The influence of genetic polymorphism in inosine triphosphate pyrophosphatase (ITPA) on thiopurine-induced adverse events has not been investigated in the context of combination chemotherapy for acute lymphoblastic leukemia (ALL). This study investigated the effects of a common ITPA variant allele (rs41320251) on mercaptopurine metabolism and toxicity during treatment of children with ALL. Significantly higher concentrations of methyl mercaptopurine nucleotides were found in patients with the nonfunctional ITPA allele. Moreover, there was a significantly higher probability of severe febrile neutropenia in patients with a variant ITPA allele among patients whose dose of mercaptopurine had been adjusted for TPMT genotype. In a cohort of patients whose mercaptopurine dose was not adjusted for TPMT phenotype, the TPMT genotype had a greater effect than the ITPA genotype. In conclusion, genetic polymorphism of ITPA is a significant determinant of mercaptopurine metabolism and of severe febrile neutropenia, after combination chemotherapy for ALL in which mercaptopurine doses are individualized on the basis of TPMT genotype.

Peptide deformylase (PDF) catalyzes the hydrolytic removal of the N-terminal formyl group from nascent ribosome-synthesized polypeptides in eubacteria. PDF represents a novel class of mononuclear iron protein, which utilizes an Fe(2+) ion to catalyze the hydrolysis of an amide bond. This Fe(2+) enzyme is, however, extremely labile, undergoing rapid inactivation upon exposure to molecular oxygen, and is spectroscopically silent. In this work, we have replaced the native Fe(2+) ion with the spectroscopically active Co(2+) ion through overexpression in the presence of Co(2+). Co(2+)-substituted PDF (Co-PDF) has an activity 3-10-fold lower than that of the Fe(2+)-PDF but is highly stable. Steady-state kinetic assays using a series of substrates of varying deformylation rates indicate that Co-PDF has the same substrate specificity as the native enzyme. Co-PDF and Fe-PDF also share the same three-dimensional structure, pH sensitivity, and inhibition pattern by various effector molecules. These results demonstrate that Co-PDF can be used as a stable surrogate of Fe-PDF for biochemical characterization and inhibitor screening. The electronic absorption properties of the Co(2+) ion were utilized as a probe to monitor changes in the enzyme active site as a result of site-directed mutations, inhibitor binding, and changes in pH. Mutation of Glu-133 to an alanine completely abolishes the catalytic activity, whereas mutation to an aspartate results in only approximately 10-fold reduction in activity. Analysis of their absorption spectra under various pH conditions reveals pK(a) values of 6.5 and 5.6 for the metal-bound water in E133A and E133D Co-PDF, respectively, suggesting that the metal ion alone is capable of ionizing the water molecule to generate the catalytic nucleophile, a metal-bound hydroxide. On the other hand, substrate binding to the E133A mutant induces little spectral change, indicating that in the E.S complex the formyl carbonyl oxygen is not coordinated with the metal ion. These results demonstrate that the function of the active-site metal is to activate the water molecule, whereas Glu-133 acts primarily as a general acid, donating a proton to the leaving amide ion during the decomposition of the tetrahedral intermediate.

The incidence of infection-related death was low. However, young age, white race, intensive chemotherapy, and lack of neutrophil surge after dexamethasone treatment were associated with infection-related complications. Close monitoring for prompt administration of antibiotics and modification of chemotherapy should be considered in these patients.

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To determine the clinical significance of minimal residual disease (MRD) in patients with prognostically relevant subtypes of childhood acute lymphoblastic leukemia (ALL), we analyzed data from 488 patients treated in St. Jude Total Therapy Study XV with treatment intensity based mainly on MRD levels measured during remission induction. MRD levels on day 19 predicted treatment outcome for patients with hyperdiploid >50 ALL, NCI standard-risk B-ALL or T-cell ALL, while MRD levels on day 46 were prognostic for patients with NCI standard-risk or high-risk B-ALL. Patients with t(12;21)/(ETV6-RUNX1) or hyperdiploidy >50 ALL had the best prognosis; those with a negative MRD on day 19 had a particularly low risk of relapse: 1.9% and 3.8%, respectively. Patients with NCI high-risk B-ALL or T-cell ALL had an inferior outcome; even with undetectable MRD on day 46, cumulative risk of relapse was 12.7% and 15.5%, respectively. Among patients with NCI standard-risk B-ALL, the outcome was intermediate overall but was poor if MRD was ≥1% on day ≥19 or MRD was detectable at any level on day 46. Our results indicate that the clinical impact of MRD on treatment outcome in childhood ALL varies considerably according to leukemia subtype and time of measurement.