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.

Synthesis of methotrexate polyglutamates in L1210 murine leukemia cells.

The ability of L1210 mouse leukemia cells and of a mutant methotrexate-resistant cell line (L1210/MTX) to synthesize methotrexate polyglutamates was studied. Host DBA/2 mice were treated with methotrexate, after which leukemic cells were harvested from ascitic fluid and levels of methotrexate and metabolites in them were determined by Sephadex G-15 gel chromatography. The level of methotrexate in L1210/MTX cells was 12.5 times greater than that in L1210 cells, reflecting the increased level of dihydrofolate reductase that characterizes this mutant cell line. Synthesis of methotrexate polyglutamates in each cell line required a dose of methotrexate (2.4 mg/kg) 10 times greater than the dose that yielded extensive methotrexate polyglutamate synthesis in rat liver and kidney in previous studies. Total methotrexate polyglutamates synthesized in 4 hr with this dose were the same in each cell line, demonstrating that this metabolism was not affected by differences in the level of dihydrofolate reductase. Methotrexate polyglutamates comprised 47+/-20% of the total methotrexate in L1210 cells. Methotrexate diglutamate was the predominant form. Levels of methotrexate monoglutamate and diglutamate were similar in L1210/MTX cells, whereas methotrexate monoglutamate was the predominant metabolite in host liver, kidney, and small intestine. These differences may reflect differences in substrate preference of pteroylpolyglutamate synthetase in these different tissues. Twenty-four hr after methotrexate administration, total methotrexate in L1210 cells was one-third of that at 4 hr; but the proportion of metabolites was the same, presumably reflecting cell death and division rather than loss of a freely exchangeable portion of intracellular methotrexate present at the earlier time. The affinity of methotrexate polyglutamates for dihydrofolate reductase was found to be similar to that of methotrexate, providing evidence that these metabolites may be as potent antagonists of folate metabolism as is methotrexate itself. Recent studies indicate that inhibition of folate metabolism in cells requires their exposure to high levels of methotrexate in order to achieve intracellular levels of methotrexate greater than needed to bind to dihydrofolate reductase. Such conditions conform to those required for synthesis of methotrexate polyglutamates. Thus, these metabolites may play a specific role in inhibiting folate metabolism, distinct from the antifolate potential that they appear to share with methotrexate.

Tissue-specific synthesis of methotrexate polyglutamates in the rat.

Purified tritium-labeled methotrexate was administered to rats and Sephadex G-15 gel chromatography was used to study the formation of poly-gamma-glutamyl metabolites of methotrexate in different tissues. These metabolities were recognized as tritium-labeled antifolate fractions and were identified by their response to serum pterolypolyglutamate hydrolases and by cochromatography with authentic standards. After doses equivalent to 15 to 20 mg for man, rat liver and kidney were found to contain both 4-amino 10-methylpterolyglutamyl-gamma-glutamic acid and 4-amino-10-methylpterolyglutamyl-gamma-glutamyl-gamma-glutamic acid. With one-third of the dose, only 4-amino 10-methylpteroylglutamyl-gamma-glutamic acid was found. Synthesis of methotrexate polyglutamates in liver and kidney was limited to the interval immediately following methotrexate administration and appeared to occur by sequential addition of single glutamyl residues to so-called "free" intracellular methotrexate. No synthesis of methotrexate polyglutamates was demonstrated in small intestine or thymus. After formation, 4-amino-10-methylpteroyglutamyl-gamma-glutamic acid disappeared from liver and kidney with a half-time of 6.5 days. The effect of these metabolites of methotrexate of cell metabolism is unknown.

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.

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.

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.

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.

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.

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.

Methotrexate metabolism in mutant Chinese hamster ovary cells lacking dihydrofolate reductase.

To study the influence of the level of dihydrofolate reductase (DHFR) on methotrexate (MTX) metabolism, the formation of methotrexate polyglutamates (MTXPGs) and the retention of the drug were examined in Chinese hamster ovary cells (DUKXB11) lacking DHFR and in control cells (CHO-UTC). Both cells accumulated MTXPGs poorly. After a 24-hr incubation with 1.0 microM [3H]MTX, the level of total MTX in DUKXB11 cells was 40% of that in CHO-UTC cells, reflecting the lack of DHFR-bound MTX and MTXPGs in the mutant cells. MTXPGs accounted for a higher proportion of the intracellular MTX in DUKXB11 than in CHO-UTC cells (25 vs 18%). Following exposure to 3.0 microM MTX for 24 hr, total drug levels were similar in both cell lines, and MTXPGs constituted even more of the intracellular drug in DUKXB11 cells compared to CHO-UTC cells (34 vs 23%). DUKXB11 cells accumulated longer MTXPGs (MTXG1u3,4) compared to CHO-UTC cells (MTXG1u2,3), following exposure to both 1.0 and 3.0 microM MTX. The longer MTXPGs in the mutant cells may have resulted from the lack of DHFR in them. Binding of MTXPGs to DHFR in CHO-UTC may interfere with their further polyglutamylation. When cells were resuspended in drug-free buffer for 1 hr following a 24-hr incubation with MTX, the retention of drug was less in DUKXB11 cells (46%) than in CHO-UTC cells (78%), due mainly to a greater loss of unmetabolized MTX in the mutant cells (89%) than in control cells (26%). Nevertheless, the amount of non-exchangeable unmetabolized MTX retained in DUKXB11 cells following exposure to 3.0 microM MTX exceeded the MTX-binding capacity. These studies demonstrate that DHFR-deficient cells accumulated more and longer MTXPGs than control cells. In addition, they suggest that some unmetabolized MTX was retained in cells not bound to DHFR.

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.

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.

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.

Measurements of red blood cell methotrexate concentrations and lymphocyte subsets during therapy of rheumatoid arthritis.

Nine patients with rheumatoid arthritis were treated with low dose oral weekly methotrexate for 6 months. Successful therapy was not associated with changes in concentrations of total circulating lymphocytes nor with alterations of T lymphocytes in the helper-inducer, OKT4, or cytotoxic-suppressor, OKT8, subpopulations. Concentrations of methotrexate in circulating erythrocytes stabilized by 1 month of therapy and this measurement did not correlate with clinical efficacy or methotrexate toxicity in the long-term patient assessments.

Methotrexate polyglutamates in cultured human cells.

Cultured human fibroblasts accumulated methotrexate polyglutamates to levels far in excess of the dihydrofolate reductase binding capacity. After four days in methotrexate-free medium the intracellular drug level dropped by 70% but nearly 80% of the remaining methotrexate was in the form of polyglutamates. Reduced folates prevented the accumulation of polyglutamates and the effects of methotrexate on deoxyuridine incorporation into DNA and cell growth if present along with methotrexate from the beginning of the incubation. However, the reduced folates were less effective if added to cells after a long exposure to methotrexate alone. Thymidine, glycine, and adenosine (GAT) prevent methotrexate toxicity only if maintained in the incubation medium. However, preincubation with methotrexate and GAT permits continued synthesis and accumulation of polyglutamates so that when the GAT and methotrexate were removed, toxicity from the retained methotrexate polyglutamates could be expressed. (2,4-diamino-5-(3'4'-dichlorophenyl)-6 methyl pyrimidine (DDMP), an antifol that does not form polyglutamate derivatives, inhibited deoxyuridine incorporation into DNA as long as the DDMP remained in the culture medium. Compared to what was seen with longer exposures to methotrexate, removal of DDMP resulted in a greater reversal of the inhibition of deoxyuridine incorporation.

Methotrexate metabolism by bone marrow cells from patients with leukemia.

Bone marrow cells from children with leukemia in remission and lymphoblasts and myeloblasts from children with early and late-stage leukemia all accumulated methotrexate during short-term culture and converted it to poly-gamma-glutamyl derivatives. This metabolism was time and dose-dependent. Leukemia cells from two patients with chronic myelocytic leukemia also synthesized methotrexate polyglutamates. Patients varied one from another in the quantity of total non-exchangeable methotrexate and methotrexate polyglutamates present in cells, but highest levels of each of these were seen in late acute lymphoblastic leukemia and in acute myeloblastic leukemia. Co-incubation of leukemic cells with both methotrexate and a ten-fold excess of folinic acid decreased accumulation and polyglutamylation of methotrexate to the same extent as achieved by reducing methotrexate concentration ten-fold. Co-incubation with methotrexate and a ten-fold excess of vincristine did not increase total cell methotrexate and methotrexate polyglutamates in leukemic cells in culture. Such an increase had been anticipated from earlier studies with other cells. Indeed, levels of methotrexate and its derivatives were modestly reduced.

Rhabdomyosarcoma of the brain.

The authors present a case of primary intracranial rhabdomyosarcoma. This is only the 10th reported case and is the only one in which the patient has survived longer than 2 years. A 9-month-old boy was found to have a large mass in the right posterior fossa. Posterior fossa craniotomy revealed an unencapsulated tumour involving almost the entire right cerebellar hemisphere and extending to the right cerebellar pontine angle. Subtotal removal was done for internal decompression. On examination of the specimen by light microscopy there were definite sarcomatous features with occasional rhabdopoietic elements and many malignant giant cells. The ultrastructural appearance confirmed the diagnosis of malignant rhabdomyosarcoma. The child was treated with combination chemotherapy and cobalt-60 teletherapy. He is alive and well 2 years after operation and has no clinical evidence of recurrent disease. His physical growth and mental development are satisfactory. The response of our patient suggests that such tumours may be controlled by subtotal removal followed by radiotherapy and chemotherapy.

Chemotherapy of childhood acute lymphoblastic leukemia.

This article reviews current chemotherapy of childhood acute lymphoblastic leukemia, with particular emphasis on the pharmacology of the drugs used. In the perspective of the overall treatment plan, the use, mode of action, toxicity and pharmacology of prednisone, vincristine, L-asparaginase, cyclophosphamide, 6-mercaptopurine, methotrexate and cytosine arabinoside are reviewed. Issues relating to central nervous system prophylaxis, drug compliance, drug resistance, and treatment failure are considered.