Purine enzymes and thiopurine methyltransferase in childhood acute lymphoblastic leukemia

This thesis aims to get more insight into the metabolism of the drug 6-mercaptopurine (6MP) in pediatric patients with ALL. 6MP is a hypoxanthine analogue. The course of three key enzymes involved in 6MP metabolism is described during ALL treatment: inosine monophosphate dehydrogenase (IMPDH), 5'-nucleotidase (5'NT) and thiopurine S-methyltransferase (TPMT). TPMT activities and genotypes have been determined in patients with ALL and in control children. Median red blood cell (RBC) TPMT activity in ALL patients at diagnosis was significantly lower than in controls. This reduction of TPMT activity in ALL patients was not due to differences in the frequency of mutations in the TPMT gene. In concordance with other authors, we found a higher TPMT activity during maintenance treatment with 6MP than at diagnosis and in controls. However, we observed that TPMT activity was already significantly increased to a maximal level after the induction therapy, before the patients received 6MP. In vitro experiments showed that the early increase of TPMT activity during treatment was presumably caused by the use of antifolates, e.g., methotrexate and trimethoprim. Monitoring IMPDH activity during ALL treatment, previously described findings showing that IMPDH activity at diagnosis was related to the number of lymphoblasts in peripheral blood of the patients with ALL were confirmed. The median IMPDH activity at diagnosis was significantly higher than for controls. IMPDH activity significantly decreased during treatment, compared to that at diagnosis, following the eradication of lymphoblasts. One year after cessation of treatment, IMPDH activity has returned to normal values. To investigate whether total purine 5'NT activity was correlated with the efficacy and toxicity of 6-thiopurine nucleotides, we studied substrate affinity of purine 5'NT for (thio)purine nucleotides in human lymphocytes. The results did not show a distinct preference. The enzyme kinetic studies furthermore revealed substrate inhibition by thio-IMP, and especially by thio-GMP as a substrate. Inhibition by thio-GMP also seems to occur in ALL patients during treatment with 6MP. We also showed a negative correlation between purine 5'NT activity and thio-GMP concentration in patients' pMNC. This may lead to hematological toxicity in thiopurine-treated patients. So, by the measurement of purine key enzyme and TPMT activities, we have gained more knowledge on 6MP metabolism in ALL treatment. This knowledge can be used for further investigations on individualisation of thiopurine treatment in order to optimise efficacy and circumvent too much toxicity.