Measurement of serum L-asparagine in the presence of L-asparaginase requires the presence of an L-asparaginase inhibitor.

The antileukemic activity of L-asparaginase (ASNase), an important component of therapy for acute lymphoblastic leukemia, is thought to result from depletion of serum L-asparagine (Asn). In studies of the pharmacological effects of ASNase, investigators have reported prolonged reduction in the serum concentration of Asn after the administration of ASNase. Such measurements may not be valid because ASNase present in the blood sample may hydrolyze Asn before its determination. We examined recovery of [U-14C]Asn from blood samples with and without various concentrations of added ASNase. In the presence of greater than or equal to 0.01 IU/ml of ASNase, the amount of [U-14C]Asn recovered was less than 15% of that without ASNase. Utilizing this assay, we studied the effect of 2 known inhibitors of ASNase in an attempt to improve Asn recovery. In the presence of aspartic beta semialdehyde (ASA), or 5-diazo-4-oxo-L-norvaline (DONV), and up to 1.0 IU/ml ASNase, Asn levels remained at greater than 90% of control. ASA prevented the hydrolysis of exogenous Asn in blood samples drawn from patients after ASNase injection. We also developed a method to determine Asn in serum utilizing high pressure liquid chromatography. Using this method, we found that the Asn level was greater than 90% of a normal level in the presence of 40 mM DONV and 1.0 IU/ml ASNase. Examination of serum from 4 patients treated with ASNase showed that Asn is detectable 7-19 days sooner when DONV is present in the blood collection system than in its absence. We conclude that: (a) as little as 0.01 IU/ml ASNase can hydrolyze Asn added to blood; (b) continued hydrolysis of Asn by ASNase ex vivo can result in falsely low serum Asn measurements; (c) ASA or DONV present in the collection tubes obviates the problem of continued ASNase activity; and (d) the degree and duration of Asn depletion after ASNase therapy is much less than previously believed. Thus, for accurate measurements of the duration and degree of Asn depletion by ASNase, an ASNase inhibitor such as ASA or DONV should be present in the blood collection system.

In vitro and in vivo killing of acute lymphoblastic leukemia cells by L-asparaginase.

L-Asparaginase (ASNase) is a potent antileukemic enzyme routinely used in the treatment of children with acute lymphoblastic leukemia. As part of investigations of the biological activity of ASNase, we have developed techniques which measure the in vitro and in vivo cell killing ability of ASNase. To study the effect of ASNase on in vitro survival of primary lymphoblasts, bone marrow mononuclear cells obtained from untreated patients with acute lymphoblastic leukemia were cultured with and without ASNase. After 5 days, viable cells were counted using trypan blue exclusion to calculate total cell kill due to ASNase. Propidium iodide exclusion, leukemia cell surface antigens, and flow cytometry were used to determine leukemia cell kill due to ASNase. Comparison of leukemia cell kill and total cell kill showed a direct linear relationship (n = 24, r = 0.7), preferential killing of leukemia cells by ASNase (slope = 0.66), and that use of leukemia cell surface markers yielded a more accurate measurement of leukemia cell killing. ASNase at concentrations from 0.0001 to 0.1 IU/ml had equal effects on extent of leukemia cell killing (P = 0.3 to 0.7), suggesting the absence of a dose response at the ASNase concentrations tested. As a measure of the in vivo response to ASNase treatment, the number of viable bone marrow leukemia cells in the patient prior to and 5 days after treatment with ASNase was measured as the product of (% of rhodamine 123 fluorescent [viable] cells) x (absolute leukemic infiltrate). The change which occurred in the viable leukemic infiltrate was the same for patients whether they received 25,000 or 2,500 IU/m2 of ASNase as a single drug. There was a linear correlation (n = 8, r = 0.9) between in vivo and in vitro leukemia cell killing by ASNase. Thus, the in vitro assay described here can be used to predict in vivo sensitivity to ASNase in acute lymphoblastic leukemia.

Comparative pharmacokinetic studies of three asparaginase preparations.

PURPOSE: As part of pharmacologic studies of asparaginase (ASNase), we determined the half-life of ASNase activity and protein, and the effect of dose, repeated doses, different drug preparations, and hypersensitivity reactions on the half-life (t1/2) of serum ASNase activity. PATIENTS AND METHODS: We measured ASNase activity (spectrophotometric assay) in serum samples obtained from patients with acute lymphoblastic leukemia (ALL) at various times during their therapy with intramuscular ASNase. ASNase protein was measured by enzyme-linked immunoadsorbent assay (ELISA). RESULTS: Studies following the initial dose of Escherichia coli-derived ASNase demonstrated no difference in apparent t1/2 following 25,000 IU/m2 versus 2,500 IU/m2 (1.24 v 1.35 days, P = .2). The apparent t1/2s following maintenance doses of E coli ASNase (middle dose t1/2, 1.28 days, or last dose t1/2, 1.14 days) showed no difference when compared with the initial dose of ASNase (P = .3 to .9). There was no significant difference between the apparent t1/2s of ASNase activity and ASNase protein (n = 8, P = .2 to .6). The serum t1/2 was 0.65 and 5.73 days for patients receiving Erwinia or polyethylene glycol (PEG)-modified E coli ASNase, respectively, as the induction dose. ASNase activity was undetectable in sera of four patients studied in the week following an anaphylactic reaction to E coli ASNase and the t1/2 was significantly shorter in five patients with a history of allergic reaction to E coli ASNase who were studied following a dose of PEG ASNase, (t1/2, 1.80 days). CONCLUSION: We conclude that (1) the apparent t1/2 of ASNase is dependent on enzyme preparation used, but is not affected by dose or by repeated use; (2) the apparent t1/2 of E coli ASNase as a protein is the same as the apparent t1/2 of enzymatic activity; and (3) patients who have had a hypersensitivity reaction to E coli ASNase have a decreased apparent t1/2 with both E coli and PEG ASNase.

Results of Dana-Farber Cancer Institute Consortium protocols for children with newly diagnosed acute lymphoblastic leukemia (1981-1995).

The Dana-Farber Cancer Institute (DFCI) ALL consortium has been conducting clinical trials in childhood acute lymphoblastic leukemia (ALL) since 1981. The treatment backbone has included intensive, multi-agent remission induction, early intensification with weekly, high-dose asparaginase, cranial radiation for the majority of patients, frequent vincristine/ corticosteroid pulses during post-remission therapy, and for high-risk patients, doxorubicin during intensification. Between 1981 and 1995, 1,255 children with newly diagnosed ALL were evaluated on four consecutive protocols: 81-01 (1981-1985), 85-01 (1985-1987), 87-01 (1987-1991) and 91-01 (1991-1995). The 5-year event-free survival (EFS) rates (+/- standard error) for all patients by protocol were as follows: 74 +/- 3% (81-01), 78 +/- 3% (85-01), 77 +/- 2% (87-01) and 83 +/- 2% (91-01). The 5-year EFS rates ranged from 78 to 85% for patients with B-progenitor phenotype retrospectively classified as NCI standard-risk, 63-82% for NCI high-risk B-progenitor patients, and 70-79% for patients with T cell phenotype. Results of randomized studies revealed that neither high-dose methotrexate during induction (protocol 87-01) nor high-dose 6-mercaptopurine during intensification (protocol 91-01) were associated with improvement in EFS compared with standard doses. Current studies continue to focus on improving efficacy while minimizing acute and late toxicities.

Toxicity, pharmacology and feasibility of administration of PEG-L-asparaginase as consolidation therapy in patients undergoing bone marrow transplantation for acute lymphoblastic leukemia.

We attempted to administer PEG-L-asparaginase (PEG-L-A) following hematologic recovery to 38 patients undergoing autologous or allogeneic marrow transplantation for acute lymphoblastic leukemia (ALL). Twenty-four patients (12 of 22 receiving allogeneic and 12 of 16 receiving autologous transplants) received between one and 12 doses of PEG-L-A, including nine who completed the planned 12 doses of therapy. The toxicities encountered were similar to those observed in non-transplanted patients undergoing therapy with PEG-L-A and included allergic reactions, pancreatitis, weight loss, hypoalbuminemia, and low levels of anti-thrombin III. Of the 24 who received the drug, eight remain in remission. Of 12 patients in second remission at the time of transplantation who received PEG-L-A, five of seven who received allogeneic and two of five who received autologous transplants remain in remission, 16+ to 46+ months from transplant. While PEG-L-A could be administered to most of the patients undergoing marrow transplantation for ALL, most patients either relapsed while receiving the drug or developed toxicities which resulted in abbreviated courses. At this time, we cannot recommend PEG-L-A as single agent, post-BMT chemotherapy.

Evaluation of tumor cell heterogeneity based on response to chemotherapy.

As the prototype for evaluation of tumor cell heterogeneity based on response to chemotherapy, several studies which examine the cells of the childhood leukemias are reviewed. These studies have examined the response to therapy of leukemic cells using a variety of clinical, in vivo and in vitro laboratory measures including: (1) response to combination chemotherapy in vivo as measured by numbers of residual leukemia cells observed within one to two weeks of initial therapy; (2) in vitro response to chemotherapy assessed in primary leukemic cell cultures exposed to antileukemic drugs.

Maternal disease as a consideration in lactation management.

Breastfeeding for mothers with chronic medical conditions presents important medical decisions for the primary physician. The issues need to be considered in light of the chronic disease, the physiological process of lactation, and the individual Mother for whom breastfeeding is very important. Management plans need to be based on adequate information and coordinated by the mother's physician and the pediatrician.

PIP: Health practitioners should not discourage mothers from breast feeding if they acquire an acute self limited illness. Depending on the treatment for a chronic disease, its severity, and its stage, mothers with some chronic diseases may breast feed. If a pregnant diabetic women has good prenatal care, continued good medical care for diabetes, and takes the needed increased insulin, the mammary gland should be able to produce ample milk for a health infant. Further, lactation induces a remission from diabetes. If pregnant women with severe asthma do not take medication, they are likely to experience severe asthma attacks which require large doses of medication to control them. 1 such medication, theophylline, passes to breast milk in a milk to plasma ratio of .6-.73. Mothers who have normal or mildly decreased renal function can breast feed successfully. In those women who experience moderate renal insufficiency, breast feeding can take place in only those cases where the infant is stable and can feed soon after delivery and the mother's condition is sound or improved after delivery. If indeed a woman with severe renal insufficiency can conceive, which is unlikely, she should not breast feed since the stress of pregnancy and delivery alone may have comprised her health. The limited research on breast feeding after a kidney transplant shows that an immunosuppressed women can produce immunocompetent milk with very little azathioprine present. Hypertensive mothers can breast feed their infants, especially if the internist treating her considers the lactation process. Diuretics may diminish milk production, but an active infant who stimulates milk production can counteract this effect. Methyldopa may suppress milk production.

The three asparaginases. Comparative pharmacology and optimal use in childhood leukemia.

Asparaginase (ASP) is a standard component of the antileukemia armamentarium. There are currently 3 preparations of asparaginase available: (1) E. coli (ASP, Elspar); (2) the enzyme derived from Erwinia chrysanthemi (ERW, Erwinase); (3) pegaspargase (PEG, Oncaspar), the E. coli enzyme modified by covalent attachment of polyethylene glycol. This report describes the findings of 3 pharmacologic end points: ASP enzyme activity in patients' sera, depletion of asparagine and the development of anti-ASP antibodies. Pharmacokinetics and pharmacodynamic studies in a group of naive children with newly diagnosed ALL demonstrate a significant difference in apparent half-life (1.24 days E. coli vs. 0.65 ERW vs. 5.73 PEG; p < 0.001) and days of asparagine depletion (14-23 E. coli vs. 7-15 ERW vs. 26-34 PEG; p < 0.01) for the 3 different preparations. Data from Pediatric Oncology Group (POG) Protocol #8866 show that high antibody levels correlated with rapid ASP clearance and a significantly lower response rate (NR = 26% vs. CR + PR + 64%). The pharmacologic characteristics of ASP in terms of clearance of enzyme activity and ability to deplete serum asparagine was dependent upon the nature of the enzyme and are significantly altered in patients who develop anti-ASP antibodies regardless of their clinical status. In addition, these data demonstrate that ASP pharmacokinetics are directly related to its anti-leukemic effect. In order to maximize the therapeutic benefits of ASP, the optimal dose and schedule of treatment should be determined based on pharmacologic testing rather than by clinical criteria alone. Future studies will focus on the role of "silent hypersensitivity" as a mechanism of resistance to ASP and strategies to maximize the therapeutic efficacy of ASP as part of ALL therapy.

Long-term results of Dana-Farber Cancer Institute ALL Consortium protocols for children with newly diagnosed acute lymphoblastic leukemia (1985-2000).

The Dana-Farber Cancer Institute (DFCI) acute lymphoblastic leukemia (ALL) Consortium has been conducting multi-institutional clinical trials in childhood ALL since 1981. The treatment backbone has included 20-30 consecutive weeks of asparaginase during intensification and frequent vincristine/corticosteroid pulses during the continuation phase. Between 1985 and 2000, 1457 children aged 0-18 years were treated on four consecutive protocols: 85-01 (1985-1987), 87-01 (1987-1991), 91-01 (1991-1955) and 95-01 (1996-2000). The 10-year event-free survival (EFS)+/-s.e. by protocol was 77.9+/-2.8% (85-01), 74.2+/-2.3% (87-01), 80.8+/-2.1% (91-01) and 80.5+/-1.8% (95-01). Approximately 82% of patients treated in the 1980s and 88% treated in the 1990s were long-term survivors. Both EFS and overall survival (OS) rates were significantly higher for patients treated in the 1990s compared with the 1980s (P=0.05 and 0.01, respectively). On the two protocols conducted in the 1990s, EFS was 79-85% for T-cell ALL patients and 75-78% for adolescents (age 10-18 years). Results of randomized studies revealed that dexrazoxane prevented acute cardiac injury without adversely affecting EFS or OS in high-risk (HR) patients, and frequently dosed intrathecal chemotherapy was an effective substitute for cranial radiation in standard-risk (SR) patients. Current studies continue to focus on improving efficacy while minimizing acute and late toxicities.

Right atrial thrombi in children with cancer and indwelling catheters.

OBJECTIVE: To determine the prevalence of right atrial thrombi in children with cancer and indwelling catheters. STUDY DESIGN: We systematically examined 156 children with cancer and indwelling catheters for the presence of a right atrial thrombus when they underwent routine screening of cardiac function by two-dimensional echocardiography. RESULTS: Thirteen children (8.8%) had right atrial thrombi. Of the 13 children, 6 had thrombi adherent to the right atrial wall, and in 5 of these 6 children the clots were considered large enough to require intervention: 2 children with obstruction of venous or tricuspid valve inflow underwent right atriotomy and thrombus removal; they recovered and remained well. The other 3 children had moderate-sized thrombi and were treated with oral anticoagulants; their clots stabilized (2 children) or regressed (1 child). The remaining 7 children had thrombus on the tip of the catheter: 5 of these children were observed; the thrombi spontaneously resolved in 2 of them and did not change in size in the other 3. In the sixth child, the catheter malfunctioned 2 weeks after discovery of the clot and the catheter was removed. The seventh child was treated with warfarin and the clot decreased in size. Thrombi were detected in a greater proportion of children with catheter tips in the right atrium versus the superior vena cava, and in a greater proportion of children with acute lymphoblastic leukemia versus other diagnoses. There was no association between the presence of a clot and duration of time the catheter was in place, the number of catheters placed, treatment with asparaginase, or treatment with total parenteral nutrition. CONCLUSION: The incidence of right atrial thrombi in children with indwelling catheters may be higher than was previously appreciated.

Prognostic significance of early response to a single dose of asparaginase in childhood acute lymphoblastic leukemia.

PURPOSE: The in vitro and in vivo efficacy of a single dose of asparaginase in children with newly diagnosed acute lymphoblastic leukemia and the correlation between in vitro and in vivo antileukemic response and long-term outcome were prospectively evaluated. PATIENTS AND METHODS: Two hundred fifty-one patients were randomized to receive 1 of 3 asparaginase preparations (Escherichia coli, Erwinia chrysanthemi [Erwinia], or pegaspargase). In vitro assessment of efficacy was expressed as the percent total cell kill (TCK), based on the number of viable cells found after 5 days of culture in the presence of asparaginase. In vivo leukemia cell kill (LCK) was calculated by comparing bone marrow cellularity and percent leukemic blasts in marrow obtained before and 5 days after treatment with a single dose of asparaginase. Acute toxicity was determined by clinical and laboratory assessment. RESULTS: There was equivalent cell kill with all three types of asparaginase. The mean in vitro TCKs for E. coli, Erwinia, and pegaspargase were 31%, 39%, and 36%, respectively (P = 0.63). The mean LCKs in marrow of patients exposed to E. coli, Erwinia, and pegaspargase were 69%, 74%, and 65%, respectively (P = 0.88). The lack of response to asparaginase in vitro predicted a higher risk for clinical relapse regardless of risk assignment (12 leukemic events among 21 in vitro nonresponders; 57%, P < 0.001). There was no difference in acute toxicity among the three asparaginase preparations. CONCLUSIONS: All three asparaginase preparations produced equivalent LCKs in in vitro and in vivo analyses. In vitro response to asparaginase provided a risk group-independent prognostic factor.

Improved outcome for children with acute lymphoblastic leukemia: results of Dana-Farber Consortium Protocol 91-01.

The Dana-Farber Cancer Institute (DFCI) acute lymphoblastic leukemia (ALL) Consortium Protocol 91-01 was designed to improve the outcome of children with newly diagnosed ALL while minimizing toxicity. Compared with prior protocols, post-remission therapy was intensified by substituting dexamethasone for prednisone and prolonging the asparaginase intensification from 20 to 30 weeks. Between 1991 and 1995, 377 patients (age, 0-18 years) were enrolled; 137 patients were considered standard risk (SR), and 240 patients were high risk (HR). Following a 5.0-year median follow-up, the estimated 5-year event-free survival (EFS) +/- SE for all patients was 83% +/- 2%, which is superior to prior DFCI ALL Consortium protocols conducted between 1981 and 1991 (P =.03). There was no significant difference in 5-year EFS based upon risk group (87% +/- 3% for SR and 81% +/- 3% for HR, P =.24). Age at diagnosis was a statistically significant prognostic factor (P =.03), with inferior outcomes observed in infants and children 9 years or older. Patients who tolerated 25 or fewer weeks of asparaginase had a significantly worse outcome than those who received at least 26 weeks of asparaginase (P <.01, both univariate and multivariate). Older children (at least 9 years of age) were significantly more likely to have tolerated 25 or fewer weeks of asparaginase (P <.01). Treatment on Protocol 91-01 significantly improved the outcome of children with ALL, perhaps due to the prolonged asparaginase intensification and/or the use of dexamethasone. The inferior outcome of older children may be due, in part, to increased intolerance of intensive therapy.