Jacob Nersting MSc PhD
Kjeld Schmiegelow MD Med Sci
Paediatrics and Adolescent Medicine,
The Juliane Marie Centre,
The University Hospital Rigshospitalet,
The Institute of Gynaecology,
Obstetrics and Paediatrics,
The Faculty of Health Sciences,
The University of Copenhagen, Denmark.
Optimal intensity of 6-mercaptopurine (6MP)-based maintenance therapy is essential for curing childhood acute lymphoblastic leukaemia, but requires extensive individualised dose adjustment because bioavailability and metabolism of 6MP vary substantially between patients.
Acute lymphoblastic leukaemia (ALL) accounts for 25% of all childhood cancers with an annual incidence of approximately four per 100,000 children below the age of 18 years. The cure rate has improved dramatically over the last decades, and contemporary treatment protocols yield overall survival rates of 85–90%.
Patients are allocated to different risk groups based on initial presentation and treatment response of the leukaemia. The treatment backbone is virtually identical for all groups, but additional anticancer agents and intensive treatment blocks are added for the higher-risk patients. The backbone generally comprises:
- a four-to-six week, three-to-four drug induction therapy to obtain morphological remission (<5% leukaemic blasts in the bone marrow)
- a consolidation phase with varying combinations of anticancer agents
- one or two delayed intensifications (generally with the same classes of drugs as in induction therapy)
- central nervous system-directed treatment with intrathecal chemotherapy with or without cranial irradiation
- one to two years’ maintenance or continuation therapy with daily oral 6-mercaptopurine (6MP) and weekly methotrexate (MTX). 6MP and the closely related 6-thioguanine (6TG) are also used frequently during the earlier phases of chemotherapy, generally in combination with high-dose MTX or low-dose cytarabine.
Most leukaemia relapses occur during or after maintenance therapy, and, if this treatment phase is truncated 12 months from diagnosis, approximately 40% of all patients will relapse.(1)
The bioavailability of oral 6MP is generally low (approximately 30%), and highly variable both between and within patients, with up to 70-fold variations in area under the curve. Xanthine oxidase (XO), which converts 6MP to 6-thiouric acid (Figure 1), is expressed in intestines and liver and is responsible for the extensive first-pass metabolism. 6MP enters cells from the plasma by passive diffusion, and is transformed from an inactive prodrug to its active metabolites (Figure 1). The major cytotoxic metabolites, 6-thioguanine nucleotides (6TGN), are formed through a thioinosine monophosphate (TIMP) intermediate by coupling 6MP with phosphoribosyl pyrophosphate (PRPP). Subsequent base modification and phosphorylation yields thioguanine triphosphate nucleotides, the deoxy form of which is incorporated into DNA. The fraudulent DNA-6TGN activates post-replication mismatch repair systems that leads to DNA strand breaks and apoptosis.
The enzyme thiopurine methyltransferase (TPMT) thio-methylates 6MP, 6TG and a number of their metabolites, and thereby reduces the amount of the drug available for 6TGN formation. With equivalent 6MP and 6TG doses, more 6TGN is formed with 6TG because the initial metabolic transformations are bypassed and less is lost to the methylation pathways. By contrast, methylated metabolites, especially methylthioinosine monophosphate (MeTIMP), are potent inhibitors of purine de novo synthesis (PDNS). Diminished PDNS reduces the endogenous nucleotide pools that compete with 6TGN for DNA incorporation. Whereas methylated metabolites themselves cause little cytotoxicity, they are important determinants of treatment outcome by potentiating the DNA incorporation of 6TGN.(2) This role of methylated metabolites should be further explored in clinical trials.
The folic acid analogue MTX, which is often co-administered with thiopurines, is an inhibitor of folate-dependent cellular processes, including purine and pyrimidine de novo synthesis. MTX also inhibits XO and thereby increases 6MP bioavailability. In addition, MTX depletes cellular stores of the reduced (activated) folates that are required for regeneration of S-adenosyl methionine, the methyl donor in TPMT-mediated reactions. Finally, inhibition by MTX of PDNS and the associated PRPP consumption preserves cellular PRPP, which is required for 6MP salvage.
Monitoring and adjusting thiopurine maintenance therapy
The thiopurines are among the most challenging agents in the ALL protocols, not least when it comes to optimisation of maintenance therapy. In most of Europe, the starting oral dose of 6MP is 50mg/day/m2, whereas in the UK, the Nordic countries, and most of the US, it is 75mg/day/m2. Although collaborative groups have relatively similar dose adjustment guidelines,(3) there are no published detailed comparisons of the actual average doses given during maintenance therapy with these two dosing strategies. Individual dose adjustment is complicated by the large inter-patient variability in bioavailability and metabolic activation of thiopurines, and there are no easy or well-established strategies for compensating this pharmacokinetic diversity, in part because methods for determination of intracellular metabolites are not generally available.
Thiopurine dose adjustment by toxicity is recommended in all current ALL protocols. This strategy assumes that the individual variation in 6MP pharmacokinetics and pharmacodynamics affects the antileukaemic effect and myelosuppression in parallel. Accordingly, the maintenance therapy dose of 6MP is targeted to a certain degree of myelosuppression, generally a white blood cell (WBC) of 1.5–3.0×109/l.3 Although the degrees of leukopenia and neutropenia during maintenance therapy are inversely related to the risk of relapse, patients vary in their normal WBC levels, and the on-treatment WBC level is thus only a weak surrogate for the treatment intensity. Hence, dosing by WBC may not secure equal treatment intensity and is not feasible for all patients. Moreover, toxicity-guided dosing relies heavily on physicians’ willingness to comply with the protocol guidelines and their experience with maintenance therapy. Finally, both difficulties in administering tablets and in accepting the toxicities induced by 6MP/MTX maintenance therapy may reduce patients’ adherence to the treatment.
In addition to leukopenia, most patients on 6MP/MTX maintenance therapy experience hepatotoxicity with a rise in aminotransferases, although rarely with affected liver function test. In general, high aminotransferases levels should not lead to dose adjustments (see below) as they have been associated with a reduced risk of relapse.(4)
Pharmacologicallyguided adjustment is an alternative or supplement to dosing by toxicity. Erythrocyte levels of free 6TGN (Ery-6TGN) is related to the risk of relapse, but dosing 6MP according to Ery-6TGN does not improve cure rates,(5) likely because Ery-6TGN levels are inadequate surrogates for the events in the nucleated target cells, where the end-point metabolite is DNA-6TGN. MeTIMP enhances DNA-6TGN incorporation, due to inhibition of purine de novo synthesis 2 and is furthermore associated with hepatotoxicity.(6) This may explain why patients with low methylated 6MP metabolite levels, for example in TPMT-deficient patients, tolerate Ery-6TGN levels up to ten-times higher than TPMT wild-type patients. Similarly, lack of methylated 6MP metabolites may explain why replacing 6MP with 6TG, as tested in three randomised studies by the US CCG, the German COALL and the British UKALL groups failed to improve ALL cure rates, even though children receiving 6TG had several-fold higher Ery-6TGN levels.
Due to the associations of leukopenia with high Ery-6TGN levels and between high aminotransferase levels and high erythrocyte MeTIMP levels, poor treatment adherence should be strongly suspected in patients who despite 6MP dose increments, develop neither leukopenia nor a rise in aminotransferases (Table 1).
Variations in the human genome significantly influence the response to leukaemia treatment. The best explored of these variants are the low activity alleles of TPMT,(7) of which the *3B/*3C/*3D variants involving the single nucleotide polymorphisms G460A and A719G account for 90% or more in Caucasian individuals; Approximately 10% are TPMT heterozygous, with one wild-type and one low-activity allele, and one in 300 patients is TPMT-deficient with two low-activity alleles and risk life-threatening myelosuppression at standard 6MP doses.(7) This far, thiopurine dosing according to the TPMT genotype is the only example of pharmacogenetically-based drug dosing in childhood ALL.(3,8) However, the benefits of this strategy remain uncertain; TPMT heterozygous patients have high intracellular 6TGN levels, reduced tolerance to 6MP, and higher cure rates,(9) but also higher risk of second cancers.(10) Unfortunately, dose increments of 6MP in TPMT wild type patients to obtain higher intracellular 6TGN levels and improved cure rates will not mirror the situation in TPMT low-activity patients, since the additional 6MP is shunted to methylated metabolites causing more liver toxicity.(6)
Measuring TPMT activity in erythrocytes is an alternative to genotyping that may also identify rare low-activity variants for which testing at the DNA level is not feasible. However, as with other erythrocyte enzymes the TPMT activity is inversely related to the erythrocyte age. Accordingly, the TPMT activity will be low at diagnosis of ALL due to reduced erythropoiesis and higher during maintenance therapy, when the erythrocyte life span is reduced. Consequently, at diagnosis, TPMT-deficient ALL patients can be identified by TPMT phenotyping, whereas heterozygous and wild type patients can be difficult to distinguish.
Variants in inosine triphosphate pyrophosphatase (ITPA), an enzyme that dephosphorylates TITP (Figure 1), may also influence 6MP pharmacodynamics. An ALL maintenance therapy study at St Jude Children’s Research Hospital demonstrated associations of low-activity ITPA variants with increased levels of methylated 6MP metabolites in children receiving 6MP doses prospectively adjusted according to TPMT status. More importantly, carriers of ITPA variants also experienced more episodes of febrile neutropenia, a potentially life-threatening toxicity. This illustrates that adjustment for major determinants in thiopurine disposition allows identification of less influential factors. Integrating these in pharmacogenetic models may lead to refined 6MP dosing guidelines which may provide a better supplement or even replace dosing strategies based on toxicity or pharmacological monitoring.
Until pharmacogenetic guidelines are available, 6MP/MTX maintenance therapy should be adjusted vigorously to obtain the targeted degree of myelosuppression. Although patients show no clinical symptoms in this phase, maintenance therapy should be taken as seriously as the other treatment phases of childhood ALL. In patients for whom leukopenia is not achieved, poor treatment adherence should be suspected if dose increments do not lead to a rise in aminotransferases.
- 6-Mercaptopurine (6MP) is one of the most important antileukaemic agents.
- The small therapeutic index and highly variable pharmacokinetics/dynamics of 6MP necessitate individual dose adjustments
- In general, dose increments will lead to either leukopenia or a rise in aminotransferases.
- If neither is experienced, poor patient adherence to the treatment should be suspected
- Patient adherence can be explored through measurements of erythrocyte levels of 6MP metabolites.
- Toyoda Y et al. Six months of maintenance chemotherapy after intensified treatment for acute lymphoblastic leukemia of childhood. J Clin Oncol 2000;18:1508–16.
- Hedeland RL et al. DNA incorporation of 6-thioguanine nucleotides during maintenance therapy of childhood acute lymphoblastic leukaemia and non-Hodgkin lymphoma. Cancer Chemother Pharmacol 2010;66:485–91.
- Arico M et al. The seventh international childhood acute lymphoblastic leukemia workshop report: Palermo, Italy, January 29–30, 2005. Leukemia 2005;19:1145–52.
- Schmiegelow K, Pulczynska M. Prognostic significance of hepatotoxicity during maintenance chemotherapy for childhood acute lymphoblastic leukaemia. Br J Cancer 1990;61:767–72.
- Schmiegelow K et al. Intensification of mercaptopurine/methotrexate maintenance chemotherapy may increase the risk of relapse for some children with acute lymphoblastic leukemia. J Clin Oncol 2003;21:1332–9.
- Nygaard U, Toft N, Schmiegelow K. Methylated metabolites of 6-mercaptopurine are associated with hepatotoxicity. Clin Pharmacol Ther 2004;75:274–81.
- Wang L, Weinshilboum R. Thiopurine S-methyltransferase pharmacogenetics: insights, challenges and future directions. Oncogene 2006;25:1629–38.
- Relling MV et al. Clinical Pharmacogenetics Implementation Consortium guidelines for thiopurine methyltransferase genotype and thiopurine dosing. Clin Pharmacol Ther 2011;89:387–91.
- Schmiegelow K et al. Thiopurine methyltransferase activity is related to the risk of relapse of childhood acute lymphoblastic leukemia: results from the NOPHO ALL-92 study. Leukemia 2009;3:557–64.
- Schmiegelow K et al. Methotrexate/6-mercaptopurine maintenance therapy influences the risk of a second malignant neoplasm after childhood acute lymphoblastic leukemia – results from the NOPHO ALL-92 study. Blood 2009;113:6077–84.