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Background: Pediatric acute lymphoblastic leukemia (ALL) therapy is accompanied by treatment-related toxicities (TRTs) and impaired quality of life. In Australia and New Zealand, children with ALL are treated with either Children's Oncology Group (COG) or international Berlin-Frankfurt-Munster (iBFM) Study Group-based therapy. We conducted a prospective registry study to document symptomatic TRTs (venous thrombosis, neurotoxicity, pancreatitis and bone toxicity), compare TRT outcomes to retrospective TRT data, and measure the impact of TRTs on children's general and cancer-related health-related quality of life (HRQoL) and parents' emotional well-being., Methods: Parents of children with newly diagnosed ALL were invited to participate in the ASSET (Acute Lymphoblastic Leukaemia Subtypes and Side Effects from Treatment) study and a prospective, longitudinal HRQoL study. TRTs were reported prospectively and families completed questionnaires for general (Healthy Utility Index Mark 3) and cancer specific (Pediatric Quality of Life Inventory (PedsQL)-Cancer Module) health related quality of life as well the Emotion Thermometer to assess emotional well-being., Results: Beginning in 2016, 260 pediatric patients with ALL were enrolled on the TRT registry with a median age at diagnosis of 59 months (range 1-213 months), 144 males (55.4%), majority with Pre-B cell immunophenotype, n = 226 (86.9%), 173 patients (66.5%) treated according to COG platform with relatively equal distribution across risk classification sub-groups. From 2018, 79 families participated in the HRQoL study through the first year of treatment. There were 74 TRT recorded, reflecting a 28.5% risk of developing a TRT. Individual TRT incidence was consistent with previous studies, being 7.7% for symptomatic VTE, 11.9% neurotoxicity, 5.4% bone toxicity and 5.0% pancreatitis. Children's HRQoL was significantly lower than population norms throughout the first year of treatment. An improvement in general HRQoL, measured by the HUI3, contrasted with the lack of improvement in cancer-related HRQoL measured by the PedsQL Cancer Module over the first 12 months. There were no persisting differences in the HRQoL impact of COG compared to iBFM therapy., Conclusions: It is feasible to prospectively monitor TRT incidence and longitudinal HRQoL impacts during ALL therapy. Early phases of ALL therapy, regardless of treatment platform, result in prolonged reductions in cancer-related HRQoL., (© 2022. The Author(s).)

Both polyamine metabolism and DNA methylation play an important role in normal and malignant growth. Specific enzyme inhibitors or drugs that interfere with these metabolic pathways have proven to be potential anticancer agents. Since DNA methylation and polyamine metabolism depend on a common substrate, i.e. S-adenosylmethionine, interaction between both pathways can be expected. Little is known about the relationship between these pathways but studies are available indicating that polyamines and DNA methylation are directly or indirectly interconnected, metabolically as well as physiologically with respect to the regulation of cell growth, differentiation and cancer development. These considerations give rise to the possibility that, by targeting both pathways, a more profound and effective inhibitory effect on the growth of malignant cells can be achieved. In previous studies we showed that 6-MP (6-mercaptopurine) as well as MTX (methotrexate), well-known drugs in the treatment of acute lymphoblastic leukaemia, inhibit DNA methylation and induce apoptosis in malignant blood cells. Our recent results show that combined treatment with 6-MP, MTX and drugs interfering with polyamine metabolism has additive/synergistic effects on the growth, cell viability and/or apoptotic death of leukaemic cells. Such a combination therapy could have great clinical value for patients in which therapy using inhibitors of thiopurines/purine metabolism has failed.

Background: Inosine 5'-monophosphate dehydrogenase (IMPDH; EC1.1.1.205) catalyzes the rate-limiting step in guanine nucleotide biosynthesis, and may play an important role in treatment of patients with antipurines., Methods: We used an HPLC method to measure the IMPDH activity in peripheral blood and bone marrow mononuclear cells (MNC). IMPDH activities were determined in children who were diagnosed with and treated for acute lymphoblastic leukemia (ALL), and in a group of control children., Results: The median IMPDH activity for control children was 350 pmol/10(6) pMNC/hr (range 97-896; n = 47). No gender or age differences were observed. IMPDH activity at diagnosis of ALL was correlated with the percentage of peripheral blood lymphoblasts (r = 0.474; P < 0.001; n = 71). The median IMPDH activity at diagnosis was 410 pmol/10(6) pMNC/hr (range 40-2009; n = 76), significantly higher than for controls (P = 0.012). IMPDH activity significantly decreased after induction treatment, and during treatment with methotrexate (MTX) infusions (median 174 pmol/10(6) pMNC/hr; range 52-516; n = 21). The activity remained low during maintenance treatment with 6-mercaptopurine (6MP) and MTX, at a significantly lower level than for controls (P < 0.004). One year after cessation of treatment IMPDH activity returned to normal values., Conclusion: The decrease of IMPDH activity at remission of ALL seems to be at least partly due to the eradication of lymphoblasts with the type 2 isoform of the enzyme.

Thiopurines are used for treatment of several diseases. Cytotoxicity is caused by the derived compounds 6-thioguanine nucleotides (TGNs) and methyl-6-thioinosine monophosphate (methylthio-IMP). The 6-thiopurine mononucleotides 6-thio-IMP (thio-IMP), 6-thio-GMP (thio-GMP) and methylthio-IMP can be catabolized by purine 5'-nucleotidase. It has been shown that the various 5'-nucleotidases are key enzymes for (6-thio)-purine metabolism. We aimed to investigate whether the overall 5'-nucleotidase (5'NT) activity is correlated with the efficacy and toxicity of 6-thiopurine nucleotides. Substrate affinity of 5'NT for IMP, GMP, AMP, thio-IMP, thio-GMP and methylthio-IMP was studied in human lymphocytes. For each of the substrates, the pH for optimal overall enzyme activity has been determined at a pH range between 6 and 10. At the optimal pH, assays were performed to establish Km and Vmax values. Optimal pH values for the various substrates were between 7 and 8.5. Km values ranged from 33 to 109 microM, Vmax ranged from 3.99 to 19.5 nmol/10(6) peripheral mononuclear cells (pMNC) h, and Vmax/Km ratios ranged from 105 to 250. The results did not show a distinct preference of 5'NT activity for any of the tested thiopurine nucleotides. The enzyme kinetic studies furthermore revealed substrate inhibition by thio-IMP and thio-GMP as a substrate. Inhibition by thio-GMP also seems to occur in patients treated with 6-mercaptopurine (6 MP); subsequently, this may lead to toxicity in these patients.

Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme, catalysing S-methylation of aromatic and heterocyclic sulphhydryl compounds. TPMT activities and genotypes have been determined in patients with acute lymphoblastic leukaemia (ALL) and in control children. Median red blood cell (RBC) TPMT activity in ALL patients at diagnosis was significantly lower than in controls (median 11.5 pmol/10(7) RBC*hr; range 1.7-30.7; n = 191 vs. 14.6 pmol/10(7) RBC*hr; range 1.6-50.7; n = 140). 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 6-mercaptopurine (6MP) than at diagnosis and in controls. However, we observed that TPMT activity was already significantly increased after the induction therapy, before the patients received 6MP (median 17.5; range 3.9-40.3 pmol/10(7) RBC*hr; n = 139). In vitro experiments indicate that the early increase of TPMT activity during treatment may be explained by the use of antifolates, e.g., methotrexate and trimethoprim.

Aberrant DNA methylation can occur early in neoplastic transformation and may lead to the development of cancer. We describe the alterations of methylation patterns at the DNA sequence level which occurred in the 5' region of the calcitonin gene in lymphoblasts from 14 pediatric patients with acute lymphoblastic leukemia (ALL). The DNA methylation status of 25 CpG sites was determined by sequence analysis after bisulfite treatment of the DNA. This method showed that 13 out of 14 patients had increased numbers of methylated CpG sites in the calcitonin gene region at initial diagnosis when compared to control DNA from healthy individuals. The 5' region of the calcitonin gene appears to be methylated to a significantly higher degree in T lineage ALL compared to B lineage ALL (P < 0.01). Each of six ALL patients who were investigated at initial diagnosis and at relapse showed alterations in DNA methylation between the two stages. These six cases were also investigated by Southern blot analysis with methylcytosine-sensitive restriction enzymes and this method showed an increase in DNA methylation in only four of the six cases. The DNA sequencing method thus appears to be better suited to assess alterations of DNA methylation than Southern blot analysis. There are marked regional differences in the frequency of methylation of individual CpG sites and in the frequency of alterations between the two stages. Our results show that alterations in DNA methylation continue to occur from the initial stage to the relapse stage of ALL, suggesting that aberrant DNA methylation may play a role in tumor progression.

6-mercaptopurine (6MP) cytotoxicity is caused by thioguanine and methylthioinosine nucleotides. Thiopurine methylation occurs to a large extent in vivo and in vitro. In this reaction, S-adenosyl-L-methionine (AdoMet), produced from methionine and ATP, is converted into S-adenosyl-L-homocysteine (AdoHcy) which, in turn, is hydrolyzed into homocysteine. Remethylation of homocysteine into methionine is inhibited by methotrexate (MTX). In cultured lymphoblasts, AdoMet: AdoHcy ratio and DNA methylation decrease after incubation with 6MP. The aim of the present study was to investigate the influence of high-dose 6MP on the methylation capacity in children with acute lymphoblastic leukemia. Five patients received 4 courses with high-dose intravenous MTX (5' g.m-2 in 24 hr) immediately followed by high-dose 6MP (1300 mg.m-2 in 24 hr). Five control patients received high-dose MTX and oral 6MP (25 mg.m -2 daily for 8 weeks). Leucovorin rescue was started at 36 hr in both groups. In the intravenous 6MP group, 6-methylmercaptopurine, its riboside, and 6-methylmercapto-8-hydroxypurine were detectable in plasma in concentrations of 0.3-2.6 muM (6MP steady state levels: 11.6 muM). In red blood cells, mean methylthioinosine nucleotide levels were one third of those of ATP (13.1 nmol/10(8)). AdoHcy levels (10 pmol/10(8)) remained constant in both groups and AdoMet was not detectable ( < 20 pmol/10(8)). In both groups, plasma homocysteine increased and methionine decreased following administration of MTX. The delay in the recovery of methionine in the intravenous 6MP group after MTX infusion is probably the result of an increased demand on methyl groups during 6MP infusion.

6-Mercaptopurine, a hypoxanthine antimetabolite, is used in the treatment of acute lymphoblastic leukemia (ALL) in children. Extensively metabolized before it exerts cytotoxic action, it is catabolized into 6-mercapto-2,8-dihydroxypurine (thiouric acid), which is excreted by the kidneys. We describe a metabolite of 6-mercaptopurine, 6-methylmercapto-8-hydroxypurine, whose presence has not been previously reported in plasma. This compound was found in high concentrations in plasma during high-dose 6-mercaptopurine infusions (1300 mg/m2 in 24 h). This previously unknown compound was identified by reversed-phase HPLC with absorbance detection and by gas chromatography-mass spectrometry. The pathways leading to 6-methylmercapto-8-hydroxypurine in vivo are not yet fully understood. In a group of 17 patients treated with four courses of high-dose 6-mercaptopurine infusions according to the ALL-8 treatment protocol of the Dutch Childhood Leukemia Study Group, the steady-state concentrations of 6-methylmercapto-8-hydroxypurine in plasma were one-fifth of the parent drug concentrations, with wide interindividual variation. The formation of high concentrations of 6-methylmercapto-8-hydroxypurine in plasma, especially during the infusion, probably indicates another catabolic pathway of high-dose 6-mercaptopurine, apart from its conversion into thiouric acid.