Optimizing thiopurine dosing based on <i>TPMT</i> and <i>NUDT15</i> genotypes: It takes two to tango
Authors: Stefania Koutsilieri, Kelly E. Caudle, Saeed Khaled Alzghari, Andrew A. Monte, Mary V. Relling, George P. Patrinos
Journal: American Journal of Hematology (2019)

The three thiopurines used in clinical practice are mercaptopurine and its prodrug azathioprine, as well as thioguanine. Although thiopurines were initially developed as chemotherapeutic agents for the treatment of childhood leukemia, nowadays they are also prescribed in a wider spectrum of clinical use as immunosuppressors.1 More specifically, mercaptopurine and azathioprine are currently used in the treatment of inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis,2 while thioguanine constitutes potential alternative in psoriasis' treatment.3 Despite the fact that thiopurines are generally considered effective medications, there have been recorded several cases of treatment discontinuation due to treatment failure or adverse drug reactions.4 The frequent dose-limiting hematopoietic toxicity which is partly explained by inherited genetic variants in drug metabolizing enzymes4 pointed to the formation of the first pharmacogenomic guideline by the Clinical Pharmacogenetics Implementation Consortium (CPIC, www.cpic.org). Here we highlight current pharmacogenomic information that needs to be taken into consideration before thiopurine administration to ensure medication's proper use. First and foremost, several metabolic transformations are to be made in order for thiopurines to exert their immunosuppressive and cytotoxic effects via their pharmacologically active metabolites, 6-thioguaninenucleotides (6-TGN).5 Breaking down thiopurines' metabolism into the most critical transformations, two thiopurines share the same metabolic pathway; azathioprine is converted to 6-mercaptopurine (6-MP) and subsequently to 6-thioinosine-monophosphate (6-TIMP), which in turn is converted into 6-thioguanine monophosphate (6-TGMP). 6-TGMP as well as its further phosphorylated forms, 6-TGDP and 6-thioguanine triphosphate metabolites (TGTP), constitute the 6-TGN pool.2 As far as thioguanine is concerned, the 6-TGN are directly generated via a one-step enzymatic process.2 Given that thiopurines are purine nucleoside analogues, they act as antimetabolites, hence their active compounds are incorporated into DNA instead of guanine, leading to the suppression of de novo synthesis of purines and cell death.6 Apart from their therapeutic effects, thiopurines are held responsible of adverse drug reactions, namely hematopoietic toxicity and myelosuppression, which affect a rather large part of the patients.7 Two enzymes have mainly been correlated with the incidence of toxicity: thiopurine S-methyltransferase (TPMT) and nucleoside diphosphate-linked moiety X-type motif (NUDT15).2 Regarding TPMT, it catalyzes the methylation of 6-MP and thioguanine to the inactive metabolites 6-methylmercaptopurine and methylthioguanine, respectively.2 Furthermore, the secondary metabolite TIMP also constitutes substrate for TPMT, leading to the methylated forms, 6-methylmercaptopurine ribonucleotides.2 As the TPMT enzyme diverts thiopurines away from the formation of the active compounds, it can be easily concluded that deficiency or low activity of this key enzyme can lead to elevated 6-TGN levels.1 In contrast, in normal or even high enzyme activity, elevated methylated thiopurine product levels are expected at the cost of 6-TGN formation.7 The more elevated the 6-TGN levels are, the higher is the likelihood of clinical response to thiopurine therapy, but so is the risk of adverse drug reactions.8 Whether the active metabolites will exceed the pharmacologic concentrations or not, is tightly linked to the TPMT activity, which in turn is related to several TPMT genetic variants.1 The three main variant TPMT alleles accounting for approximately 90% of low-activity phenotypes are *2, *3A, and *3C.6 As for the other key enzyme in thiopurines' metabolism, NUDT15 catalyzes the dephosphorylation of the TGTP to the TGMP, preventing the incorporation of thiopurine active metabolites into the DNA.8 Notably, although the actual concentration of TGNs is generally not affected, the ratio of TGMP against TGTP is increased.5 In case of NUDT15 genetic defects, the increase in the TGTP availability for incorporation into the DNA leads to an elevated risk of clinical toxicity.1, 5 Given that the activity of this nudix hydrolase is modulated by genetic variants, recently identified coding variants in NUDT15 have emerged as important determinants of thiopurine toxicity.2 More specifically, the p.R139C (rs116855232; c.415C>T) was the first variant to be identified, leading to thiopurine toxicity, as a result of an almost complete deactivation and in vivo instability of the NUDT15 enzyme.1 Diving into the effect of genetic alterations regarding these two enzymes, the aforementioned variants leading to decreased TPMT activity are correlated with hematologic and gastrointestinal toxicity after thiopurine administration.6 Indeed, it is well established that patients with two loss-of-function TPMT alleles have a very high risk for life-threatening myelosuppression, due to the high concentration of TGN, if treated with standard thiopurine doses.6 Similarly, heterozygotes for a variant TPMT allele are also prone to thiopurine metabolism and thus, they cannot tolerate full thiopurine doses either.6 The severity of the adverse drug reactions in combination with thiopurines' narrow therapeutic index necessitates frequent dose adjustments.9 Therefore, pre-emptive TPMT genotyping may allow the optimization of treatment approaches,5 as it substantially reduces the risk of adverse drug reactions without compromising therapeutic outcomes.1, 9 Individuals with normal TPMT activity can be prescribed with full thiopurine doses, whereas TPMT-deficient patients are recommended to start with drastically reduced doses.2, 5 Although TPMT constitutes a well-established pharmacogenomic biomarker for thiopurine drug dose individualization, it could not explain why a significant portion of patients with wildtype TPMT still experienced excessive toxicity.1 It has recently become evident that loss-of-function variants in the NUDT15 gene have also been associated with hematopoietic toxicity.5 Particularly, various studies both regarding IBD and acute lymphoblastic leukemia (ALL) have demonstrated that carriers of NUDT15 deficient alleles cannot tolerate standard doses of thiopurines, bearing excessive risk of drug toxicity.1, 9 Therefore, there was an urgent need to develop NUDT15-based dosing algorithm in order to individualize thiopurine therapy.5 As a result, and based on a growing body of evidence, the CPIC has recently updated the existing azathioprine, mercaptopurine, and thioguanine prescribing guideline to be based not only on the TPMT but also on the NUDT15 genotypes (10; https://cpicpgx.org/guidelines/guideline-for-thiopurines-and-tpmt). Given that NUDT15 deficient alleles result in the excessive incorporation of thiopurines' active compounds to DNA and hence, toxicity, dose reduction based on this allele would likely tailor thiopurines' exposure to a level that is similar to wildtype patients receiving standard thiopurine doses.1 Alongside the fact that NUDT15 does not affect the concentration of the pharmacologically active compounds,5 it seems rational to assume that dose reduction based on NUDT15 would not eliminate the desired therapeutic outcome,1 remark that is also in concordance with the CPIC guideline.10 Of note, this CPIC guideline has introduced some important innovative aspects; first of all, taking into account the different diseases and/or initial doses in which thiopurines are administered, there is a reciprocal differentiation with regard to the recommended drug dosing.10 In other words, in case of malignancy, substantial dose reduction is highly recommended in poor TPMT or NUDT15 metabolizers, whereas in nonmalignant conditions, physicians should consider prescribing an alternative agent other than thiopurines.10 Furthermore, depending on the level of the starting dose, patients who are intermediate metabolizers for the TPMT or the NUDT15 enzyme, may or may not be prescribed with a drastic reduction of thiopurine dose.10 With regard to patients with intermediate metabolizer status for both TPMT and NUDT15 (ie, compound intermediate metabolizers) compared to intermediate metabolizers for only TPMT or NUDT15, there is a tendency for a lower thiopurine tolerance and as such, an even greater dose reduction might be necessary.1, 9, 10 Another important remark made in this revised guideline is the reference to the prevalence of the two genes in different racial groups, so as to properly guide physicians in those cases where genetic testing results are available for either TPMT or NUDT15.10 It is highlighted that the TPMT-based dose adjustment cannot be universally informative, given the scarcity of deficient variants in certain ancestral groups.5 More specifically, testing for NUDT15 variants may be particularly relevant for Asian populations, in which the frequency of NUDT15 loss-of-function variants exceeds that of TPMT variant alleles.2, 9 In addition, the higher prevalence of decreased function NUDT15 gene variants among individuals of Hispanic backgrounds5 and of TPMT variants in those with European and African origin should be considered.1, 9 Obviously, in those cases where test results are available for only one of the aforementioned genes (TPMT or NUDT15), prescribing recommendations based on that gene's results must be implemented, bearing in mind that variants in the other gene may have important implications in the final dose adjustment.1, 10 The introduction of the "possible intermediate metabolizer" phenotype constitutes the other hallmark in the CPIC guideline.10 The assignment refers to the possibility for a patient to carry one uncertain/unknown function allele in combination with one known no function allele and the subsequent implications this may have in the dosing regimen.10 Actually, TPMT gene is highly polymorphic with more than 20 TPMT deficient variants already been identified, which are expected to induce toxicity due to the increase in 6-TGN concentration.6 Moreover, although the p.R139C NUDT15 variant (rs116855232; c.415C>T) is mainly held responsible for thiopurine toxicity, there is growing evidence that additional functional variants for NUDT15 exist,5 which remain to be thoroughly studied for their significance in clinical implementation.1 Therefore, if TPMT and/or NUDT15 genotyping is only based on a genetic screening approach of certain variant alleles, a false negative result may be generated, when neither variant alleles are included in the genotyping panel or novel deleterious variants are present. As such, TPMT and/or NUDT15 gene resequencing coupled with in silico variant prediction can be reassuring for the TPMT and/or NUDT15 gene-based metabolizing status of a patient. Moreover, complementary phenotype laboratory tests (eg, measurement of thiopurine metabolites in erythrocytes or measurement of erythrocyte TPMT activity) may serve as helpful adjuncts to genotyping tests, confirming TPMT phenotype and also testing for patient adherence to the appropriate medication regimen.2 This updated CPIC guideline also underlines the significance of clinical monitoring on top of genotype-guided dosing regarding thiopurines.10 Observing the dosing recommendations carefully, we can easily conclude that there is some variability in the tolerated thiopurine dosages, especially within TPMT or NUDT15 intermediate metabolizers with a small fraction of individuals who do not seem to require significant dose reduction.9, 10 Moreover, there may be other reasons underlying poor tolerance to thiopurines that are not related to TPMT or to NUDT15 genetic variation; concomitant therapy with agents that lead to overlapping adverse effects (like methotrexate leading to myelosuppression) or which can exert their effect on the pharmacokinetics of thiopurines (like allopurinol minimizing methylated active metabolites) are indicative examples.2, 10 Hence, further investigation for disease progression and/or toxicity biomarkers is required in order to more precisely adjust thiopurine doses on the basis of the genotype-directed starting doses.5, 8 Based on data derived from the published literature, the Pharmacogenomics Working Party and the Pharmacovigilance Risk Assessment Committee (PRAC) of the European Medicines Agency (EMA; www.ema.europa.eu) considered that there is currently sufficient available evidence to highlight the need of NUDT15 genotyping in the aforementioned cases. In particular, sections 4.2 and 4.4 of the summary of medicinal product characteristics were updated in order to include a new warning on the increased risk of severe toxicity in patients with an inherited NUDT15 gene variant that are treated with 6-MP [European Medicines Agency; https://ec.europa.eu/health/documents/community-register/2017/20170713138228/anx_138228_en.pdf (assessed 28 March 2019)]. There is currently no data available to support mandating physicians to initiate genetic testing prior to use of the product. However, the PRAC agreed to include a statement that pharmacogenomic testing of NUDT15 prior to initiating treatment may be considered [European Medicines Agency; https://ec.europa.eu/health/documents/community-register/2017/20170713138228/anx_138228_en.pdf (assessed 28 March 2019)]. On the other hand, the US Food and Drug Administration Agency (FDA) clearly recommends pharmacogenomic testing regarding TPMT and NUDT15 genes before the initiation of mercaptopurine's and thioguanine's treatment [https://www.fda.gov/downloads/Drugs/ScienceResearch/UCM578588.pdf (assessed 28 March 2019)]. In essence, although the discovery and clinical implementation of NUDT15 variant alleles in individualizing thiopurine dosing are relatively recent and the exact impact of NUDT15 genotype-guided dose adjustments on thiopurine toxicity and efficacy are less studied compared to the well investigated TPMT-based dose adjustments, it is clear that the addition of NUDT15 genotyping would provide additional insights as far as thiopurine dose adjustment is concerned. Currently, there is one prospective randomized clinical trial that is addressing TPMT and NUDT15-guided treatment individualization, using azathioprine for IBD patients to prevent adverse drug reactions (https://clinicaltrials.gov/ct2/show/NCT03719118). As far as existing retrospective studies are concerned, it is strongly suggested that patients with loss-of-function NUDT15 alleles are at excessive risk of thiopurine toxicity if the standard dose is administered. As such, the inclusion of the NUDT15 gene in the arsenal of pharmacogenomic biomarkers for thiopurine dose adjustment would significantly contribute toward further reducing the incidence of potentially lethal adverse drug reactions, hence improving the quality of life of the patients, while reducing the costs derived from the unnecessary hospitalization of these patients. The authors declare no conflict of interests. G.P.P. is a full member and National Representative at the European Medicines Agency, CHMP-Pharmacogenomics Working Party. S.K. is a recipient of an Onassis Foundation scholarship.