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Purpose: Many molecular pathways, including cell cycle control, angiogenesis, and drug resistance, mediate tumor growth and survival. Vascular endothelial growth factor-A (VEGF-A) serum levels 100 pg/mL have been associated with good and poor prognoses, respectively. Experimental Design: The hypothesis was that serum VEGF-A levels in standard-risk acute lymphoblastic leukemia pediatric patients at induction are predictive of event-free survival (EFS). One hundred seventeen patients were entered in CCG-1962 study and randomized into the native and polyethylene glycolated asparaginase arms. VEGF-A levels were quantified by an ELISA assay. Results: All patients had a decrease in VEGF-A levels by day 14 of induction, but they later dichotomized; EFS group levels remained low and event group levels increased. A correlation exists between high VEGF-A levels at entry to induction and time to event. Moreover, 6-year EFS patients have lower end of induction VEGF-A levels (28 ± 6 pg/mL) than event patients (>100 pg/mL; P < 0.01). Kaplan-Meier curves using various VEGF-A values were produced; with ≤30 at entry into induction (day 0) and ≤60 pg/mL at the end of induction (day 28), patients with low VEGF-A levels had superior EFS (P < 1e−4). Furthermore, patients who had an increase in VEGF-A during induction (ΔVEGF-positive, days 0-28) were more likely to have an event (P < 1e−4). Bifurcation by asparaginase treatment arm did not alter these results. Conclusions: These observations strongly support that high VEGF-A levels in induction are an asparaginase treatment–independent predictive marker for EFS. Hence, an anti-VEGF-A therapy should be tested in acute lymphoblastic leukemia.

Standard therapy in the United States for malignancy-associated hyperuricemia consists of hydration, alkalinization, and allopurinol. Urate oxidase catalyzes the enzymatic oxidation of uric acid to a 5 times increased urine soluble product, allantoin. Rasburicase is a new recombinant form of urate oxidase available for clinical evaluation. This multicenter randomized trial compared allopurinol to rasburicase in pediatric patients with leukemia or lymphoma at high risk for tumor lysis. Patients received the assigned uric acid-lowering agent for 5 to 7 days during induction chemotherapy. The primary efficacy end point was to compare the area under the serial plasma uric acid concentration curves during the first 96 hours of therapy (AUC0-96). Fifty-two patients were randomized at 6 sites. In an intent-to-treat analysis, the mean uric acid AUC0-96 was 128 ± 70 mg/dL.hour for the rasburicase group and 329 ± 129 mg/dL.hour for the allopurinol group (P

PURPOSE In preclinical studies, thioguanine (TG) has been shown to be more potent than the standard acute lymphoblastic leukemia (ALL) maintenance agent, mercaptopurine (MP), suggesting that TG may be more efficacious than MP in the treatment of childhood ALL. As part of a pilot trial in which TG was used in place of MP, we studied the plasma pharmacokinetics of oral TG and measured steady-state plasma and CSF TG concentrations during a continuous intravenous infusion (CIVI) in children with newly diagnosed standard-risk ALL. METHODS Nine plasma samples were collected after each patient's first 60 mg/m2 oral TG dose during maintenance. CIVI TG (20 mg/m2/h over 24 h) was administered during the consolidation phase of therapy, and simultaneous plasma and CSF samples were collected near the end of the infusion. TG was measured by reverse-phase HPLC with ultraviolet detection. Erythrocyte TG nucleotide (TGN) concentrations were measured 7 days after a course of CIVI TG and prior to the start of each maintenance cycle. RESULTS After oral TG (n = 35), the mean (+/- SD) peak plasma concentration was 0.46 +/- 0.68 microM and the AUC ranged from 0.18 to 9.5 microM.h (mean 1.5 microM.h). Mean steady-state plasma and CSF TG concentrations during CIVI (n = 33) were 2.7 and 0.5 microM, respectively. The mean (+/- SD) TG clearance was 935 +/- 463 ml/min per m2. Plasma TG concentrations did not correlate with erythrocyte TGN concentrations after oral or CIVI TG. The 8-OH-TG metabolite was detected in plasma and CSF. CONCLUSIONS TG concentrations that are cytotoxic to human leukemia cell lines can be achieved in plasma after a 60 mg/m2 oral dose of TG and in plasma and CSF during CIVI of TG.

Background In patients with persistent fever and neutropenia, amphotericin B is administered empirically for the early treatment and prevention of clinically occult invasive fungal infections. However, breakthrough fungal infections can develop despite treatment, and amphotericin B has substantial toxicity. Methods We conducted a randomized, double-blind, multicenter trial comparing liposomal amphotericin B with conventional amphotericin B as empirical antifungal therapy. Results The mean duration of therapy was 10.8 days for liposomal amphotericin B (343 patients) and 10.3 days for conventional amphotericin B (344 patients). The composite rates of successful treatment were similar (50 percent for liposomal amphotericin B and 49 percent for conventional amphotericin B) and were independent of the use of antifungal prophylaxis or colony-stimulating factors. The outcomes were similar with liposomal amphotericin B and conventional amphotericin B with respect to survival (93 percent and 90 percent, respectively), resolution of fever (58 percent and 58 percent), and discontinuation of the study drug because of toxic effects or lack of efficacy (14 percent and 19 percent). There were fewer proved breakthrough fungal infections among patients treated with liposomal amphotericin B (11 patients [3.2 percent]) than among those treated with conventional amphotericin B (27 patients [7.8 percent], P=0.009). With the liposomal preparation significantly fewer patients had infusion-related fever (17 percent vs. 44 percent), chills or rigors (18 percent vs. 54 percent), and other reactions, including hypotension, hypertension, and hypoxia. Nephrotoxic effects (defined by a serum creatinine level two times the upper limit of normal) were significantly less frequent among patients treated with liposomal amphotericin B (19 percent) than among those treated with conventional amphotericin B (34 percent, P Conclusions Liposomal amphotericin B is as effective as conventional amphotericin B for empirical antifungal therapy in patients with fever and neutropenia, and it is associated with fewer breakthrough fungal infections, less infusion-related toxicity, and less nephrotoxicity.

We prospectively assessed the pharmacokinetics of methotrexate, mercaptopurine, and erythrocyte thioguanine nucleotide levels in a homogenous population of children with lower risk acute lymphoblastic leukemia and correlated pharmacokinetic parameters with disease outcome. The maintenance therapy regimen included daily oral mercaptopurine (75 mg/m2) and weekly oral methotrexate (20 mg/m2). One hundred ninety-one methotrexate doses and 190 mercaptopurine doses were monitored in 89 patients. Plasma drug concentrations of both agents were highly variable. The area under the plasma concentration-time curve (AUC) of methotrexate ranged from 0.63 to 12 μmol•h/L, and the AUC of mercaptopurine ranged from 0.11 to 8 μmol•h/L. Drug dose, patient age, and duration of therapy did not account for the variability. Methotrexate AUC was significantly higher in girls than boys (P = .007). There was considerable intrapatient variability for both agents. Erythrocyte thioguanine nucleotide levels were also highly variable (range, 0 to 10 pmol/g Hgb) and did not correlate with mercaptopurine dose or AUC. A Cox regression analysis showed that mercaptopurine AUC was a marginally significant (P = .043) predictor of outcome, but a direct comparison of mercaptopurine AUC in the remission and relapsed patient groups failed to show a significant difference. Methotrexate and mercaptopurine plasma concentrations and erythrocyte thioguanine nucleotide levels were highly variable, but measurement of these pharmacokinetic parameters at the start of maintenance will not distinguish patients who are more likely to relapse.

PURPOSE Because there is a compelling need to develop new agents for intrathecal use, we investigated the safety, efficacy, and CSF pharmacokinetics of diaziquone (AZQ) following intrathecal administration in patients with refractory meningeal malignancies. PATIENTS AND METHODS Thirty-nine patients received 45 courses of intrathecal AZQ. Two schedules were studied; twice-weekly administration of a 1- or 2-mg dose and "concentration times time" (C x T) administration of 0.5 mg every 6 hours for three doses, administered once weekly. RESULTS Dose-limiting toxicity consisting of headache, nausea, or vomiting occurred in only three patients and only at the 2-mg, twice weekly dose. The schedules of 1 mg twice-weekly and 0.5 mg every 6 hours for three doses were well tolerated. Thirty-seven courses were assessable for response. The overall response rate was 62%. Complete responses (CRs) occurred in 14 of 37 courses (38%) and partial responses (PRs) occurred in nine of 37 courses (24%). Among patients with meningeal leukemia, CRs were observed in 11 of 26 courses (42%) and PRs in nine of 26 courses (35%). There was no difference in response rate related to dose or schedule. The pharmacokinetic behavior of intrathecally administered AZQ was characterized by biexponential disappearance from ventricular CSF, with mean half-lives of 18.2 and 78.6 minutes. The mean clearance rate was 0.37 mL/min. CONCLUSION Intrathecal AZQ is safe, well tolerated, and highly active against refractory meningeal malignancies.

Radiation nephritis is the principle late toxicity seen after total body irradiation in barrier-maintained rats when hematologic toxicity is prevented by bone marrow transplantation. Renal dysfunction is observed for single doses as low as 7.5 Gy. Hepatic blood flow, as measured by indocyanine green clearance, is decreased after 8.5–9.5 Gy single-dose total body irradiation. Serum albumin levels are decreased after 9.5 Gy single-dose total body irradiation. Hypoalbuminemia is a symptom of hepatic damage, but can also be caused by renal damage or edema. No decrease in total serum protein is observed, indicating that proteinuria resulting from renal damage is not the cause of hypoalbuminemia. No edema and some dehydration are observed. These data indicate that hepatic damage as well as renal damage may be occurring after total body irradiation plus bone marrow transplantation. Animals given total body irradiation plus bone marrow transplantation show decreased tolerance to a wide variety of immunosuppressive and cytotoxic drugs, even when these drugs are given months after total body irradiation. Altered drug clearance after total body irradiation plus bone marrow transplantation is observed for cis-platinum, vincristine, and adriamycin. The increase in cis-platinum toxicity after total body irradiation plus bone marrow transplantation is caused by decreased renal drug clearance. The decrease in vincristine tolerance and the alterations in adriamycin and vincristine pharmacokinetics are caused by altered drug distribution after total body irradiation plus bone marrow transplantation. These results indicate that bone marrow transplant survivors may show altered clearance of, and decreased tolerance to, a wide variety of drugs that are used after bone marrow transplantation.

Background Although mercaptopurine (MP) is conventionally used to treat childhood acute lymphoblastic leukemia (ALL), thioguanine (TG) is a more potent thiopurine in vitro and, when administered orally to patients, achieves cytotoxic drug concentrations in the cerebrospinal fluid (CSF). We performed a pilot study incorporating oral and 24-hr continuous IV infusion (CIVI) TG in children with newly diagnosed standard-risk ALL. Procedure Children with newly diagnosed standard-risk ALL (age 1–10 years, WBC

Purpose: To determine the antileukemic activity of weekly oral aminopterin in patients with refractory acute leukemia; to describe the pharmacodynamic properties of aminopterin; and to contrast the intracellular metabolism of aminopterin and methotrexate by patients' blasts in vitro. Experimental Design: Forty-six patients were enrolled in three strata: children with acute lymphoblastic leukemia (ALL), adults with ALL, and patients with acute myeloid leukemia (AML). Aminopterin was given weekly, in two doses of 2 mg/m2, 12 hours apart. Limited sampling pharmacokinetic analysis was done during the first week of therapy. Accumulation of [3H]aminopterin and [3H]methotrexate by leukemic blasts was studied in vitro. Results: Six of 22 children with ALL (27%; 95% confidence interval, 8-47%) had clinically significant responses. None of those with AML and only two of 11 adults with ALL had responses meeting protocol definitions, although peripheral blast counts tended to decrease with therapy in all groups. Mucosal toxicity was minimal, even with limited use of leucovorin rescue. Complete bioavailability of aminopterin was confirmed, with a mean area under the curve of 0.52 ± 0.03 μmol hour/L after oral dosing. No relationship between aminopterin pharmacokinetics and response was seen. In vitro, aminopterin showed more consistent metabolism by leukemic blasts to polyglutamates than methotrexate. Lineage-specific differences in the pattern of intracellular antifolylpolyglutamates were observed. Conclusions: Weekly oral aminopterin has significant activity among children with refractory ALL. With greater cellular accumulation and metabolism, more reliable bioavailability than methotrexate, and tolerable toxicity at this dose and schedule, aminopterin deserves further study as a potent alternative to methotrexate.

Purpose: Asparaginase therapy is an important component in the treatment of children with acute lymphoblastic leukemia. Polyethylene glycol-conjugated asparaginase (PEG-ASNase) has significant pharmacological advantages over native Escherichia coli asparaginase. We investigated the pharmacokinetics of PEG-ASNase, presence of antibodies to PEG-ASNase, and concentrations of asparagine in serum and cerebrospinal fluid (CSF) in combination chemotherapy for relapsed pediatric acute lymphoblastic leukemia. Experimental Design: Twenty-eight pediatric patients with relapsed medullary (n = 16) and extramedullary (n = 11) acute lymphoblastic leukemia were enrolled at three pediatric institutions and had at least two serum and CSF samples obtained for analysis. Patients received induction therapy (including PEG-ASNase 2500 IU/m2 intramuscularly weekly on days 2, 9, 16, and 23) and intensification therapy (including PEG-ASNase 2500 IU/m2 intramuscularly once on day 7). Serum samples were obtained weekly during induction and intensification. CSF samples were obtained during therapeutic lumbar punctures during induction and intensification. Results: Weekly PEG-ASNase therapy resulted in PEG-ASNase activity of >0.1 IU/ml in 91–100% of patients throughout induction. During intensification, PEG-ASNase on day 7 resulted in PEG-ASNase activity >0.1 IU/ml in 94% and 80% of patients on days 14 and 21, respectively. Serum and CSF asparagine depletion was observed and maintained during induction and intensification in the majority of samples. PEG-ASNase antibody was observed in only 3 patients. Conclusions: Intensive PEG-ASNase therapy in the treatment of relapsed acute lymphoblastic leukemia reliably results in high-level serum PEG-ASNase activity, and asparagine depletion in serum and CSF is usually achieved. Incorporation of intensive PEG-ASNase in future trials for recurrent acute lymphoblastic leukemia is warranted.

Purpose To determine the maximum tolerated dose, toxicity, and pharmacokinetics of gemcitabine in children with refractory solid tumors. Patients and Methods Gemcitabine was given as a 30-minute infusion for 2 or 3 consecutive weeks every 4 weeks, to 42 patients aged 1 to 21 years. Doses of 1,000, 1,200 and 1,500 mg/m2 were administered for 3 weeks. Subsequently, gemcitabine was given for only 2 consecutive weeks at 1,500, 1,800, and 2,100 mg/m2. Plasma concentrations of gemcitabine and its metabolite, 2′2′-difluorodeoxyuridine, were measured in 28 patients. Results Forty patients who received 132 courses of gemcitabine were assessable for toxicity. The maximum tolerated dose of gemcitabine given weekly for 3 weeks was 1,200 mg/m2. Dose-limiting toxicity was not seen in one-third of children treated at any doses given for 2 weeks. The major toxicity was myelosuppression in three of five patients at 1,500 mg/m2 for 3 weeks, and one of seven patients at 1,800 mg/m2 for 2 weeks. Other serious adverse events were somnolence, fever and hypotension, and rash in three patients. Gemcitabine plasma concentration–time data were fit to a one- (n = 5) or two-compartment (n = 23) open model. Mean gemcitabine clearance and half-life values were 2,140 mL/min/m2 and 13.7 minutes, respectively. One patient with pancreatic cancer had a partial response. Seven patients had stable disease for 2 to 17 months. Conclusion Gemcitabine given by 30-minute infusion for 2 or 3 consecutive weeks every 4 weeks was tolerated well by children at doses of 2,100 mg/m2 and 1,200 mg/m2, respectively.

Since the first report on cDNA cloning in 1972 (1), this technology has been developed into a powerful and universal tool in the isolation, characterization, and analysis of both eukaryotic and prokaryotic genes. But the conventional methods of cDNA cloning require much effort to generate a library that is packaged in phage or plasmid and then to survey a large number of recombinant phages or plasmids. There are three major limitations of those methods. First, a substantial amount (at least 1 μg) of purified mRNA is needed as starting material to generate libraries of sufficient diversity (2). Second, the intrinsic difficulty of multiple sequential enzymatic reactions required for cDNA cloning often leads to low yields and truncated clones (3). Finally, screening of a library with hybridization technique is time-consuming.

Isolation of a full-length gene on the basis of a limited sequence information is often troublesome and challenging. Tremendous effort is needed to isolate a specific gene by screening cDNA or genomic libraries by oligonucleotide or nucleic acid probes. In those methods, basically nucleic acid probes are used in a screening process to check whether or not a plaque or a colony contains the sequence of interest. There have been attempts to isolate specific DNA fragments using immobilized DNA, in which particular DNA fragments were enriched by hybrid selection and then the concentrated library was screened by a specific DNA probe (1,2). Recently, polymerase chain reaction (PCR) has been applied to the cloning of genes. Friedmann et al. (3) first used PCR to screen λgt11 library with two gene-specific primers. This protocol can be effectively used to isolate a particular DNA fragment between two specific primers or to generate nucleic acid probe from cDNA libraries. The unknown sequences flanking the fragment between the two specific primers cannot be amplified by this method.

The purpose of this pharmacoeconomic analysis was to compare pegaspargase. a newer chemotherapeutic agent used for treating acute lymphoblastic leukemia, with native Escherichia coli L-asparaginase in induction, delayed intensification 1 and delayed intensification 2.A subset of patients with newly diagnosed, standard-risk, acute lymphoblastic leukemia enrolled in the Children's Cancer Group (CCG) study CCG-1962 at seven participating institutions gave consent and was enrolled in our pharmacoeconomic analysis study. Societal (transportation, lodging, missed workdays, food, babysitter) and payer (frequency of encounters) cost data were collected from diaries (n = 27). Additional payer costs, such as drug costs, cost per clinic visit, and cost per inpatient day stay were collected from patients in CCG-1962 and participating institutions. We considered costs of therapy, including higher pegaspargase costs when comparing regimens of pegaspargase versus native E. coli L-asparaginase in induction, delayed intensification 1, and delayed intensification 2.Our results showed that the costs of the two therapies were similar from the payer perspective, with pegaspargase costing 1.8% more than E. coli L-asparaginase. The difference between groups also was small (1%) from the societal perspective. Inpatient stay accounted for 88% of pegaspargase payer costs and 91% of the native E. coli L-asparaginase costs.We recommend that pegaspargase not be withheld from treatment protocols solely because of its higher pharmacy costs.

For this study, 118 children with standard-risk acute lymphoblastic leukemia (ALL) were given randomized assignments to receive native or pegylated Escherichia coli asparaginase as part of induction and 2 delayed intensification phases. Patients treated with pegaspargase had more rapid clearance of lymphoblasts from day 7 and day 14 bone marrow aspirates and more prolonged asparaginase activity than those treated with native asparaginase. In the first delayed intensification phase, 26% of native asparaginase patients had high-titer antibodies, whereas 2% of pegaspargase patients had those levels. High-titer antibodies were associated with low asparaginase activity in the native arm, but not in the pegaspargase arm. Adverse events, infections, and hospitalization were similar between arms. Event-free survival at 3 years was 82%. A population pharmacodynamic model using the nonlinear mixed effects model (NONMEM) program was developed that closely fit the measured enzyme activity and asparagine concentrations. Half-lives of asparaginase were 5.5 days and 26 hours for pegaspargase and native asparaginase, respectively. There was correlation between asparaginase enzymatic activity and depletion of asparagine or glutamine in serum. In cerebrospinal fluid asparagine, depletion was similar with both enzyme preparations. Intensive pegaspargase for newly diagnosed ALL should be tested further in a larger population.

Cancer cells proliferate, invade surrounding tissues, and metastasize because they have escaped the normal mechanisms that control these processes [1,2]. Many of the genes that regulate these processes have now been isolated and characterized. Pharmacologic control of the expression of these genes would provide exciting new approaches to cancer chemotherapy.

An escalating-dose trial of idarubicin, used weekly for 3 doses in combination with vincristine, prednisone, and L-asparaginase (VPLI), to reinduce remission of childhood ALL at first bone marrow relapse was conducted by the Childrens Cancer Study Group (CCSG). The maximum tolerated dose (MTD) of idarubicin, used in the manner, was determined to be 12.5 mg/m2/dose. Twelve of 16 (75%) evaluable patients in first marrow relapse of ALL treated at a dose of 10 or 12.5 mg/m2 entered a second complete remission, compared to 41 of 69 evaluable patients (59%) treated in a comparable way with daunorubicin (30 mg/m2) (VPLD). Prolonged myelosuppression was observed in both groups, but the frequency of documented bacterial sepsis and the duration of required hospitalization were greater among patients treated with idarubicin. No additional toxicity, specifically attributable to idarubicin, was observed at these doses.

A potential new therapeutic approach to control gene expression is the use of double-stranded (ds) oligodeoxyribonucleotides (oligos) to compete for the binding of nuclear factors to specific promoter and enhancer elements. As a model, we have tested the effect of oligo length, sequence and number of nuclear factor binding sites on in vitro transcription of adenovirus (Ad) Elb. Short ds oligos containing an SPI-binding sequence (spl) inhibited transcription of Elb by more than 90%. Oligos containing multiple spl sequences were more effective inhibitors than would be expected for a comparable number of unlinked spl sites. A ds oligo with phosphorothioate (PS) linkages inhibited transcription at one-tenth the concentration needed for its normal homologue. An oligo with spl and a consensus TATA site was no more effective than one with spl alone. The stability of the PS-linked oligos will allow testing of this approach in vivo if they are adequately corporated into whole cells.

Introduction: The mechanisms of treatment failure in ALL are poorly understood. Attention has focused on the intrinsic resistance of the leukemic blast. ASNase contributes importantly to response and outcome in ALL. In about 1/3 of 1st relapse patients, we found inadequate ASN depletion, i.e., ASN > 3 mmoles on Day 14 of Induction (11 days following a single dose of pegylated ASNase) and an inferior re-induction rate (Pediatr Blood Cancer2006; 47:141). Patients with relapse have a superior re-induction rate with qwk rather than conventional qow pegylated ASNase (Blood2000; 96:1709). In the present study, we hypothesized that inadequate ASN depletion with standard ASNase dosing leads to relapse in newly diagnosed children with ALL. Methods: Between 1997 and 1998, 118 children with NCI standard risk ALL were enrolled on Children’s Cancer Group CCG-1962 study after informed consent (Blood2002; 99:1986). By random allocation, patients received either native ASNase 6,000 IU/m2 tiw × 9 in Induction and × 6 in each of 2 Delayed Intensification (DI) phases (n=59, 15 relapses, 1 2nd malignancy) or a single dose of pegylated ASNase 2500 IU/m2 (n=59, 10 relapses) in Induction and each of 2 DI phases. All ASNase was administered intramuscularly and begun on Day 3–5 of Induction and each DI phase. Multiple samples were obtained for each patient and cooled promptly to prevent ex-vivo deamination, which may lead to falsely low, not falsely high ASN values. We examined the incidence of relapse by 8 years in patients ranked by ASN concentration and ASNase activity. Results: The overall 4-year EFS is 84%. Overall, 17/ 31 patients (55%) in the highest Day 14 ASN tertile relapsed compared to 7/ 75 (9%) in the lower two tertiles (p Conclusion: In vivo, asparagine (ASN) depletion reflects a balance between ASNase activity and ASN production, which may vary for an individual over time and among individuals. ASN depletion on Induction Day 14, 9–11days after ASNase administration, predicts outcome in children with SR ALL receiving either native or pegylated ASNase. ASNase activity is not predictive. Failure to achieve adequate ASN depletion in Induction may contribute to relapse in childhood ALL. ASN Concentration Induction Day Pegylated ASNase Native ASNase Day 7 ASN μmoles Relapses/ total μmoles Relapses/ total 1st tertile > 1.14 3/ 16 > 0.99 6/ 16 2nd tertile 0.03–1.12 4/ 16 0.04–0.73 3/17 3rd tertile 0.01 2/ 19 0.01 4/23 DAy 14 ASN 1st tertile > 2.78 9/ 17 > 2.62 8/ 14 2nd tertile 0.18–2.68 0/ 17 0.23–0.27 3/ 15 3rd tertile 0.01–0.07 1/ 17 0.01 3/ 26 ASNase Activity Induction Day Pegylated ASNase Native ASNase Day 7 Activity IU/ml Relapses/ total IU/ml Relapses/ total 1st tertile ≤0.64 3/17 ≤0.23 5/19 2nd tertile 0.65–0.92 2/18 0.24–0.51 3/18 3rd tertile 0.93–1.63 4/17 0.52–1.13 5/19 DAy 14 Activity 1st tertile ≤ 0.31 2/17 ≤ 0.23 5/18 2nd tertile 0.34–0.66 4/18 0.24–0.42 4/19 3rd tertile 0.68–1.25 4/17 0.42–1.24 5/18

9026 Background: Many molecular pathways including cell-cycle control, angiogenesis & drug resistance mediate tumor growth and survival. Activation of Akt, PI3K and Ras can be induced by the activation of receptor tyrosine kinases, such as the VEGF-R and related growth factor receptors. VEGF-A serum levels 100 pg/ml have been associated with good and poor prognoses, respectively. The higher the VEGF-A levels the greater the chance of MRD blasts surviving chemotherapy. ALL blasts secrete VEGF-A and express VEGF-R. The hypothesis was that serum VEGF-A levels in SR ALL pediatric patients at entry of Induction (IND) are predictive of Event-Free Survival (EFS). Methods: 117 patients were entered in CCG-1962 study and were randomized into the Native & PEG-ASNase arms. VEGF-A serum levels in Induction were quantified by an ELISA assay. Results: . All patients had a decrease in VEGF-A levels by Day 14 of IND, but they later dichotomized; with EFS group levels remained low and Event group increasing. The VEGF-A levels at the end of IND remained unchanged in all patients in the DI1 and DI2 treatment phases. A linear relationship exists between high VEGF-A levels at entry to IND and time to Event. Moreover, 7 yr EFS patients have lower VEGF-A average levels of 28±2 pg/ml than Event patients, >100 pg/ml (P No significant financial relationships to disclose.

8539 Background: Although mercaptopurine (MP) is the thiopurine antimetabolite conventionally used in the treatment of childhood acute lymphoblastic leukemia (ALL), thioguanine (TG) is more potent ...

8556 Background. Recent reports demonstrated the incomplete Asn depletion in CSF (Appel et al., Leukemia, 2003), which verified our earlier findings (Avramis et al., Blood 2002). Methods. We examined the measured by HPLC amino acid assay CSF and concomitant serum Asn deamination in 20 out of 24 relapsed plus 28 randomly selected event-free (CCR) patients (pts) from CCG-1962 SR ALL study. Results. These analyses are shown below. Asn levels in CSF or serum are lower significantly in both arms after ASNase treatment, however neither ASNase formulation resulted in complete Asn depletion in these biological fluids. In addition, specific PD analyses were conducted to evaluate the impact of the Input of Asn in the PEG-ASNase arm pts, characterized by the biochemical parameters of Imax plus additive error from the host, i.e., nutrients & the de-novo biosynthesis by the liver. The Imax of Asn in serum was estimated to be statistically higher in pts with events (8.9E-3 ±6E-3 nmoles/ml/min), than in the randomly sel...

High-dose methotrexate (HDMTX) with leucovorin (CF) "rescue" is being investigated for treatment of many malignant tumors. CF is usually begun 2 hours after ending the HDMTX infusion. However, since CF and methotrexate compete for the same cellular transport system, at high extracellular methotrexate concentrations it may be impossible to "rescue" cells with CF. A regimen of HDMTX with delayed leucovorin "rescue" was therefore designed. In this program, a 6-hour infusion of methotrexate (7.5 gm/m2) was followed 24 hours later by leucovorin "rescue." Nine patients with osteogenic sarcoma received 115 courses of this treatment. Toxicity was minimal. Plasma methotrexate values were identical to those following early CF 'rescue" regimens. HDMTX with delayed "rescue" is well tolerated. Although theoretically sound, further studies are needed to determine its efficacy in comparison to standard early "rescue" regimens.

The analytical methodologies for the determination of free amino acids in plasma, serum, erythrocytes and leukemic cells are described. Deproteinization of the sample by methanol or organic acids is followed by derivatization with phenylisothiocyanate to form stable phenylthiocarbamylamino acid derivatives. The derivatives are separated by reversed-phase high-performance liquid chromatography in 80 min using a 5-microns C18 column (250 X 4 mm I.D.) and monitored by ultraviolet detection at 254 nm. Twenty physiological amino acids are resolved and quantified in plasma and erythrocyte samples. The resolution and sensitivity of the analytical method permitted unequivocal quantification of very low asparagine and glutamine levels in leukemic cells and growth media following treatment with asparaginase and glutaminase enzymes despite the presence of high aspartic and glutamic acid levels.

Cultured human pancreatic carcinoma cells (MIA PaCa-2) have been shown previously to be very sensitive to E. coli L-asparaginase (EC II). The present studies have demonstrated that another enzyme, Acinetobacter glutaminase-asparaginase (AGA) is much more effective in inhibiting cell growth. At the concentration of 0.0025 U/ml of AGA activity the enzyme totally inhibited cell growth, whereas the EC II with the same concentration did not show any effect. The inhibition of cell growth correlated well with inhibition of protein and glycoprotein synthesis. The addition of L-glutamine at the concentration of 1 mM completely reversed the inhibition of protein synthesis. Similarly, the addition of L-glutamine at the concentration of 3 mM daily on 3 successive days after adding AGA resulted in significant reversal of growth inhibition. The results of this study indicate that the action of AGA on MIA PaCa-2 is, to a great extent, exerted through its L-glutaminase activity.

Fourteen children (aged 3 to 14 years) with average-risk acute lymphoblastic leukemia were studied after an oral dose of 6-mercaptopurine (6-MP) (75 mg/m2) administered alone and, on the next day, concurrently with oral methotrexate (20 mg/m2). When 6-MP was administered alone, both the peak plasma concentration (15 to 150 ng X ml-1) and the AUC (36 to 340 ng X ml-1 X hr) were highly variable. Concurrent methotrexate resulted in a 31% increase in the AUC (P less than 0.01) and a 26% increase in peak plasma levels (P less than 0.05) of 6-MP. The AUC of methotrexate correlated with the degree of increase in 6-MP plasma concentrations. These findings are consistent with previous in vitro studies demonstrating that methotrexate is an inhibitor of xanthine oxidase, the enzyme that catabolizes 6-MP to the inactive metabolite thiouric acid. Although the increases in 6-MP AUC and peak plasma concentrations resulting from concurrent methotrexate administration were statistically significant, this interaction is probably not clinically significant at standard low oral doses of methotrexate in light of the wide interpatient variability in these pharmacokinetic parameters of 6-MP.

The effect of hepatic irradiation on adriamycin toxicity and pharmacokinetics was studied in 10 children who received adriamycin with concurrent abdominal irradiation for Wilms' tumor. Hepatic irradiation to 2400–2700 rad at 100–150 rad per fraction did not alter the clinical toxicity or plasma pharmacokinetics of adriamycin.

Ineffective erythropoiesis occurred during desacetyl vinblastine amide sulfate (VDS) therapy of a patient with metastatic Ewing's sarcoma. The peripheral blood was characterized by anisocytosis, poikilocytosis and reticulocytopenia. Bone marrow showed megaloblastic red cell hyperplasia with nuclear fragmentation, binuclearity and abnormal metaphases. Radioiron incorporation into red cells was markedly decreased. Electron microscopy showed red cell surface changes previously called ropalocytosis. Normal erythropoiesis promptly resumed when VDS treatment was withheld. Although less common than leukopenia, red cell abnormalities are part of the spectrum of vinca alkaloid toxicity.

Acinetobactor glutaminase-asparaginase was treated with [6-14C]diazo-5-oxonorleucine, reduced with sodium borohydride, and cleaved with cyanogen bromide. Radioactivity was present only in a 96-residue-N-terminal peptide which eluted as the second peptide peak on Sephadex G-50. Radioactivity was released with the threonine in position 12 during automatic sequencing of this peptide. The amino acid sequence of a 60-residue tn-terminal segment and a 16-residue C-terminal segment of this peptide was determined. Pseudomonas 7 A glutaminase-asparaginase was treated with [6-14C]diazo-5-oxonorleucine and reduced with sodium borohydride. Radioactivity was released with the threonine in residue 20 during automatic sequencing of the whole enzyme. Analysis of 26 N-terminal residues showed that an 8-residue segment containing the radioactive threonine was identical with that in Acinetobacter glutaminase-asparaginase and in Escherichia coli asparaginase. Additional identical residues were noted in the N-terminal regions of these enzymes.

Single crystals of glutaminase-asparaginase from two sources have been grown. A new crystal form (III) of Acinetobacter enzyme belongs to the space group I222, a = 96·7 , b = 112·4and c = 70·9 , with one subunit in the asymmetric unit. Crystals of Pseudomonas 7A enzyme belong to the space group P212121, a = 118·0 , b = 131·2and c = 85·1 , with a tetrameric molecule in the asymmetric unit. Both types of crystals are suitable for high resolution structure determination.

High concentrations of Escherichia coli asparaginase (80 U/ml) altered the binding of concanavalin A (Con A) to L 5178Y murine lymphoma cells that are sensitive to the cytotoxic action of this enzyme. Incubation of the asparaginase sensitive line in asparagine-free media or media containing Acinetobacter glutaminase-asparaginase did not alter the Con A binding of these cells. Escherichia coli asparaginase had no effect on Con A binding of two asparaginase resistant L5178Y cell lines that were isolated and maintained in asparagine depleted or asparaginase containing medium. The E. coli asparaginase preparation inhibited protein and glycoprotein biosynthesis to comparable degrees. It did not have proteolytic or glycolytic activity. Escherichia coli asparaginase did not alter the binding of wheat germ, soybean or ricin agglutinins to any of these cell lines. These data suggest that high concentrations of E. coli asparaginase have a specific effect on the Con A receptor in the sensitive line.

Acute lymphocytic leukemia (ALL) is the most common malignancy in childhood accounting for 35--40% of cancers in this age group. At the time of diagnosis approximately a trillion ( 1012) leukemic cells are present throughout the body (1). The bone marrow has been replaced and sup­ pressed by leukemic cells causing a cessation of normal blood cell produc­ tion. Previously both physicians and parents equated a diagnosis of acute lymphocytic leukemia with a certainty of rapid death. This dire prognosis has changed dramatically in the last 20 yeats. Now a complete bone marrow remission can be induced in 95% of children with ALL. Moreover, one half of these children will have a prolonged leukemia-free survival and probable cure. This chapter reviews the therapeutic advances that have led to this great improvement in survival, the current problems associated with this treat­ ment, and approaches to achieve more specific and effective therapy. Recent articles have summarized the first two topics (1-7), so we concentrate on new directions for therapy. Innovative new approaches are needed since the percentage of patients who achieve long-term leukemia·free survival has not increased appreciably in the last 10 years.

A highly sensitive and rapid assay for the detection of 6-mercaptopurine metabolites in the red blood cells of leukemic patients receiving the drug has been developed. The method employs a batch-chromatographic procedure using a mercurial cellulose resin to selectively absorb thiol compounds combined with separation by high-performance liquid chromatography using a Partisil-SAX column and uv detection. This method permits detection of 6-thioinosine monophosphate, 6-thiouric acid, and 6-thioguanosine mono-, di-, and triphosphates in patient samples with a sensitivity of 5–10 pmol. No 6-thioinosine di- or triphosphates were detected in patient samples. The results of our study indicate that 6-thioguanosine triphosphate is the major metabolite of 6-mercaptopurine retained by red blood cells after oral or iv administration of the drug.

Deoxycytidine kinase (dC kinase) is the rate-limiting enzyme in the anabolism of important anticancer and retroviral nucleoside derivatives. Its activity is often decreased in resistance to these drugs. To analyze the structure, function, and control of this clinically important enzyme we isolated 15 cDNA clones for human deoxycytidine kinase from lambda gt11 thymus and Molt 4 libraries. Four clones were sequenced. The largest clone is 2.9 kilobases and codes for a 626-amino acid open reading frame. The DNA and deduced amino acid sequence of the human dC kinase clones are homologous with a previously unidentified murine cDNA clone p3.4J (EMBL:MM34j) reported to be related to granulocyte-macrophage colony-stimulating factor. Deoxycytidine kinase also has cysteine-rich regions that are homologous with thioredoxin, the beta subunit of prolyl 4-hydroxylase, phosphoinositide-specific phospholipase C, thyroid hormone-binding protein, and protein disulfide isomerase. No differences were seen in the amount and size of deoxycytidine kinase protein and mRNA between CCRF/CEM and L1210 leukemic cell lines that express and do not express enzyme activity. Genomic restriction fragments were similar between the active and inactive CCRF/CEM cell lines. These data suggest that the cells deficient in dC kinase activity have a small defect in the structural gene.

A 2 1/2-year-old girl with acute lymphoblastic leukemia received chemotherapy and prophylactic cranial irradiation. After six years of remission, including three years off therapy, metastatic thyroid carcinoma appeared in the cervical lymph nodes. The predisposing factors for the development of thyroid carcinoma as a second malignancy in this case are discussed. It is suggested that thyroid carcinoma should be added to the growing list of second malignancies in acute lymphoblastic leukemia and that careful thyroid examination be included in the follow-up of long-term survivors.

Crystalline glutaminase-asparaginase with antitumor activity was prepared from an Achromobacteraceae soil isolate organism. This enzyme has l-glutaminase and l-asparaginase activity in a ratio of 1.2:1. The purification procedure provides an over-all yield of 40 to 60% from crude cell-free extract to homogeneous glutaminase-asparaginase and is adaptable to large scale isolation of the enzyme. Glutaminase-asparaginase is crystallizable from aqueous alcohol solutions in the absence of heavy metal cations. The highest yields of enzyme were obtained when cells were grown aerobically in a basal synthetic medium composed of l-glutamic acid, ammonium sulfate, trace minerals, and phosphate buffer. Glutaminase-asparaginase content remained relatively constant when the organism was at temperatures between 15 and 25°. Above 25° the enzyme content decreased with increasing temperature. The isoelectric point by isoelectric focusing of glutaminase-asparaginase on ampholytes is 8.43. The specific activity of homogeneous enzyme is 190 ± 20 i.u. per mg of protein and the E1%280 is 10.2. No carbohydrate or phospholipid was detected in the enzyme. No disulfide or sulfhydryl groups appear to be present on the enzyme. The Km values for l-glutamine and l-asparagine are 5.8 ± 1.5 x 10-6 and 4.8 ± 1.4 x 10-6 m, respectively. Glutaminase-asparaginase catalyzes the hydrolysis of the d isomers of glutamine and asparagine at about one-third the rate of the l isomers. The enzyme is not inhibited by ethyl-enediaminetetraacetate (0.1 mm), ammonia (10 mm), l-glutamate (30 mm), or l-aspartate (30 mm). 6-Diazo-5-oxo-l-norleucine which is not a substrate for the enzyme irreversibly inactivates glutaminase-asparaginase at very low concentrations. In contrast the l and d isomers of 5-diazo-4-oxonorvaline are attacked by the enzyme and are considerably poorer inhibitors.

The transformation of lymphocytes by phytohemagglutinin and the uptake of thymidine by DNA are completely inhibited by 17 mlU/ml L-glu taminase GA:1.2. The transformation is also inhibited by 1,700 ml

Acinetobacter glutaminase-asparaginase has been shown to consist of 4 subunits (molecular weight 33,000) by sedimentation equilibrium in 5.5 m guanidine HCl and electrophoresis in sodium dodecyl sulfate on polyacrylamide gels after cross-linking the protein with dimethyl suberimidate. Moving boundary velocity experiments showed that most of the native enzyme sediments as the tetramer (s20,w = 7.42 ± 0.03 S). On the other hand, equivalent boundary calculations always showed a smaller s20,w. Analytic sedimentation equilibrium experiments revealed a tetramerdimer dissociation with a dimer molecular weight of 69,000 ± 3000. The molecular weight on calibrated Sephadex G-200 and Bio-Gel P-200 was 97,000 and 93,000, respectively, which may indicate reversible dissociation. The hydrolysis of 5-diazo-4-oxonorvaline was used to determine the sedimentation coefficient of the active species. Sedimentation of the enzyme in 5-diazo-4-oxonorvaline showed complex patterns with ultraviolet optics due to protein absorbance. A new double sector cell was devised which allows layering of enzyme into both sectors simultaneously, cancelling the absorbance of the enzyme. The s20,w value for the species which degraded 5-diazo-4-oxonorvaline was 7.6 ± 0.2 S. By matching zone sedimentation and active enzyme experiments, enzyme species smaller than tetramer were shown to have 4% or less of the activity of the tetramer.

An enzyme that catalyzes the reversible hydroxylation of nicotinic acid to 6-hydroxynicotinic acid has been purified from extracts of a nicotinic acid-fermenting clostridium. The enzyme appears to be a flavin adenine dinucleotide-containing non-heme iron protein and utilizes triphosphopyridine nucleotide as the ultimate electron acceptor. The purified enzyme also exhibits reduced triphosphopyridine nucleotide oxidase and diaphorase activity.

Active enzyme sedimentation of five asparaginase and glutaminase-asparaginase enzymes with antitumor activity was studied. The catalytically active species of each enzyme appeared to have a molecular weight greater than 100,000 g/mole. Gel filtration and disc gel electrophoresis confirmed the absence of catalytically active smaller species.

The characteristics and partial purification of a clostridial enzyme that catalyzes the reversible reduction of 6-hydroxynicotinic acid to 6-oxo-1,4,5,6-tetrahydronicotinic acid are described. This enzyme requires reduced ferredoxin or reduced methyl viologen dye as the electron donor. The enzyme is extremely sensitive to inactivation by air and iron-chelating reagents.

The development of acquired resistance to antitumor agents is a major impediment to the cure of pediatric cancers and leukemias. The causes of drug resistance include: (1) defective drug transport, (2) defective drug metabolism to active species, (3) altered intracellular pools of nucleotides, (4) increased drug inactivation, (5) altered DNA repair, (6) gene amplification and (7) altered target proteins [1–3]. These mechanisms may be due to enhanced expression of specific genes as seen in cells that have amplification of dihyrofolate dehydrogenase (DHFR), adenosine deaminase, ribonucleotide reductase, and possibly pleotropic drug resistance, a condition where cells are resistant to anthracyclines, vinca alkaloids and dactinomycin [4–6]. Alternatively, resistant cells may have altered genes or reduced expression of the genes that code for transport proteins, drug receptors and enzymes that activate certain antitumor agents. In most cases of drug resistance, we do not know the mechanisms for this altered gene expression.

Publisher Summary This chapter presents the general assay methods and the properties of a representative, well-characterized enzyme from an Acinetobacter soil organism. Four assay methods are commonly used to measure the hydrolysis of glutamine or asparagine: (1) release of ammonia and direct nesslerization; (2) release of ammonia, distillation, and assay with a phenol-hypochlorate reagent; (3) isolation of the radiolabeled dicarboxylic acid on ion-exchange columns; and (4) coupling the formation of dicarboxylic acid to the disappearance of NADH. Asparagine is usually used as a substrate because of its greater stability. The purification procedure consists of several steps: (1) preparation of the crude extracts, (2) CM-Sephadex chromatography, (3) ammonium sulfate fractionation, and (4) DEAE-Sephadex chromatography. Several enzymes with glutaminase activity, optimal activity at physiologic pH, and antitumor activity have been isolated. These enzymes have an identical 8 amino acid segment that contains the threonine residue binding site for 6-diazo-5-oxonorleucine.

Desialised orosomucoid (α-1-acidic glycoprotein) was coupled to Pseudomonas 7A glutaminase-asparaginase by glutaraldehyde, iodinated and injected into mice. The half-life of radioactivity and glutaminase activity in plasma was about 7 min. Radioactivity and glutaminase activity in the liver reached a peak at about 20 min. The radioactivity in liver then declined with a half-life of about 20 min. Enzyme activity in liver declined with a half-life of about 10 min. The ratio of enzyme activity to radioactivity was lower in the liver than in plasma at all times during the experiment, indicating rapid hepatic inactivation of the enzyme. Uptake into the liver could be blocked by excess desialised orosomucoid. Glutamine levels in the liver were about 10% of normal for 44 min but returned to 50% of normal by 93 min. Intestines, kidney and spleen failed to exhibit any appreciable uptake of desialated orosomucoid glutaminase-asparaginase.