Low or absent SPARC expression in acute myeloid leukemia with MLL rearrangements is associated with sensitivity to growth inhibition by exogenous SPARC protein.

Secreted protein, acidic and rich in cysteine (SPARC), is a matricellular glycoprotein with growth-inhibitory and antiangiogenic functions. Although SPARC has been implicated as a tumor suppressor in humans, its function in normal or malignant hematopoiesis has not previously been studied. We found that the leukemic cells of AML patients with MLL gene rearrangements express low to undetectable amounts of SPARC whereas normal hematopoietic progenitors and most AML patients express this gene. SPARC RNA and protein levels were also low or undetectable in AML cell lines with MLL translocations. Consistent with its tumor suppressive effects in various solid tumor models, exogenous SPARC protein selectively reduced the growth of cell lines with MLL rearrangements by inhibiting cell cycle progression from G1 to S phase. The lack of SPARC expression in MLL-rearranged cell lines was associated with dense promoter methylation. However, we found no evidence of methylation-based silencing of SPARC in primary patient samples. Our results suggest that low or absent SPARC expression is a consistent feature of AML cells with MLL rearrangements and that SPARC may function as a tumor suppressor in this subset of patients. A potential role of exogenous SPARC in the therapy of MLL-rearranged AML warrants further investigation.

Pediatric Oncology Group (POG) studies of acute myeloid leukemia (AML): a review of four consecutive childhood AML trials conducted between 1981 and 2000.

From 1981 to 2000, a total of 1823 children with acute myeloid leukemia (AML) enrolled on four consecutive Pediatric Oncology Group (POG) clinical trials. POG 8101 demonstrated that the induction rate associated with the 3+7+7 combination of daunorubicin, Ara-C, and 6-thioguanine (DAT) was greater than that associated with an induction regimen used to treat acute lymphoblastic leukemia (82 vs 61%; P=0.02). Designed as a pilot study to determine the feasibility of administration of noncross-resistant drug pairs and later modified to assess the effect of dose intensification of Ara-C during the second induction course, POG 8498 confirmed the high initial rate of response to DAT (84.2%) and showed that dose intensification of Ara-C during the second induction course resulted in a trend toward higher event-free survival (EFS) estimates than did standard-dose DAT (2+5) during the second induction course (5 year EFS estimates, 22 vs 27%; P=0.33). Age <2 years and leukocyte count <100 000/mm3 emerged as significantly good prognostic factors. The most significant observation made in the POG 8498 study was the markedly superior outcome of children with Down's syndrome who were treated on the high-dose Ara-C regimen. POG 8821 compared the efficacy of autologous bone marrow transplantation (BMT) with that of intensive consolidation chemotherapy. Intent-to-treat analysis revealed similar 5-year EFS estimates for the group that underwent autologous BMT (36+/-4.7%) and for the group that received only intensive chemotherapy (35+/-4.5%) (P=0.25). There was a high rate of treatment-related mortality in the autologous transplantation group. The study demonstrated superior results of allogeneic BMT for patients with histocompatible related donors (5-year EFS estimate 63+/-5.4%) and of children with Down's syndrome (5-year EFS estimate, 66+/-8.6%). The POG 9421 AML study evaluated high-dose Ara-C as part of the first induction course and the use of the multidrug resistance modulator cyclosporine. Preliminary results showed that patients receiving both high-dose Ara-C for remission induction and the MDR modulator for consolidation had a superior outcome (5-year EFS estimate, 42+/-8.2%) than did patients receiving other treatment; however, the difference was not statistically significant. These four studies demonstrate the importance of dose intensification of Ara-C in the treatment of childhood AML; cytogenetics as the single most prognostic factor and the unique curability of AML in children with Down's syndrome.

Normal sister chromatid exchange frequency in long-term survivors with acute leukemia.

We hypothesized that the sister chromatid exchanges assay in acute leukemia long-term survivors may detect: (a) long-term effects of combined chemo- and radiotherapy; and possibly (b) those individuals with inherently deficient DNA repair. Accordingly, we determined the sister chromatid exchanges frequency in 26 blood specimens from 24 acute leukemia long-term survivors (patients) and 14 blood specimens from 13 control subjects (controls). The patients consisted of 23 children with acute lymphocytic and one child with acute myelocytic leukemia. The median length of chemotherapy was 5 years. Eighteen of the 24 patients also received prophylactic fractional central nervous system irradiation for the first 3 years of treatment, and one patient received therapeutic irradiation to the central nervous system. The median off-therapy period at the time of study was 2.5 years with a range of 0 to 7.5 years. The controls consisted of the parents of the patients and laboratory personnel. A mean exchange score per cell was established for each specimen (25 to 30 cells/specimen were scored), and it ranged from 3.0 to 9.7 in the patients and from 3.0 to 11.5 in the controls. A mean +/- S.D. calculated from those means was 6.0 +/- 1.8 for the patients and 6.9 +/- 2.8 for the controls. They were not significantly different. We conclude that chemo- and radiotherapy produced no persistent DNA alterations detectable by this method.

Levels of p53 protein increase with maturation in human hematopoietic cells.

Transfection of the wild-type p53 gene into malignant cell lines usually results in an inhibition of proliferation. However, the physiological function of the endogenous p53 gene product has been difficult to ascertain. In order to examine whether p53 is involved in the regulation of proliferation and/or differentiation of hematopoietic tissue, we modified a recently developed flow cytometric assay to assess p53 protein expression in normal human hematopoietic cells, primary leukemias, and selected leukemia cell lines. In normal human bone marrow, p53 protein was not detected in the proliferative, progenitor cell populations identified by the cell surface antigens CD34 (progenitor cells of multiple lineages) or glycophorin (erythroid precursors). In contrast, low but detectable levels of p53 protein were observed in the nonproliferative, mature lymphoid, granulocytic, and monocytic cell populations. Similarly, p53 levels increased and DNA synthesis decreased during 12-O-tetradecanoylphorbol-13-acetate-induced differentiation of ML-1 myeloblastic leukemia cells. Both of these results suggest that endogenous, wild-type p53 protein may play a role in hematopoietic cell maturation, possibly by contributing to the inhibition of proliferation that occurs during terminal differentiation. Leukemia cells deviated from this pattern of expression: (a) in contrast to the normal, proliferative bone marrow progenitor cells, a significant percentage of patient leukemia samples expressed detectable levels of p53 protein; and (b) leukemia cell lines exhibited lineage-specific abnormalities in p53 expression, with overexpression in lymphoid cell lines and lack of expression in myeloid cell lines.

Cystathionine-beta-synthase cDNA transfection alters the sensitivity and metabolism of 1-beta-D-arabinofuranosylcytosine in CCRF-CEM leukemia cells in vitro and in vivo: a model of leukemia in Down syndrome.

The significantly higher event-free survival rates of Down syndrome (DS) children with acute myeloid leukemia compared with non-DS children is linked to increased sensitivity of DS myeloblasts to 1-beta-D-arabinofuranosylcytosine (ara-C) and the enhanced metabolism of ara-C to ara-C triphosphate (J. W. Taub et al., Blood, 87: 3395-3403, 1996). The cystathionine-beta-synthase (CBS) gene (localized to chromosome 21q22.3) may have downstream effects on reduced folate and S-adenosylmethionine pathways; ara-C metabolism and folate pools are linked by the known synergistic effect of sequential methotrexate and ara-C therapy. We have shown that relative CBS transcripts were significantly higher in DS compared with non-DS myeloblasts, and CBS transcript levels correlated with in vitro ara-C sensitivity (J. W. Taub et al., Blood, 94: 1393-1400, 1999). A leukemia cell line model to study the relationship of the CBS gene and ara-C metabolism/sensitivity was developed by transfecting CBS-null CCRF-CEM cells with the CBS cDNA. CBS-transfected cells were a median 15-fold more sensitive in vitro to ara-C compared with wild-type cells and generated 8.5-fold higher [3H]ara-C triphosphate levels after in vitro incubation with [3H]ara-C. Severe combined immunodeficient mice implanted with CBS-transfected CEM cells demonstrated greater responsiveness to therapy, reflected in significantly prolonged survivals after ara-C administration compared with mice implanted with wild-type cells and treated with the same dosage schedule. The transfected cells also demonstrated increased in vitro and in vivo sensitivity to gemcitabine. Deoxycytidine kinase (dCK) activity was approximately 22-fold higher in transfected CEM cells compared with wild-type cells. However, levels of dCK transcripts on Northern blots and protein levels on Western blots were nearly identical between CBS-transfected and wild-type cells. Collectively, these results suggest a posttranscriptional regulation of dCK in CBS-overexpressing cells that contributes to increased ara-C phosphorylation and drug activity. Further elucidating the mechanisms of increased sensitivity of DS cells to ara-C related to the CBS gene may lead to the application of these novel approaches to acute myeloid leukemia therapy for non-DS patients.

Cord blood lead level in an urban inner-city hospital.

Lead levels were measured by inductively coupled plasma mass spectrometry (ICP-MS) in umbilical cord blood samples of 150 neonates in an urban inner-city hospital. The mean (SD) gestation and birth weight of our cohort were 38.8 (1.7) weeks and 3,217 (519) grams. There were 89% African-Americans, 53% males and 79% were born via vaginal delivery. Mean (SD) maternal age was 24.5 (5.8) years. History of drug abuse and smoking was reported in 8.7% and 10.7% respectively, with only 1 mother reporting a history of high lead level in childhood. Prenatal vitamin intake was reported in 99.3%. Cord blood lead level was available in 144 patients, with lead level of <1µg/dL seen in 141 (97.9%) and>1 in 3 (2.1%) patients. No patient had cord blood lead level of >2µg/dL. High lead levels during childhood in high-risk urban population, however, suggest the need for intensive efforts for prevention of environmental exposure to lead in early childhood.

Comparison of two schedules of intermediate-dose methotrexate and cytarabine consolidation therapy for childhood B-precursor cell acute lymphoblastic leukemia: a Pediatric Oncology Group study.

PURPOSE: To compare efficacy and toxicity of two schedules of intermediate-dose methotrexate (IDM) and cytarabine (Ara-C) in remission consolidation of childhood acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS: In 1986, the Pediatric Oncology Group (POG) began a randomized trial to test two schedules of consolidation chemotherapy in children with newly diagnosed B-precursor cell ALL. MTX and Ara-C were given as overlapping 24-hour infusions. The dose and sequence of MTX and Ara-C administration were based on a preclinical model that had demonstrated synergism between these two agents. Two hundred fifteen patients in complete remission were randomized to front-loading consolidation therapy in which six MTX/Ara-C infusions were administered at 3-week intervals from the 7th through the 19th week of therapy. Two hundred thirteen patients in complete remission were randomized to receive standard consolidation therapy in which the six MTX/Ara-C infusions were given every 12 weeks from the 7th through the 67th week of therapy. RESULTS: Both regimens produced similar rates of adverse side effects, except for a higher incidence of CNS toxicity in individuals randomized to the front-loading arm (32 of 215 v 12 of 213 patients, P = .002). Leukoencephalopathy occurred in three patients on the front-loading regimen and was permanent in one. By Kaplan-Meier analysis, the probability of continuing in complete remission for 5 years was 79% (SE = 5%) and 85% (SE = 5%) for good-risk patients, and 66% (SE = 6%) and 61% (SE = 7%) for poor-risk patients randomized to front-loading and standard regimens, respectively. CONCLUSION: Although differences in complete remission durations were not statistically significant by log-rank analysis (P = .62 for good-risk patients, .89 for poor-risk patients, and .99 overall), the results are comparable to those in previous studies using more toxic agents as components of remission consolidation therapy.

Phase I trial of paclitaxel in children with refractory solid tumors: a Pediatric Oncology Group Study.

PURPOSE: A phase I study was performed to describe the principal toxicities and identify the maximum-tolerated dose (MTD) of Taxol (paclitaxel; Bristol-Myers Squibb Co, Wallingford, CT) in children with therapy-resistant solid tumors. Additionally, the pharmacokinetic disposition of Taxol in children was studied, and preliminary evidence of the activity of Taxol against pediatric solid tumors was assessed. PATIENTS AND METHODS: Twenty-four-hour continuous infusions of Taxol were administered every 21 days to children (median age, 12 years; range, 2 to 22) with refractory solid tumors. Doses ranged from 200 to 420 mg/m2, there was no intrapatient dose escalation. RESULTS: A total of 62 courses of Taxol were administered to 31 patients. Two patients developed acute anaphylaxis during their second infusion of taxol at doses of 200 mg/m2 and 350 mg/m2, respectively. No other allergic reactions were documented. Myelosuppression occurred at all dose levels, but was of short duration (< or = 7 days) and did not appear to increase with consecutive courses or at higher dosage levels. A stocking-and-glove peripheral neuropathy became evident at doses > or = 290 mg/m2. Dose-limiting neurotoxicity occurred at 420 mg/m2 and comprised a significant fine-motor and peripheral neuropathy in one patient, and a tonic-clonic seizure in another. End-of-infusion plasma concentrations ranged from 0.40 to 6.4 mumol/L, and were not found to be dose-dependent over the range of doses studied. A complete response was documented in one patient, partial response in two, and minimal response in one for an overall response rate of 13%. CONCLUSION: Neurotoxicity was dose-limiting when Taxol was administered by 24-hour continuous infusion to pediatric patients with relapsed solid tumors. In this population, the recommended dose for phase II trials is 350 mg/m2/d.

A comparison of induction and maintenance therapy for acute nonlymphocytic leukemia in childhood: results of a Pediatric Oncology Group study.

Two hundred fifty-six children with previously untreated acute nonlymphocytic leukemia (ANLL) were evaluated on a Pediatric Oncology Group (POG) phase III randomized trial of both induction and continuation chemotherapies. Induction therapy compared vincristine, cytarabine, and dexamethasone (VADx) with daunorubicin, cytarabine, and thioguanine (DAT). The complete remission (CR) rate using DAT was superior (82% v 61%, P = .02). Postremission therapy consisted of either "standard" two-cycle therapy or a more intensive four-cycle regimen given for 2 years. Overall, there was no difference in outcome for patients randomized to either continuation regimen. The overall complete continuous remission rate (CCR) for the "best" induction/continuation therapy combination at 2 years was .50 (SE = .06), at 3 years was .35 (.04), and at 4 years was .34 (.05). Analysis of selected clinical and laboratory parameters demonstrated differences in induction responses favoring DAT induction but did not impact eventual disease-free survival. There were two subgroups of patients who responded better to four-cycle continuation therapy. These were patients with French-American-British (FAB) M1/M2 (2-year CCR was .20 v .44, P = .01) and patients older than 10 years at diagnosis (.32 v .62, P = .004).

High-dose cytarabine for intensification of early therapy of childhood acute myeloid leukemia: a Pediatric Oncology Group study.

In June 1984, the Pediatric Oncology Group (POG) initiated a pilot study (8498) using high-dose cytarabine (HdA; 3 g/m2) for intensification of early therapy in childhood acute myelogenous leukemia (AML) (group I). Remission induction therapy consisted of two courses of daunorubicin, cytarabine (Ara-C), and thioguanine (DAT). Postremission therapy consisted of four sequential courses, each consisting of (1) four doses of HdA (HdA4) followed by asparaginase (L-Asp), (2) etoposide (VP) plus azacytidine (Az), (3) prednisone, vincristine, methotrexate, and mercaptopurine (POMP), and (4) Ara-C daily for 5 days by continuous infusion. Six doses of intrathecal Ara-C were given for CNS prophylaxis. In December 1986, the protocol was amended (group II) to substitute six doses of HdA (HdA6) for the second DAT (two + five) induction course; postinduction, a single course of HdA6 was given instead of four HdA/L-Asp courses, and the remainder of the therapy was unchanged. One hundred forty group I patients and 145 group II patients were assessable. The two groups were similar with regard to clinical prognostic groups. No significant differences were noted in the two groups with regard to remission induction (85% [SE = 2%] in each group), induction deaths (6.5% v 7.0%), or deaths in remission (one in each group). Cerebellar toxicity was reported in three patients in group II (with HdA6) but none in group I (HdA4). At present, patients who received HdA6 (group II) had higher event-free survival than patients in group I (EFS at 3 years, 34% [SE = 11%] v 29% [SE = 4%]), and disease-free survival (DFS at 3 years, 42% [SE = 14%] v 34% [SE = 4%]), but the differences were not statistically significant. In both groups, children less than 2 years and those with WBCs less than 100,000/microL had significantly better outcome (EFS of 55% [SE = 10%] and 36% [SE = 5%] at 3 years, respectively) than children greater than or equal to 2 years and those with WBCs greater than or equal to 100,000/microL (EFS of 27% [SE = 5%] and 20% [SE = 9%] at 3 years, respectively.

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.

Combined etoposide and 5-azacitidine in children and adolescents with refractory or relapsed acute nonlymphocytic leukemia: a Pediatric Oncology Group Study.

An intensive regimen of combined etoposide (VP-16) and 5-azacitidine (5-Az) was used to treat 96 children and adolescents with refractory or relapsed acute nonlymphocytic leukemia (ANLL). Patients were given two sequential five-day courses of VP-16, 250 mg/m2 for three days, followed by 5-Az, 300 mg/m2 for two days. An additional five-day course was administered if marrow aplasia was not evident by day 13. A complete remission rate of 45% was achieved with a median of two courses of VP-16 and 5-Az. The outcome of induction therapy was not influenced by prior treatment, blast cell morphology, or disease status on study entry (refractory or relapsed). Twenty-seven patients have relapsed after remission periods of 35 to 920 days (median, 110 days); seven others are free of leukemia for up to 519 days. The effectiveness of VP-16/5-Az combination therapy in patients refractory to anthracyclines and cytarabine indicates a potential role for these compounds in first-line treatment of patients with ANLL.

Quality-adjusted survival after treatment for acute myeloid leukemia in childhood: A Q-TWiST analysis of the Pediatric Oncology Group Study 8821.

PURPOSE: To describe quality-of-life considerations in post-remission therapies for children with acute myelogenous leukemia. PATIENTS AND METHODS: A quality-adjusted survival analysis, using the quality-adjusted time without symptoms or toxicity (Q-TWiST) method, was applied to Pediatric Oncology Group Trial 8821, which compared randomized assignment with intensive consolidation chemotherapy (CC) or autologous bone marrow transplantation (ABMT). Nonrandomized assignment to allogeneic bone marrow transplantation (allo BMT) on the basis of availability of a matched related donor was also evaluated. A 25-patient cohort provided data for modeling chronic graft-versus-host disease. The Q-TWiST analysis was performed based on the intent-to-treat principle. RESULTS: As previously reported, the 3-year event-free survival was not significantly different between the randomized arms (CC v ABMT). At a median follow-up of 5 years (of the censoring distribution), the CC group had less time in toxicity (TOX) and more time without symptoms or toxicity (TWiST), relapse-free time, and alive time than patients assigned to ABMT (none of these were statistically significant). Compared with the CC group, allo BMT patients spent more time in TOX (P <.001), more time in TWiST (P =.06), and had more relapse-free time (P =.03) and time alive (P =.07). Allo BMT was superior to ABMT with greater time in TWiST (P =.02), relapse-free time (P =.01), and time alive P =.002). CONCLUSION: The Q-TWiST analysis is a powerful decision aid in choosing among alternative therapies. Prospective information on patient preferences will facilitate future trials evaluating treatment outcomes. Refinements in the Q-TWiST method could be included to further enhance the power of this patient care decision-making tool.

Expression of myeloid-associated and lymphoid-associated cell-surface antigens in acute myeloid leukemia of childhood: a Pediatric Oncology Group study.

PURPOSE: Although the expression of both myeloid- and lymphoid-associated cell-surface antigens in acute myeloid leukemia (AML) has been described, the clinical significance of such antigen expression remains unknown in the pediatric population. We sought to define an antibody panel for optimal diagnostic antigenic analysis and to test associations among antigen expression and a number of clinical features at presentation and prognosis in pediatric AML. PATIENTS AND METHODS: We reviewed the extensive immunophenotypic analysis performed at the time of diagnosis on 132 assessable patients registered on a single Pediatric Oncology Group AML protocol between 1984 and 1988. RESULTS: Eighty-eight percent of patients were identified by testing for expression of CD33 and CD13. Overall, 61% of patients expressed at least one lymphoid-associated antigen, most commonly CD4, CD7, or CD19. Expression of CD5, CD10, CD20, or CD22, commonly detected in T- or B-lineage pediatric acute lymphoid leukemia (ALL), was uncommon; coexpression of multiple lymphoid-associated antigens was also uncommon. Expression of the monocyte-associated antigen CD14 correlated with French-American-British (FAB) M4 or M5 morphology. Otherwise, no correlation between antigen expression and FAB classification was noted. None of the myeloid, lymphoid, natural-killer (NK), or progenitor-associated antigens were associated with significant differences in the likelihood of remission induction or event-free survival when expressor versus nonexpressor groups were compared. CONCLUSIONS: The distribution of cell-surface antigen expression in pediatric acute leukemia usually permitted the discrimination of AML from ALL by using a limited panel of antibodies. Although the expression of lymphoid-associated antigens was common, such expression did not seem to be associated with an adverse prognosis in pediatric AML.

Racial differences in the survival of childhood B-precursor acute lymphoblastic leukemia: a Pediatric Oncology Group Study.

PURPOSE: We conducted a historic cohort study to test the hypothesis that, after adjustment for biologic factors, African-American (AA) children and Spanish surname (SS) children with newly diagnosed B-precursor acute lymphoblastic leukemia had lower survival than did comparable white children. PATIENTS AND METHODS: From 1981 to 1994, 4,061 white, 518 AA, and 507 SS children aged 1 to 20 years were treated on three successive Pediatric Oncology Group multicenter randomized clinical trials. RESULTS: AA and SS patients were more likely to have adverse prognostic features at diagnosis and lower survival than were white patients. The 5-year cumulative survival rates were (probability +/- SE) 81.9% +/- 0.6%, 68.6% +/- 2.1%, and 74.9% +/- 2.0% for white, AA, and SS children, respectively. Adjusting for age, leukocyte count, sex, era of treatment, and leukemia blast cell ploidy, we found that AA children had a 42% excess mortality rate compared with white children (proportional hazards ratio [PHR] = 1.42; 95% confidence interval [CI], 1.12 to 1. 80), and SS children had a 33% excess mortality rate compared with white children (PHR = 1.33; 95% CI, 1.19 to 1.49). CONCLUSION: Clinical presentation, tumor biology, and deviations from prescribed therapy did not explain the differences in survival and event-free survival that we observed, although differences seem to be diminishing over time with improvements in therapy. The disparity in outcome for AA and SS children is most likely related to variations in chemotherapeutic response to therapy and not to compliance. Further improvements in outcome may require individualized dosing based on specific pharmacogenetic profiles, especially for AA and SS children.

Preponderance of thiopurine S-methyltransferase deficiency and heterozygosity among patients intolerant to mercaptopurine or azathioprine.

PURPOSE: To assess thiopurine S-methyltransferase (TPMT) phenotype and genotype in patients who were intolerant to treatment with mercaptopurine (MP) or azathioprine (AZA), and to evaluate their clinical management. PATIENTS AND METHODS: TPMT phenotype and thiopurine metabolism were assessed in all patients referred between 1994 and 1999 for evaluation of excessive toxicity while receiving MP or AZA. TPMT activity was measured by radiochemical analysis, TPMT genotype was determined by mutation-specific polymerase chain reaction restriction fragment length polymorphism analyses for the TPMT*2, *3A, *3B, and *3C alleles, and thiopurine metabolites were measured by high-performance liquid chromatography. RESULTS: Of 23 patients evaluated, six had TPMT deficiency (activity < 5 U/mL of packed RBCs [pRBCs]; homozygous mutant), nine had intermediate TPMT activity (5 to 13 U/mL of pRBCs; heterozygotes), and eight had high TPMT activity (> 13.5 U/mL of pRBCs; homozygous wildtype). The 65.2% frequency of TPMT-deficient and heterozygous individuals among these toxic patients is significantly greater than the expected 10% frequency in the general population (P <.001, chi(2)). TPMT phenotype and genotype were concordant in all TPMT-deficient and all homozygous-wildtype patients, whereas five patients with heterozygous phenotypes did not have a TPMT mutation detected. Before thiopurine dosage adjustments, TPMT-deficient patients experienced more frequent hospitalization, more platelet transfusions, and more missed doses of chemotherapy. Hematologic toxicity occurred in more than 90% of patients, whereas hepatotoxicity occurred in six patients (26%). Both patients who presented with only hepatic toxicity had a homozygous-wildtype TPMT phenotype. After adjustment of thiopurine dosages, the TPMT-deficient and heterozygous patients tolerated therapy without acute toxicity. CONCLUSION: There is a significant (> six-fold) overrepresentation of TPMT deficiency or heterozygosity among patients developing dose-limiting hematopoietic toxicity from therapy containing thiopurines. However, with appropriate dosage adjustments, TPMT-deficient and heterozygous patients can be treated with thiopurines, without acute dose-limiting toxicity.

Hemoglobin autoxidation and regulation of endogenous H2O2 levels in erythrocytes.

Red cells from mice deficient in glutathione peroxidase-1 were used to estimate the hemoglobin autoxidation rate and the endogenous level of H2O2 and superoxide. Methemoglobin and the rate of catalase inactivation by 3-amino-2,4,5-triazole (3-AT) were determined. In contrast with iodoacetamide-treated red cells, catalase was not inactivated by 3-AT in glutathione peroxidase-deficient erythrocytes. Kinetic models incorporating reactions known to involve H2O2 and superoxide in the erythrocyte were used to estimate H2O2, superoxide, and methemoglobin levels. The experimental data could not be modeled unless the intraerythrocytic concentration of Compound I is very low. Two additional models were tested. In one, it was assumed that a rearranged Compound I, termed Compound II*, does not react with 3-AT. However, experiments with an NADPH-generating system provided evidence that this mechanism does not occur. A second model that explicitly includes peroxiredoxin II can fit the experimental findings. Insertion of the data into the model predicted a hemoglobin autoxidation rate constant of 4.5 x 10(-7) s(-1) and an endogenous H2O2 and superoxide concentrations of 5 x 10(-11) and 5 x 10(-13) M, respectively, lower than previous estimates.

Further elucidation of mechanism of resistance to vincristine in myeloid cells: role of hypochlorous acid in degradation of vincristine by myeloperoxidase.

Inherent resistance of myeloblasts to vincristine (VCR) has been related to the activity of myeloperoxidase (MPO) which can degrade VCR in the presence of hydrogen peroxide (H2O2). We investigated the relationship between VCR degradation and hypochlorous acid (HOCl) generation from the reaction of H2O2 with chlorine (Cl) as catalyzed by MPO. A cell-free system, three human leukemia cell lines (CEM/CCRF, HL-60, U937) and 15 bone marrow samples from children with acute myeloid leukemia (AML) were studied. VCR cytotoxicity was evaluated by MTT assay and by quantitative measurement of apoptosis. In vitro levels of VCR in cell-free systems were measured by high performance liquid chromatography (HPLC), and intracellular HOCl levels by oxidation of 5-thio-2-nitrobenzoic acid with the accompanying decrease in the absorbency at 412 nm. VCR was degraded by increasing concentrations of HOCl in cell-free systems and this activity was inhibited by taurine, which is known to block HOCl activity. This finding was confirmed by the VCR cytotoxicity studies on cell lines. The HOCl-producing myeloblasts from patients were resistant to VCR. In five samples out of eight HOCl was also detected extracellularly. These results suggest that oxidation by HOCl may be the final step in VCR degradation catalyzed by MPO through its action on intracellular H2O2 and Cl. Leukemia (2000) 14, 47-51.

Prognostic factors in children and adolescents with acute myeloid leukemia (excluding children with Down syndrome and acute promyelocytic leukemia): univariate and recursive partitioning analysis of patients treated on Pediatric Oncology Group (POG) Study 8821.

The purpose of the paper was to define clinical or biological features associated with the risk for treatment failure for children with acute myeloid leukemia. Data from 560 children and adolescents with newly diagnosed acute myeloid leukemia who entered the Pediatric Oncology Group Study 8821 from June 1988 to March 1993 were analyzed by univariate and recursive partitioning methods. Children with Down syndrome or acute promyelocytic leukemia were excluded from the study. Factors examined included age, number of leukocytes, sex, FAB morphologic subtype, cytogenetic findings, and extramedullary disease at the time of diagnosis. The overall event-free survival (EFS) rate at 4 years was 32.7% (s.e. = 2.2%). Age > or =2 years, fewer than 50 x 10(9)/I leukocytes, and t(8;21) or inv(16), and normal chromosomes were associated with higher rates of EFS (P value = 0.003, 0.049, 0.0003, 0.031, respectively), whereas the M5 subtype of AML (P value = 0.0003) and chromosome abnormalities other than t(8;21) and inv(16) were associated with lower rates of EFS (P value = 0.0001). Recursive partitioning analysis defined three groups of patients with widely varied prognoses: female patients with t(8;21), inv(16), or a normal karyotype (n = 89) had the best prognosis (4-year EFS = 55.1%, s.e. = 5.7%); male patients with t(8;21), inv(16) or normal chromosomes (n = 106) had an intermediate prognosis (4-year EFS = 38.1%, s.e. = 5.3%); patients with chromosome abnormalities other than t(8;21) and inv(16) (n = 233) had the worst prognosis (4-year EFS = 27.0%, s.e. = 3.2%). One hundred and thirty-two patients (24%) could not be grouped because of missing cytogenetic data, mainly due to inadequate marrow samples. The results suggest that pediatric patients with acute myeloid leukemia can be categorized into three potential risk groups for prognosis and that differences in sex and chromosomal abnormalities are associated with differences in estimates of EFS. These results are tentative and must be confirmed by a large prospective clinical trial.

Pharmacokinetic interactions of cyclosporine with etoposide and mitoxantrone in children with acute myeloid leukemia.

The purpose of this study was to assess the effect of the multidrug resistance modulator cyclosporine (CsA) on the pharmacokinetics of etoposide and mitoxantrone in children with de novo acute myeloid leukemia (AML). Serial blood samples for pharmacokinetic studies were obtained in 38 children over a 24-h period following cytotoxin treatment with or without CsA on days 1 and 4. Drug concentrations were quantitated using validated HPLC methods, and pharmacokinetic parameters were determined using compartmental modeling with an iterative two-stage approach, implemented on ADAPT II software. Etoposide displayed a greater degree of interindividual variability in clearance and systemic exposure than mitoxantrone. With CsA treatment, etoposide and mitoxantrone mean clearance declined by 71% and 42%, respectively. These effects on clearance, in combination with the empiric 40% dose reduction for either cytotoxin, resulted in a 47% and 12% increases in the mean AUC for etoposide and mitoxantrone, respectively. There were no differences in the rates of stomatitis or infection between the two groups. CsA treatment resulted in an increased incidence of hyperbilrubinemia, which rapidly reversed upon conclusion of drug therapy. The variability observed in clearance, combined with the empiric 40% dose reduction of the cytotoxins, resulted in statistically similar systemic exposure and similar toxicity.