitochondrial and cytosolic serine hydroxymethyltransferases and chromosomal localization.

Human cDNAs for cytosolic and mitochondrial serine hydroxymethyltransferase (SHMT) were cloned by functional complementation of an Escherichia coli glyA mutant with a human cDNA library. The cDNA for the cytosolic enzyme encodes a 483-residue protein of M(r) 53,020. The cDNA for the mitochondrial enzyme encodes a mature protein of 474 residues of M(r) 52,400. The deduced protein sequences share a high degree of sequence identity to each other (63%), and the individual isozymes are highly homologous to the analogous rabbit liver cytosolic (92% identity) and mitochondrial (97% identity) SHMT isozymes (Martini, F., Angelaccio, S., Pascarella, S., Barra, D., Bossa, F., and Schirch, V. (1987) J. Biol. Chem. 262, 5499-5509; Martini, F., Maras, B., Tanci, P., Angelaccio, S., Pascarella, S., Barra, D., Bossa, F., and Schirch, V. (1989) J. Biol. Chem. 264, 8509-8519). SHMT is a highly conserved protein with the human isozymes retaining about 43% sequence identity with the E. coli protein. The human cytosolic and mitochondrial SHMT genes were localized to chromosome regions 17p11.2 and 12q13, respectively. The high degree of nucleotide sequence identity between the two isozymes, and the presence of keratin genes in both chromosomal regions, is consistent with these regions of chromosome 12 and 17 arising by a duplication event.

Accumulation of high levels of methotrexate polyglutamates in lymphoblasts from children with hyperdiploid (greater than 50 chromosomes) B-lineage acute lymphoblastic leukemia: a Pediatric Oncology Group study.

Hyperdiploidy (greater than 50 chromosomes, or a DNA index greater than 1.16) confers a favorable prognosis in B-lineage acute lymphoblastic leukemia of childhood. Children with B-lineage acute lymphoblastic leukemia whose lymphoblasts at diagnosis accumulate high levels of methotrexate (MTX) and MTX polyglutamates (MTXPGs) in vitro experience a better event-free survival than those whose lymphoblasts do not (Blood 76:44, 1990). Lymphoblasts from 13 children with hyperdiploidy (greater than 50 chromosomes) accumulated high levels of MTX-PGs (1,095 and 571 to 2,346 pmol/10(9) cells [median and 25% to 75% intraquartile range]). These levels were higher than those in B-lineage lymphoblasts from 19 children with other aneuploidy (326 and 159 to 775 pmol/10(9) cells) and 15 children with diploidy (393 and 204 to 571 pmol/10(9) cells) (P = .0015). Chromosomal trisomies in hyperdiploid cases were highly nonrandom. Chromosome 9 was not one of the chromosomes involved in trisomies, even though this chromosome contains the gene for folate polyglutamate synthetase, which is the enzyme required for MTXPG synthesis. The correlation between MTXPG level and percentage of S-phase cells was weak, suggesting that increased levels of MTXPGs could not be attributed to elevated proportions of cells in active DNA synthesis. The ability of hyperdiploid lymphoblasts to accumulate high levels of MTXPGs may increase their sensitivity to MTX cytotoxicity, accounting in part for the improved outlook for hyperdiploid patients treated with regimens that emphasize MTX as a primary component of continuation therapy.

Red blood cell methotrexate and folate levels in children with acute lymphoblastic leukemia undergoing therapy: a Pediatric Oncology Group pilot study.

We enrolled children with acute lymphoblastic leukemia (ALL) in a Pediatric Oncology Group (POG) pilot study to monitor erythrocyte (RBC) methotrexate (MTX) and folate (F) levels before and during treatment. The mean value for RBCF at diagnosis was 0.86 +/- 0.46 nmol/ml RBC in the 214 patients who achieved remission and 1.21 +/- 0.74 nmol/ml RBC in the 10 patients who did not (P = 0.020). Folate levels tended to increase during remission induction, but they dropped following an intensive consolidation with methotrexate to levels that were sustained throughout chemotherapy treatment. Methotrexate levels reached mean values of approximately 0.15 nmol/ml RBC at the end of an intensive methotrexate consolidation, then fell to levels that were sustained throughout maintenance therapy. There was a weak correlation between improved event-free survival and higher RBCMTX levels after consolidation, but no correlation was found between improved survival and the level of RBCMTX or RBCF during maintenance therapy. A larger study with more complete data is needed to determine whether RBCMTX or RBCF might be useful in predicting event-free survival in patients with ALL.

Recombinant interferon alfa given before and in combination with standard chemotherapy in children with acute lymphoblastic leukemia in first marrow relapse: a Pediatric Oncology Group pilot study.

Recombinant interferon alfa (rIFN-alpha) was given to 31 children with acute lymphoblastic leukemia (ALL) in first on-therapy marrow relapse as the sole treatment (30 megaunits/m2/d intravenously x 10 days) before standard four-drug reinduction and during multiagent continuation therapy (30 megaunits/m2 subcutaneously x 3 consecutive days every 3 weeks). After 10 days of rIFN-alpha, there were two partial remissions (PRs); seven additional patients had either greater than or equal to 25% reduction in the percentage of marrow blast cells or hypoplastic marrow. Two patients had progressive disease with an increase in leukocyte counts. All patients experienced influenza-like symptoms, and there were isolated instances of severe abdominal pain and personality change. Dose-limiting toxicity comprised grade III/IV transaminase elevation (two patients) and syncope with personality change (one patient). Twenty-three of 31 children (74%) subsequently achieved marrow remission using standard agents. One patient was taken off study during teniposide (VM-26) and cytarabine (ara-C) consolidation due to toxicity. Continuation therapy including rIFN-alpha pulse was well tolerated in the remaining children; only one patient required rIFN-alpha dosage reduction (for CNS toxicity). rIFN-alpha toxicity did not necessitate reductions in doses of standard chemotherapy agents or significant delays in therapy. Five patients remain in remission at 26+ to 36+ months; 13 patients relapsed in marrow, one in the meninges (7 months), and one in meninges, mediastinum, and lymph nodes (2 months). Two children were removed from study for marrow transplant. In summary, high-dose rIFN-alpha alone had a modest antileukemic effect. In contrast to the clinical experience with combined rIFN-alpha and chemotherapy in adults, rIFN-alpha given in a pulse-like manner throughout continuation therapy did not compromise the intensity of the standard chemotherapy regimen.

Phase II study of 13-cis-retinoic acid in pediatric patients with acute nonlymphocytic leukemia--a Pediatric Oncology Group study.

Forty-one patients with refractory acute non-lymphocytic leukemia (ANLL) in relapse were treated with 13-cis-retinoic acid (cRA) as salvage therapy. The cRA was given as a single oral dose of 100 mg/m2/day for 4 weeks. One patient achieved a complete remission and two patients achieved a partial remission with reduction of the bone marrow blast count from 40 to 20% after the first course. We recommend further study of cRA in combination with other agents in the treatment of ANLL in children.

Accumulation of methotrexate and methotrexate polyglutamates in lymphoblasts at diagnosis of childhood acute lymphoblastic leukemia: a pilot prognostic factor analysis.

Lymphoblasts in bone marrow samples, obtained from 43 children with acute lymphoblastic leukemia at diagnosis, were incubated with 1.0 mumols/L [3H] methotrexate for 24 hours in vitro. Nonexchangeable methotrexate and methotrexate polyglutamates were separated and quantitated. Event-free survival at 5 years was 38% +/- 9% for all 43 patients (27 failures), and 44% +/- 10% for the 35 with non-T, non-B-cell acute lymphoblastic leukemia (20 failures). Of these 35 children, those whose lymphoblasts accumulated more than 100 pmol methotrexate and 500 pmol methotrexate polyglutamates per billion cells experienced better 5-year event-free survival than those whose lymphoblasts did not (65% +/- 12% v 22% +/- 9%, P = .010). This difference characterized "good-risk" patients who were female (P = .014), less than age 7 at diagnosis (P = .005), or had low initial white blood cell counts (less than 20 X 10(9)/L, P = .018). Findings were similar for the 43 children with acute lymphoblastic leukemia and for the "good-risk" children in this total group. Thus, the ability of lymphoblasts to accumulate methotrexate and form methotrexate polyglutamates may be important to the curative properties of current therapy of acute lymphoblastic leukemia in children, particularly for "good-risk" patients. In such patients, inherent rather than acquired drug resistance may be the initial event leading to treatment failure.

Phase I study of a 120-hour continuous intravenous infusion of 5-fluorouracil in pediatric patients with recurrent solid tumors: a Pediatric Oncology Group study.

To determine the maximum tolerated dose of 5-fluorouracil administered as a 120-hour continuous intravenous infusion to pediatric patients, we performed a phase I study using a starting dosage of 900 mg/m2/day. The maximum tolerated dosage (MTD) was 1,100 mg/m2/day. At this dosage level 40% of courses were complicated by grade 3 mucositis. Three additional patients were treated at the dosage level of 1,000 mg/m2/day after the MTD was determined. We recommend the dosage level of 1,000 mg/m2/day for phase II studies of 5-fluorouracil administered as a 120-hour continuous intravenous infusion to pediatric patients.

Phase I trial of indicine-N-oxide in children with leukemia and solid tumors: a Pediatric Oncology Group study.

A phase I trial of indicine-N-oxide was carried out in 12 children with solid tumors and in 16 with leukemia. Doses of 5, 6, and 7.5 g/m2 were given parenterally as a 15-min infusion every 3 weeks. The maximum tolerated dose in patients with solid tumors was 7.5 g/m2 and the dose-limiting toxicity was myelosuppression. In leukemia, the maximum tolerated dose was 6.0 g/m2 and hepatotoxicity was dose-limiting. Half of the children with leukemia showed elevations in transaminase levels and one child died of massive hepatic necrosis. This hepatotoxicity limits the use of indicine-N-oxide in children with leukemia. Antineoplastic activity was limited to a transient reduction in the numbers of circulating leukemic cells.