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Background: 6-mercaptopurine has been a standard component of long-term continuing treatment for childhood lymphoblastic leukaemia, whereas 6-thioguanine has been mainly used for intensification courses. Since preliminary data have shown that 6-thioguanine is more effective than 6-mercaptopurine, we compared the efficacy and toxicity of the two drugs for childhood lymphoblastic leukaemia., Methods: Consecutive children with lymphoblastic leukaemia diagnosed in the UK and Ireland between April, 1997, and June, 2002, were randomly assigned either 6-thioguanine (750 patients) or 6-mercaptopurine (748 patients) during interim maintenance and continuing therapy. All patients received 6-thioguanine during intensification courses. We analysed event-free and overall survival on an intention-to-treat basis. We obtained toxicity data using an adverse-event reporting system, with follow-up questionnaires to seek detailed information for specific toxicities. This trial is registered with the International Standard Randomised Controlled Number 26727615 with the name ALL97., Findings: After a median follow up of 6 years, there was no difference in event-free or overall survival between the two treatment groups. Although 6-thioguanine conferred a significantly lower risk of isolated CNS relapse than did 6-mercaptopurine (odds ratio [OR] 0.53, 95% CI 0.30-0.92, p=0.02), the benefit was offset by an increased risk of death in remission (2.22, 1.20-4.14, p=0.01), mainly due to infections during continuing therapy. Additionally, 95 patients developed veno-occlusive disease of the liver. Of these, 82 were randomly assigned 6-thioguanine, representing 11% of all 6-thioguanine recipients. On long-term follow-up, about 5% of 6-thioguanine recipients have evidence of non-cirrhotic portal hypertension due to periportal liver fibrosis or nodular regenerative hyperplasia., Interpretation: Compared with 6-mercaptopurine, 6-thioguanine causes excess toxicity without an overall benefit. 6-mercaptopurine should remain the thiopurine of choice for continuing therapy of childhood lymphoblastic leukaemia.

Concern about late adverse effects of cranial radiotherapy (XRT) has led to alternative approaches to eliminate leukaemia from the central nervous system (CNS) in childhood acute lymphoblastic leukaemia (ALL). The Medical Research Council UKALL XI trial recruited 2090 children with ALL between 1990 and 1997. Median follow-up is 7 years 9 months; event-free survival (EFS) and overall survival were 63.1% and 84.6%, respectively, at 5 years and 59.8% and 79.4% at 10 years. The isolated CNS relapse rate was 7.0% at 10 years. Patients were randomized for CNS-directed therapy within white blood cell (WBC) groups. For WBC <50 x 10(9)/l, high-dose intravenous methotrexate (HDMTX) (6-8 g/m2) with intrathecal methotrexate (ITMTX) was compared with ITMTX alone, and was significantly better at preventing isolated and combined CNS relapse, but non-CNS relapses were similar. There was no significant difference in EFS at 10 years, 64.1% [95% confidence interval (CI) 60.4-67.8] with HDMTX plus ITMTX, and 63.0% (95% CI 59.5-66.5) with ITMTX alone. For WBC >/=50 x 10(9)/l, HDMTX with ITMTX was compared with XRT and a short course of ITMTX. CNS relapses were significantly fewer with XRT, but there was a non-significant increase in non-CNS relapses. EFS was not significantly different, being 55.2% (95% CI 47.8-62.6) at 10 years with XRT and 52.1% (95% CI 44.8-59.4) with HDMTX plus ITMTX.

The case records of 99 consecutive children with acute lymphoblastic leukaemia who received either 6-thioguanine (6-TG) or 6-mercaptopurine (6-MP) as maintenance therapy for at least 1 year were reviewed for hepatic veno-occlusive disease (VOD). Overall, 12% of those on 6-TG developed VOD (all boys). Isolated persistent thrombocytopenia appeared to be the earliest indicator of incipient VOD. Multivariate analysis identified male sex and 6-TG as risk factors. In all cases, VOD was mild and reversible on withdrawing 6-TG or replacing it with 6-MP. The data implicate a sex-linked polymorphic variation in xenobiotic pathways of thiopurine metabolism in the pathogenesis of VOD.

1. The aim of this study was to investigate 6-mercaptopurine (6MP) metabolism by human liver cytosol in vitro. 2. Cytosol was prepared from seven human livers (A-G). A single cytosol (C) was used to optimize incubation conditions. 3. Cytosols A-G were incubated with 6MP at 2, 10 and 500 microM for two fixed times (5 and 48 h). Parent drug, thiopurine and thionucleotide metabolites were quantitated by high performance liquid chromatography at all time points. 4. At 5 and 48 h the 2 microM and 10 microM 6MP incubations contained both 6MP and its initial nucleotide metabolite, thioinosine 5'-monophosphate (TIMP). In addition, the 10 microM 6MP 48 h incubates contained small amounts of 6-thioguanine (6TG, median 0.12 microM). At 500 microM 6MP all seven liver incubates produced a range of metabolites. At 48 h these included thiouric acid, 8-hydroxy-6-mercaptopurine and 6-methylmercaptopurine (median 31, 19.5 and 8.8 microM respectively), with TIMP, 6TG, thioxanthine and thioxanthine nucleotide at median concentrations of 61, 0.79, 2.11 and 0.80 microM respectively. Thioguanine nucleotides, major metabolites measured in vivo, were not detected. 5. These results indicate that the human liver 6MP metabolic profile is dependent upon drug concentration.

As a prelude to a nationwide randomized trial of thioguanine (TG) versus mercaptopurine (MP) for childhood lymphoblastic leukaemia we compared a pilot group of 23 children taking TG with a matched group taking MP. We assessed drug tolerance based on haematological toxicity and measured erythrocyte (RBC) concentrations of thioguanine nucleotides (TGN). The median tolerated dose of TG was 30 mg/m2 compared to 55 mg/m2 for MP. There was no difference in the pattern of anaemia or neutropenia between the two groups, but dose-limiting thrombocytopenia was more evident in the TG children (P< 0.001), four of whom had a decrease in platelet count to <20 x 10(9)/l compared to only one on MP. The median RBC TGN concentration for those on 40 mg/m2 TG was 1726 pmol/8 x 10(8) RBCs compared with 308 pmol/8 x 10(8) RBCs for those on 75 mg/m2 MP (P< 0.0001). There was an inverse correlation between RBC TGNs and neutrophil count in the MP group but not in those on TG. No correlation between metabolite concentration and thrombocytopenia was found in either group. These results provide further evidence that TG has a selective effect on platelets. They also showed that RBC TGN were, on average, 5-fold higher in those taking TG but did not obviously relate to myelotoxicity as found in children on MP. The higher concentrations seen may partly reflect the erythrocyte's ability to metabolize TG directly to TGN by pathways not open to MP.

This paper describes a specific and sensitive reversed-phase HPLC assay for the measurement of 6-methylthioguanine (methyl-TG) and methyl-TG nucleotides (methyl-TGNs) in red blood cells (RBCs), which is suitable for routine clinical use. Briefly, an ethyl acetate extract of RBCs is evaporated and reconstituted in 0.1 M HCl. The methyl-TG is separated from other thiopurines by reversed-phase HPLC and quantitated using UV detection. For the measurement of methyl-TGNs the free base (methyl-TG) is obtained by acid hydrolysis of the nucleotide back to the parent thiopurine. The intra-assay C.V. over the concentration range of 0.055-1.10 nmol methyl-TG per 4x10(8) (100 microl) RBCs ranged from 2.8 to 8.5%, and the mean recovery of methyl-TG over the calibration range was 61.6% (coefficient of variation, C.V., 3.8%). The lower limit of reproducibility was 0.055 nmol extracted from 100 microl RBCs. Analysis of blood samples from children with leukaemia receiving 6TG chemotherapy, revealed RBC methyl-TGNs at concentrations ranging from 323 to 1365 pmol per 8x10(8) RBCs. No methyl-TG was detected in any of the patient samples.

Aims: The response to 6-mercaptopurine (6MP) is highly variable. Its antileukaemic effect can be related to drug derived 6-thioguanine nucleotides (TGNs). The inherited level of thiopurine methyltransferase (TPMT) activity may be a major factor in the clinical response to 6MP because TPMT forms methylmercaptopurine metabolites (MeMPs) at the expense of TGNs. The aim of this study was to explore the clinical importance of TPMT phenotype., Methods: Thiopurine metabolism was studied in a consecutive cohort of children with acute lymphoblastic leukaemia (ALL) treated according to the Medical Research Council trial UK ALL XI. TPMT phenotype was measured in 38 children at diagnosis, and thiopurine metabolites were measured at defined times during 2 years treatment in 29 of these children., Results: TPMT activities at diagnosis ranged from 5.5 to 18.5 units ml(-1) packed RBCs, no different from the range of activities reported in healthy children. TGNs and MeMPs measured during the first 6MP cycle at 75 mg m(-2) ranged from 187 to 594 pmol 6TGNs, median 327, and 0.5 to 22.0 nmol MeMPs, median 4.5, per 8 x 10(8) RBCs. TPMT activity was not significantly related to the generation of MeMPs (r(s) = 0.06), but was negatively correlated to 6TGNs (r(s) = -0.44, P<0.025, n=29). TGNs were related to neutropenia at the point of dose reduction (r(s) = -0.5, P<0.01). TPMT activity was also inversely related to the duration of cytopenia driven 6MP withdrawal (r(s)= -0.41, P<0.05)., Conclusions: These findings support the suggestion that the inherited activity of TPMT in a given individual can modulate the cytotoxic effect of 6MP, and this information may help in clinical management.

Daily 6-mercaptopurine (6MP) forms the backbone of continuing chemotherapy for childhood lymphoblastic leukaemia (ALL). A major metabolic route is catalysed by thiopurine methyltransferase (TPMT). TPMT deficiency occurs in 1 in 300 individuals and results in high concentrations of thioguanine nucleotides (TGNs), cytotoxic 6MP metabolites. A leukaemic child taking 6MP repeatedly developed profound pancytopenias. TPMT deficiency was confirmed. TGN formation was then studied on attenuated 6MP dosages. Four weekly oral doses of 75 mg/m2 6MP produced TGNs of 2348 pmol/8 x 10(8) red cells, nearly double the maximum TGNs recorded in ALL children with TPMT activity taking long term daily 75 mg/m2 6MP. Grossly elevated TGN concentrations were also produced at 10% standard 6MP dosage (7.5 mg/m2 daily), accompanied by unacceptable 6MP toxicity (neutropenia, diarrhoea, vomiting). The child was eventually stabilised on 10% alternate day therapy and after 15 weeks TGNs were 1670 pmol, just above the upper end of the TGN range for ALL children with TPMT activity.

Measurement of red cell 6-mercaptopurine (MP) derived 6-thioguanine nucleotide (TGN) and methylmercaptopurine metabolites (MeMPs) can be used to monitor therapy in children who are administered MP for childhood lymphoblastic leukemia. Red cell TGNs are not influenced by the time of blood sampling in relation to the last MP dose. The purpose of this study was to find out whether the same is true for the MeMPs. Plasma MP and red cell MP metabolite pharmacokinetics were studied in seven children immediately before and for 4 hours after a protocol standardized dose of MP. Duplicate blood samples were taken, one was processed immediately whereas one was left at an ambient temperature for 24 hours. The variation in TGN and MeMP metabolites over the 0- to 4-hour period (10 time points per child) was within the error of the assays used. The coefficients of variation for the TGNs ranged from 2.7% to 7% and for the MeMPs, 4% to 10.7%. There was no difference in the TGN and MeMP concentrations measured when the blood samples were left for 24 hours. If a child takes a MP tablet immediately before a clinic appointment, it has no major influence on MeMP measurements.

The current U.K. trial protocol (UKALL XI) for childhood lymphoblastic leukaemia demands mercaptopurine (MP) dose escalation in children who tolerate daily 75 mg/m2 MP (100% dose) without cytopenias. The previous trial (UKALL X) did not. MP metabolism was studied in a group of UKALL XI children (n = 21) who tolerated 100% dosages and who were matched in this respect with a similar group of UKALL X children. Red blood cell MP derived thioguanine nucleotide (TGN) concentrations were measured in both groups under comparable conditions; at 75 mg/m2 MP there was no significant difference. MP dose escalation in the UKALL XI children produced higher TGN concentrations (TGNs at 100% vs 125% dosages, median difference 90 pmol/8 x 10(8) RBCs, 95% CI 25 to 165 pmol, P < 0.02). Assayed at the time of cytopenia induced dose reduction, the UKALL XI children had accumulated significantly higher TGN concentrations than the UKALL X children (median difference 78 pmol/8 x 10(8) RBCs, 95% CI 20 to 144, P < 0.02). These findings indicate that dose escalation in children tolerant of 100% MP dosages produces higher peak TGN concentrations.

The formation of intracellular thionucleotides are a prerequisite for mercaptopurine (MP) cytotoxicity, and interindividual variations in the inherited level of thiopurine methyltransferase (TPMT) activity regulate their formation. Measurement of pretreatment TPMT activities can identify the TPMT "deficient" patient and, conversely, the individual with very high enzyme activities. The former are at higher risk of acute toxicity and potentially fatal bone marrow failure and the latter of suboptimal treatment. Leukaemic children taking MP therapy who form inadequate amounts of thioguanine nucleotides (TGNs) do not experience drug toxicity and are at an increased risk of disease relapse. When low TGNs are due to very high TPMT activities, thioguanine may be a more appropriate thiopurine. Another cause of inadequate TGN concentrations is partial or noncompliance with oral chemotherapy. Compliance problems can be identified by the measurement of both TGNs and methylated drug metabolites.

Daily oral 6-mercaptopurine (6MP) is important in the treatment of childhood lymphoblastic leukaemia (ALL), but there is great inter-patient variability in the pattern of evident drug effect (myelosuppression) seen at a standard dose. In an attempt to reduce that variability the current practise in the United Kingdom for the last 4 years has been to escalate the amount prescribed in patients who do not experience cytopenias at 75 mg/m2. We undertook a study to see whether that strategy would increase the total dose of 6MP prescribed in such patients and whether it would alter the pattern of myelosuppression. Over a 6-month period we studied 44 children treated conventionally (without escalation) and compared them with another 44 (matched for sex) who were treated on the same protocol but where doses were increased in monthly 25% steps if 75 mg/m2 was tolerated without cytopenias. We then compared the two groups for the total dose of drug prescribed and the frequency and duration of neutropenia or thrombocytopenia. The median cumulative dose of 6MP received by the conventionally treated children (10,002 mg/m2) was not significantly different from that of the children treated with dose escalation (9,429 mg/m2). In a comparison of the 30 children who actually received inflated doses of 6MP with the 37 from the conventional cohort who would have been eligible to do so, it was again found that the cumulative median doses were similar (10,460 versus 10,916 mg/m2). There was a difference between the two groups in the pattern of myelosuppression -- the escalated group spent significantly more time off 6MP than did the non-escalated group (median 4.5 versus 3 weeks; P<0.005, 95% CI from -1 to -3). These findings imply that the method of dose escalation employed does not allow more 6MP to be prescribed in children tolerant of the standard dose. The chief effect seems to be to generate longer periods off therapy, and this could paradoxically decrease the anti-neoplastic activity of the drug. Alternative ways of prescribing should be explored.