The folate analog methotrexate (MTX) and the thio-substituted purine analog 6-mercaptopurine (6MP) became pioneering anticancer agents more than half a century ago, when first Farber et al1 and then Burchenal2,3 demonstrated that such drugs can induce temporary remissions in childhood leukemia. Soon cure became the goal,4,5 and through a series of trials it was shown that the chance of long-term remission of childhood acute lymphoblastic leukemia (ALL) was significantly improved, when patients received several years of postremission maintenance therapy with daily 6MP and weekly MTX.4–8 Today, ALL protocols include an induction regimen with 3 or 4 antileukemic drugs followed by several months of consolidation therapy.9–15 Then oral 6MP/MTX maintenance therapy is given until 2 to 3 years from diagnosis, the longer duration being reserved for boys due to their inferior prognosis with shorter therapy.16–20 Despite its long history, the antileukemic mechanisms of maintenance therapy remain to be revealed. Recent studies of NT5C2 mutations in relapsed ALL clones emerging during maintenance therapy21,22 support a direct antileukemic effect of maintenance therapy.23,24 ALL stem cells may be uniquely sensitive to inhibition of de novo pathways in nucleotide synthesis crucial for DNA repair, methylation, and mitotic duplication.25 In addition, maintenance therapy could modulate apoptotic pathways,26 or induce changes in the microenvironment of the leukemic stem cells,27–30 for example, by impeding antiangiogenesis.31,32 IS YEARS OF 6MP/MTX THERAPY NEEDED FOR ALL PATIENTS? Maintenance therapy seems to be important for most ALL subsets, including T-cell ALL21 and other patients with hyperleukocytosis at diagnosis,33 adolescents,34 and Down syndrome patients with ALL.35 Observational studies support that 6MP/MTX maintenance therapy is superior to other drug combinations,33 and that poor physician compliance or poor patient adherence significantly increase the risk of relapse.36–39 As intensification of induction and consolidation therapy have improved cure rates of ALL, currently being >80%,9,15,40 the necessity of several years of the less toxic maintenance therapy has been questioned. A systematic review of 42 randomized studies with 12,000 childhood ALL cases indicated that longer maintenance therapy gave a slightly (although statistically significantly) lower risk of relapse, but with no difference in survival due to a higher risk of death in remission.19 Furthermore, longer duration of maintenance therapy as well as higher 6MP/MTX drug doses have in 3 recent studies been associated with an increased risk of second malignancies,41–43 but so far the subset of patients at risk of developing second cancers has not been identified. Shortening ALL therapy from 24 to 18 months significantly reduces the probability of event-free survival (pEFS),44 and if all chemotherapy is truncated at 52 weeks from diagnosis, the pEFS5y may be as low as 60%, even for non–high-risk ALL patients.45 The fact that some children with ALL are cured after only a few months of chemotherapy7,45 is not surprising, as monitoring of minimal residual disease (MRD) has shown that patients may have 28,000 registered drug doses and calculated by weighting each registered dose according to the time interval to the next measurement. The median m6MP dose for all patients is 59.4 mg/m2/d. FIGURE 2 Distribution of mean white blood cell count (mWBC) and prescribed mean 6-mercaptopurine (m6MP) doses during maintenance therapy for 538 patients included in the NOPHO ALL-92 maintenance therapy study.61 Means are based on a total of >28,000 registered drug doses and blood counts and calculated by weighting each registration according to the time interval to the next registration. The median m6MP (59.4 mg/m2/d) and median mWBC (3.3×109/L) are significantly correlated (r S=0.20; P 90%, but is significantly reduced at doses >40 mg/m2.57 Similar to natural folates, MTX is converted into MTX polyglutamates (MTXPG, with 2 to 7 glutamyl residues) by the enzyme folylpolyglytamyl transferase, which enhances intracellular retention, inhibition of the target enzymes in purine and pyrimidine de novo synthesis, and treatment efficacy (Fig. 3).96 The propensity for MTX to undergo polyglutamation is higher for B-cell precursor ALL subtypes (not least the high-hyperdiploid cases) than for T-cell ALL.95,97,98 Accordingly, many groups offer high-dose MTX at doses of 5 g/m2/24 h during consolidation therapy to cure T-cell ALL. MTXPG bind tightly to and inhibit dihydrofolate reductase, the enzyme responsible for reducing folates to their bioactive tetrahydrofolate form.23 During weekly low-dose oral MTX therapy, MTXPG accumulates in red blood cell precursors in the bone marrow, and MTXPG with longer glutamyl chains are then retained in the erythrocytes (Ery-MTXPG) throughout their life span.99 Steady-state Ery-MTXPG is achieved after 4 to 8 weeks.100,101 High Ery-MTXPG levels have been associated with increased risk of myelotoxicity,100,102 but only a single Nordic study has found Ery-MTXPG levels significantly related to remission duration,102 and this association could not be confirmed in later studies,61,103 potentially due to more intensive use of intravenous MTX in these studies. No study has explored the impact on relapse rates of various Ery-MTX polyglutamate chain lengths. At steady state, Ery-MTXPG is both interindividually and intraindividually related to the dose of oral MTX and may thus be used for monitoring treatment adherence.34,100 PHARMACOGENETICS Single nucleotide polymorphisms in genes that affect the disposition of anticancer agents influence the outcome of childhood ALL.104,105 However, so far only TPMT variants have influenced drug dosing,76,96 and it is poorly explored which host genome variants that ultimately determine the complex metabolism and efficacy of thiopurines and MTX, how this influences the toxicity profiles across ethnic groups,106–110 and how such data should be applied for dose adjustments. TPMT The normal substrate for TPMT is not known, and, in the absence of thiopurines, TPMT-deficient individuals are clinically and biochemically normal. In white individuals, the most common variants are *3A,*3B, and *3C all involving G460A and/or A719G and accounting for at least 90% of low-activity alleles among white individuals of North European decent.63,76 Approximately 5% to 10% are TPMT heterozygous carrying 1 wild-type and 1 low-activity allele, and 1 in 300 is TPMT deficient and at risk of life-threatening myelosuppression at standard 6MP doses.111,112 Although thiopurine dosing according to the TPMT genotype has been implemented by a few ALL study groups,48,79,113 the benefits of this strategy remain uncertain. Compared with TPMT wild-type patients, heterozygous patients experience higher intracellular 6TGN levels, more myelotoxicity, higher cure rates,63,113,114 but probably also a higher risk of second cancers.41,115,116 The German BFM group that administered lower starting doses of 6MP (50 mg/m2) failed to confirm the association with second cancers.117 It is noteworthy that, a recent study indicated that reduction of oral 6MP starting doses from 75 to 50 mg/m2/d, reflecting these BFM data, did reduce the risk of second cancers among TPMT heterozygous patients, but at the same time lead to an increased risk of relapse.43 Measuring TPMT activity in erythrocytes is an alternative to genotyping and may also identify rare low-activity variants missed by routine allele testing. However, as TPMT activity is inversely related to the erythrocyte age,118 the TPMT activity will in general be increased during maintenance therapy when the erythrocyte life span is shortened, and be low at diagnosis of ALL due to reduced erythropoiesis, hampering reliable discrimination of heterozygous and wild-type TPMT phenotypes. Inosine Triphosphate Pyrophosphatase (ITPA) Low-activity alleles of ITPA, the enzyme that dephosphorylates thioinosine triphosphate (Fig. 3), may increase methylated thiopurine metabolite levels,119,120 the risk of hepatotoxicity121,122 and of bone marrow toxicity123 with febrile neutropenia,119,124 and potentially also the risk of relapse.110 The frequency of ITPA low-activity alleles show wide interethnic variability being 1% to 2% among Hispanics, but almost 20% in Asian populations, which may influence tolerance to thiopurine therapy.125 Other 6MP Metabolizing Genes Other 6MP metabolizing enzymes, such as xanthine oxidase and hypoxanthine guanine phosphoribosyl transferase (HGPRT) may vary among individuals,67,126 in part determined by genetic polymorphisms, and at least low HGPRT in B-cell precursor ALL has been associated with an inferior cure rate, although this association was not related to increased in vitro thiopurine resistance.127 MTX Several groups have demonstrated that MTX treatment efficacy is associated with polymorphisms in dihydrofolate reductase,128 thymidylate synthetase,129,130 reduced folate carrier,131 5,10-methylenetetrahydrofolate reductase, and methylenetetrahydrofolate dehydrogenase132 (for reviews on childhood ALL and rheumatoid arthritis, see Davidsen and colleagues105,133–135). However, the results of these studies are often contradictory with some studies demonstrating improved cure rates for a specific genetic polymorphism, whereas others demonstrate the opposite, many of the studies are small, most only address 1 or a few of the many genetic polymorphisms involved in the disposition of MTX, and in general they address responses to high-dose MTX rather than low-dose MTX maintenance therapy. Furthermore, it is impossible to evaluate whether a specific polymorphism exert its modifying effect on relapse rate and/or toxicities directly through changed MTX disposition or indirectly by modifying endogenous folate levels. So far no groups have adjusted their MTX treatment strategies based on polymorphisms in the MTX/folate pathway. TOXICITY AND RELAPSE RATE Leukopenia Dose adjustments guided by toxicity assumes that the individual variations in 6MP/MTX pharmacokinetics and/or pharmacodynamics affect leukemic and normal cells in parallel.136 For maintenance therapy, 6MP/MTX dosage is targeted to a preset degree of myelosuppression, generally a WBC of 1.5 to 3.0 (or 3.5)×109/L,48 but randomized studies demonstrating benefits hereof are lacking.137 Most observational studies have shown low WBC and/or ANC during maintenance therapy to be related to red blood cell levels of cytotoxic 6MP/MTX metabolites and/or to a reduced relapse rate.38,61,82,100,138–144 However, ANC correlates so closely with WBC, that it is virtually impossible to determine which of these 2 parameters is superior as guidance for dose adjustment (Fig. 4A). In the Nordic Society for Paediatric Haematology and Oncology (NOPHO) ALL92 maintenance therapy study,61 patients with an average ANC 28,000 blood counts and calculated by weighting each measurement according to the time interval to the next measurement. Each dot represents 1 patient. mWBC and mANC are highly correlated (r S=0.77; P 3.0 to 3.5×109/L despite prescribed 6MP dose increments, and in patients with a TPMT wild-type genotype/phenotype, if dose increments do not lead to a rise in aminotransferases. The randomized Brazilian ALL99 study indicated that intermittent oral high-dose 6MP with IV MTX 200 mg/m2/6 h not only improved adherence but also gave better pEFS than oral 6MP (50 mg/m2/d) with IM MTX 25 mg/m2/wk, although only for boys.185 However, the extent of patient adherence in the oral 6MP arm is difficult to assess, as 6MP/MTX metabolite measurements were not done, and it is also unclear whether the difference in 6MP and/or MTX dosing in the 2 treatment arms caused the difference in EFS. CIRCADIAN SCHEDULE The circadian schedule has a strong impact on efficacy and toxicity of a number of anticancer agents.186 Two maintenance therapy studies from the 1980s and 1990s found that the risk of relapse was several-fold higher for patients who reported taking 6MP and MTX in the morning compared with patients on evening schedule.187,188 It was speculated that differences in biological activity between malignant lymphoid cells and normal bone marrow cells determined these chronochemotherapeutical findings,189,190 but whatever the biological mechanism, changing patients from morning to evening schedule seemed a simple procedure to improve outcome, and this has become the general standard.48 However, a recent large study of 526 children on maintenance therapy with almost 10,000 E-6TGN/MTXpg measurements, found no association between relapse rates and the cumulative duration of evening dosage for the individual patient, when adjusting for 6MP and MTX doses, WBC levels during maintenance therapy, and Ery-6TGN and Ery-MTXPG levels.191 FOOD AND MAINTENANCE THERAPY Several small studies,56,59,192,193 although not all,194–196 have demonstrated reduced bioavailability for both MTX and 6MP, when the drugs are administered together with food, and for 6MP specifically with milk due to its content of xanthine oxidase.197 Accordingly, nearly all study groups recommend 6MP and MTX to be taken without concomitant ingestion of food.48 Still, titrating the dose of MTX and 6MP by toxicity should counterbalance lower bioavailability, and restricting the individual patients’ choices of drug administration could reduce adherence. Only 1 large clinical study has explored the prognostic impact on administering 6MP/MTX with food, and this study demonstrated no significant influence of concomitant food ingestion on relapse rate, this also being the case within subgroups defined by their circadian schedule.188 COADMINISTRATION OF OTHER DRUGS It is unproven that alternative or additive components of maintenance therapy such as intravenous 6MP,198,199 6TG, allopurinol, high-dose MTX,9 vincristine/glucocorticoid,200,201 or more intensive reinductions19 significantly reduce relapse rates with contemporary ALL therapy, although they can add to the burden of myelotoxicity202 and hepatotoxicity, which may necessitate 6MP and MTX dose reductions.155,203,204 Specifically, vincristine/glucocorticoid pulses during 6MP/MTX have been applied by many groups, but so far most, although not all,205 randomized studies have failed to demonstrate benefits of such pulses.200,201,206,207 Folate supplementation has been widely used to counteract MTX-induced toxicity without compromising efficacy in rheumatoid arthritis208 or posttransplantation.209 However, folate supplementation should probably be avoided during maintenance therapy, as it has been shown to influence both 6MP metabolism210 and myelotoxicity.211 Finally, trimethoprim-sulfamethoxazole given as Pneumocystis jiroveci pneumonia prophylaxis212 interferes with MTX213 and 6MP pharmacokinetics,214 and also enhances myelotoxicity leading to lower prescribed 6MP and MTX doses,215 but in spite hereof does not seem to increase relapse rates,215 and thus seems safe to prescribe to avoid this life-threatening infection. RELAPSE DURING MAINTENANCE THERAPY Several high-risk ALL subsets such as T-cell ALL,216 patients with hyperleukocytosis,217 and patients with t(1;19)[E21-PBX1], MLL rearrangements,218 or hypodiploidy219 nearly always relapse during maintenance therapy. In contrast, the majority of other B-cell precursor ALL relapses occurs within 2 to 3 years after cessation of treatment.220 Relapse during maintenance therapy has been associated with insufficient 6MP treatment intensity as indicated by low Ery-6TGN levels,61 or it may simply reflect poor treatment compliance/adherence.39,221 Two recent studies demonstrated activating mutations in the NT5C2 gene, which plays a role in nucleotide homeostasis, in approximately 15% to 20% of both B-cell precursor22 and T-cell ALL patients21 that relapse during 6MP/MTX maintenance therapy. It is noteworthy that, such mutations are rare among B-cell precursor ALL patients that relapse off therapy,22 indicating that such patients relapse rapidly when NT5C2 mutations emerge, or that the survival advantage for NT5C2-mutated clones at the cost of normal hematopoietic cells disappears once 6MP therapy is discontinued. In the future, targeted deep sequencing may allow routine screening for emerging clones with mutations that hamper the efficacy of thiopurines or MTX, which would allow modification of maintenance therapy to counteract such resistance mechanisms. CONCLUSIONS AND FUTURE DIRECTIONS During the last decades more attention has been paid to dose titration by myelotoxicity, and some groups even monitor 6MP and MTX metabolites to reveal poor treatment adherence. However, until it has been determined that such therapeutic drug monitoring eases dose adjustments, improves cure rates, and/or reduce toxicity, maintenance therapy should be adjusted according to the WBC, and lack of myelotoxicity and hepatotoxicity regarded as a surrogate marker for nonadherence. Future research should address the applicability of DNA-TG monitoring, extensive host single nucleotide polymorphism profiling, screening methods for resistant leukemic subclones, and alternative thiopurine dosing regimens to improve maintenance therapy for the individual patient.
