Thiopurine S-methyltransferase Status Research Articles

TPMT and NUDT15 Variants Predict Discontinuation of Azathioprine for Myelotoxicity in Patients with Inflammatory Disease: Real-World Clinical Results.

Azathioprine is used frequently to treat several inflammatory conditions. However, treatment is limited by adverse events-in particular, myelotoxicity. Thiopurine-S-methyltransferase (TPMT) and nudix hydrolase-15 (NUDT15) are enzymes involved in azathioprine metabolism; variants in the genes encoding these enzymes increase the risk for azathioprine myelotoxicity. The Clinical Pharmacogenetics Implementation Consortium (CPIC) has recommended dose adjustments based on the results of TPMT and NUDT15 genotyping. However, little is known about the importance of this genetic information in routine clinical care. We hypothesized that in patients with inflammatory diseases, TPMT and NUDT15 genotype data predict the risk of discontinuing azathioprine due to myelotoxicity. This was a retrospective cohort study in 1,403 new adult azathioprine users for the management of inflammatory conditions for whom we had genetic information and clinical data. Among patients who discontinued azathioprine, we adjudicated the reason(s). Genotyping was performed using the Illumina Infinium Expanded Multi-Ethnic Genotyping Array plus custom content. We used CPIC guidelines to determine TPMT and NUDT15 metabolizer status; patients were grouped as either: (i) poor/intermediate, or (ii) normal/indeterminate metabolizers. We classified 110 patients as poor/intermediate, and 1,293 patients as normal/indeterminate metabolizers. Poor/intermediate status was associated with a higher risk for azathioprine discontinuation due to myelotoxicity compared to normal/indeterminate metabolizers (hazard ratio (HR)=2.90, 95% confidence interval (CI): 1.58-5.31, P=0.001). This association remained significant after adjustment for race, age at initiation, sex, primary indication, and initial daily dose of azathioprine (adjusted HR (aHR)=2.67, 95% CI: 1.44-4.94, P=0.002). In conclusion, TPMT and NUDT15 metabolizer status predicts discontinuation due to myelotoxicity for patients taking azathioprine for inflammatory conditions.

Relationship Between Thiopurine S-Methyltransferase Genotype/Phenotype and 6-Thioguanine Nucleotide Levels in 316 Patients With Inflammatory Bowel Disease on 6-Thioguanine.

Background:In inflammatory bowel disease (IBD), conventional thiopurine users cease treatment in 60% of cases within 5 years, mostly because of adverse events or nonresponse. In this study, the authors aimed to investigate the role of 6-thioguanine nucleotide (TGN) measurements, geno/phenotyping of thiopurine S-methyltransferase (TPMT), and their mutual relationship with TG therapy in IBD.Methods:An international retrospective, multicenter cohort study was performed at 4 centers in the Netherlands (Máxima Medical Centre) and the United Kingdom (Guy's and St. Thomas' Hospital, Queen Elizabeth Hospital, and East Surrey Hospital).Results:Overall, 526 6-TGN measurements were performed in 316 patients with IBD. The median daily dosage of TG was 20 mg/d (range 10–40 mg/d), and the median duration of TG use was 21.1 months (SD, 28.0). In total, 129 patients (40.8%) had a known TPMT status. In the variant-type and wild-type TPMT genotype metabolism groups, median 6-TGN values were 1126 [interquartile range (IQR) 948–1562] and 467.5 pmol/8 × 10E8 red blood cells (RBCs) (IQR 334–593). A significant difference was observed between the 2 groups (P = 0.0001, t test). For TPMT phenotypes, in the slow, fast, and normal metabolism groups, the median 6-TGN values were 772.0 (IQR 459–1724), 296.0 (IQR 200–705), and 774.5 pmol/8 × 10E8 RBCs (IQR 500.5–981.5), with a significant difference observed between groups (P < 0.001, analysis of variance).Conclusions:Our findings indicated that TPMT measurements at TG initiation can be useful but are not necessary for daily practice. TPMT genotypes and phenotypes are both associated with significant differences in 6-TGN levels between metabolic groups. However, the advantage of TG remains that RBC 6-TGN measurements are not crucial to monitor treatments in patients with IBD because these measurements did not correlate with laboratory result abnormalities. This presents as a major advantage in countries where patients cannot access these diagnostic tests.

A randomised, double-blind, parallel-group trial to assess mercaptopurine versus placebo to prevent or delay recurrence of Crohn’s disease following surgical resection (TOPPIC)

BackgroundCrohn’s disease (CD) is a chronic, relapsing, inflammatory bowel disease. Up to 65% of patients with CD require an operation to control the disease within 10 years. Both endoscopic and clinical recurrence is common within 2 years of operation, with re-operation rates cumulating at 5% of patients per year.ObjectivesThis study assessed if the use of mercaptopurine (MP) can prevent or delay postoperative recurrence in CD.DesignAn individually randomised, multicentre, double-blind, placebo-controlled trial with follow-up at 6, 13, 31, 49, 67, 85, 103, 121, 139 and 157 weeks.SettingTwenty-nine tertiary referral hospitals in the UK.ParticipantsThose aged ≥ 16 years in Scotland (or aged ≥ 18 years in England and Wales) with a histologically confirmed diagnosis of CD (according to the Lennard-Jones criteria) and surgical intervention ≤ 3 months prior to randomisation to remove all observable disease at ileocolonic or small bowel resections. Patients were excluded if they had a known intolerance of or hypersensitivity to thiopurines; were known to require further surgery; underwent strictureplasty alone; had a stoma; or had an active or untreated malignancy or absent thiopurineS-methyltransferase (TPMT) activity. Prior to randomisation any postoperative infections were fully treated and existing treatments for CD were stopped.InterventionDaily oral dose of MP or placebo, with dose adjusted according to body weight (kg) and TPMT status. Blood samples for genetic and serological analysis were taken at randomisation with additional blood and stool samples collected at weeks 0, 13, 49, 103 and 157 for central analysis of drug metabolite and faecal calprotectin levels, with endoscopic assessment at weeks 49 and 157.Main outcome measuresThe primary end point was clinical recurrence of CD (Crohn’s Disease Activity Index score of &gt; 150 points plus 100-point rise) and the need for anti-inflammatory rescue therapy or primary surgical intervention. Secondary end points included faecal calprotectin and thioguanine levels, and assessment of endoscopic recurrence. The primary analysis was adjusted for baseline values of previous treatment with MP and azathioprine, with the adjusted analysis considered to be the primary analysis.ResultsBetween June 2008 and April 2012, 240 patients were enrolled and received at least one dose of the study drug. A total of 128 (53%) participants were randomised to receive MP and 112 (47%) to receive placebo. No randomised patients were excluded from the analysis. More patients achieved the primary end point in the placebo group (n = 26, 23.2%) than in the MP group (n = 16, 12.5%), with an adjustedp-value of 0.073 [hazard ratio (HR) 0.535, 95% confidence interval (CI) 0.27 to 1.06]. Of the smokers on MP, 3 out of 29 (10.3%) had clinical recurrence versus 12 out of 26 (46.2%) on placebo, demonstrating that MP was effective at preventing postoperative recurrence in smokers (HR 0.127, 95% CI 0.04 to 0.46) but not in non-smokers (HR 0.898, 95% CI 0.42 to 1.94). The proportion of patients experiencing adverse events was similar in the treatment and placebo groups.LimitationsThere was a lower than anticipated primary event rate (12.5% in the treatment group vs. 23.2% in the placebo group, as opposed to expected rates of 30% vs. 50%).ConclusionsThe Trial Of Prevention of Post operative Crohn’s disease (TOPPIC) is the largest single, double-blind trial assessing the use of thiopurines to prevent postoperative recurrence in CD. From the trial itself, MP was not effective in reducing the frequency of clinical postoperative recurrence of CD overall, but the data suggest that it has clinically meaningful effect among the subgroup of patients who continue to smoke after surgery.Future workExploratory analyses of possible predictors of disease recurrence using collected data and samples.Trial registrationCurrent Controlled Trials ISRCTN89489788 and EudraCT 2006-005800-15.FundingThis project was funded by the Efficacy and Mechanism Evaluation programme, a Medical Research Council and National Institute for Health Research (NIHR) partnership, and will be published in full inEfficacy and Mechanism Evaluation; Vol. 4, No. 4. See the NIHR Journals Library website for further project information.

Thiopurine S-Methyltransferase as a Pharmacogenetic Biomarker: Significance of Testing and Review of Major Methods.

Background: Thiopurine S-methyltransferase (TPMT) enzyme metabolizes thiopurine drugs which are widely used in various disciplines as well as in leukemias. Individual enzyme activity varies depending on the genetic polymorphisms of TPMT gene located at chromosome 6. Up to 14% of popu-lation is known to have a decreased enzyme activity, and if treated with standard doses of thiopurines, these individuals are at a high risk of severe Adverse Drug Reactions (ADR) as myelosuppression, gas-trointestinal intolerance, pancreatitis and hypersensitivity. However, TPMT-deficient patients can suc-cessfully be treated with decreased thiopurine doses if enzyme status is identified by a prior testing. TPMT status identification is a pioneering experience in application of pharmacogenetic testing in clini-cal settings. 4 TPMT (*2, *3A, *3B, *3C) alleles are known to account for 80-95% of a decreased en-zyme activity, and therefore, identifying the presence of these alleles supported by phenotypic measure-ment of the enzyme activity can reveal patient’s TPMT status. Evaluation of the levels of thiopurine me-tabolites further supports the practice of appropriate dose adjustment by providing the efficient monitor-ing of drug cytotoxicity.Conclusion: We hereby review the thiopurine pharmacogenetics and the methods applied in common practice to evaluate patient’s TPMT status.

Testing for thiopurine methyltransferase status for safe and effective thiopurine administration: a systematic review of clinical guidance documents.

A common pharmacogenomic test is for thiopurine S-methyltransferase (TPMT) status prior to treatment with thiopurine drugs, used to treat auto-immune conditions and pediatric cancer. Guidelines assist practitioners with decisions regarding testing and treatment. The objectives were to conduct a systematic review and critical appraisal of guidance documents with statements regarding TPMT testing and thiopurine dosing. Guidelines, clinical protocols and care pathways from all disciplines were eligible. A quality appraisal was carried out by three appraisers using the Appraisal of Guidelines for Research and Evaluation II. Of the 20 documents found, 5 recommended genotyping while 4 recommended phenotyping. Thirteen documents provided dosing recommendations based on TPMT status. The quality appraisal revealed wide variation across documents. The National Institute for Health and Clinical Excellence and Cincinnati Children's Hospital guidelines demonstrated the highest overall quality with scores of 79 and 76, respectively. Low-scoring documents failed to use systematic methods to develop recommendations or to provide evidence to support recommendations. Guidance documents that included dosing recommendations demonstrated higher quality.

Review article: the benefits of pharmacogenetics for improving thiopurine therapy in inflammatory bowel disease

Thiopurines represent an effective and widely prescribed therapy in inflammatory bowel disease (IBD). Concerns about toxicity, mainly resulting from a wide inter-individual variability in thiopurine metabolism, restrict their use. Optimal thiopurine dosing is challenging for preventing adverse drug reactions and improving clinical response. To review efficacy and toxicity of thiopurines in IBD. To provide pharmacogenetic-based therapeutic recommendations. We conducted a query on PubMed database using 'inflammatory bowel disease', 'thiopurine', 'azathioprine', '6-mercaptopurine', 'TPMT', 'pharmacogenetics', 'TDM', and selected relevant articles, especially clinical studies. Thiopurine metabolism - key enzyme: thiopurine S-methyltransferase (TPMT) - modulates clinical response, as it results in production of the pharmacologically active and toxic metabolites, the thioguanine nucleotides (6-TGN). Adjusting dosage according to TPMT status and/or metabolite blood levels is recommended for optimising thiopurine therapy (e.g. improving response rate up to 30% or decreasing haematological adverse events of 25%). Other enzymes or transporters of interest, as inosine triphosphatase (ITPase), glutathione S-transferase (GST), xanthine oxidase (XO), aldehyde oxidase (AOX), methylene tetrahydrofolate reductase (MTHFR) and ATP-binding cassette sub-family C member 4 (ABCC4) are reviewed and discussed for clinical relevance. Based on the literature data, we provide a therapeutic algorithm for thiopurines therapy with starting dose recommendations depending on TPMT status and thereafter dose adjustments according to five metabolite profiles identified with therapeutic drug monitoring (TDM). This algorithm allows a dosage individualisation to optimise the management of patients under thiopurine. Furthermore, identification of new pharmacogenetic biomarkers is promising for ensuring maximal therapeutic response to thiopurines with a minimisation of the risk for adverse events.

Use of thiopurine testing in the management of inflammatory bowel diseases in clinical practice: A worldwide survey of experts

We performed a worldwide survey to evaluate the extent to which gastroenterologists who are experts in the field of inflammatory bowel diseases (IBDs) are utilizing thiopurine metabolism in practice. This was a Web-based cross-sectional survey consisting of 12 multiple-choice and open-ended questions. Between December 2009 and April 2010, 175 questionnaires were received. The proportion of practitioners with access and reimbursement for thiopurine S-methyltransferase (TPMT) genotype, TPMT phenotype, 6-thioguanine nucleotides (6-TGN) levels, and 6-methylmercaptopurine ribonucleotides (6-MMP) levels was 48%, 54%, 44%, and 35%, respectively. Before azathioprine initiation, TPMT genotype and phenotype were performed by only 30% and 43% of responders, respectively. In patients on thiopurine therapy, 6-TGN and 6-MMP levels were determined by 54% and 44% of responders, respectively. Only 27% of physicians always wait for TMPT activity/genotype results before initiating azathioprine and 81% do not routinely recheck metabolite levels after dose escalation or reduction. In cases of very high or low TPMT activity, 75% and 74% of practitioners take into account TMPT activity result, respectively. If access to all azathioprine metabolite measurements was available and if all these tests were reimbursed by public health insurance, 47% of responders would use these tests more often in their practice. The availability and reimbursement of TPMT status and azathioprine metabolites strongly influenced experts' attitudes. Thiopurine testing is relatively underutilized by IBD gastroenterologists. The availability and reimbursement of TPMT status and azathioprine metabolites strongly influence the management of IBD patients treated with thiopurines.

Thiopurine S-methyltransferase (TPMT) assessment prior to starting thiopurine drug treatment; a pharmacogenomic test whose time has come

Thiopurine S-methyltransferase (TPMT) is involved in the metabolism of thiopurine drugs. Patients that due to genetic variation lack this enzyme or have lower levels than normal, can be adversely affected...

Improving pharmacovigilance in Europe: TPMT genotyping and phenotyping in the UK and Spain

Thiopurine S-methyltransferase (TPMT) is the rate-limiting step in the conversion of thiopurine drugs including azathioprine (AZA) to inactive metabolites. Heritable deficiency of TPMT activity increases risk for adverse events, most notably, myelosuppression leading to leukopenia and neutropenic sepsis. The reported European Commission study was undertaken to identify current evidence for the clinical utility of testing for TPMT status and extent of uptake, by either genotyping or phenotyping, in the clinical setting. Data presented here for the UK and Spain indicate that there has been a considerable increase in the uptake of TPMT testing in recent years. There are some data that support routine TPMT testing before AZA prescribing for reducing AZA-related adverse events. Key data include evidence in favor of TPMT testing in addition to the current practice of routine monitoring for reducing the number of AZA-related episodes of myelosuppression, averting deaths from neutropenic sepsis and improving health-related quality of life. Further data are needed for determining the cost-effectiveness of routine TPMT testing.

Side effects from azathioprine in a patient with thiopurine S-methyl transferase deficiency

Azathioprine is an immunosuppressant and cytotoxic pro-drug which acts to impair purine synthesis. Side effects including nausea, rash, hepatotoxicity and bone marrow suppression frequently limit its use. Thiopurine S-methyl transferase (TPMT) is an important enzyme which metabolises thiopurines such as azathioprine to inactive metabolites. TPMT activity varies greatly between individuals, and has a trimodal distribution. Individuals may be homozygous for the highly active wild type allele, heterozygous with a low activity variant allele, or be homozygous for the low activity variant. Reduced TPMT activity conveys a greater risk of drug related side effects from thiopurines. We report a case in which azathioprine used to treat non specific interstitial pneumonia resulted in marked pancytopaenia. Azathioprine treatment had been commenced without first checking the TPMT status. The subsequent discovery of TPMT deficiency in this patient explained the development of complications. Limited therapeutic options led to the patient being treated with an extremely low azathioprine dose regime. The drug was tolerated without further toxicity. This case highlights the benefits of screening the TPMT activity of all patients where azathioprine treatment is being considered. It also gives further evidence that TPMT deficient individuals are able to tolerate a very low dose azathioprine regime without side effects.

Paradoxical Elevated Thiopurine S-Methyltransferase Activity After Pancytopenia During Azathioprine Therapy: Potential Influence of Red Blood Cell Age

There is an increased risk of developing bone marrow depression and infections during azathioprine therapy for inflammatory bowel disease. Patients with low or absent thiopurine S-methyltransferase (TPMT) activity have an increased risk of developing myelotoxicity. We describe a patient who developed pancytopenia combined with cytomegalovirus pneumonia after several years of azathioprine use. The bone marrow depression was probably caused by the viral infection, as all others causative factors were unlikely. Surprisingly, we observed grossly elevated TPMT activity (182 nmol/g/h) during the recovery phase, following the pancytopenic period. After complete recovery of the bone marrow suppression, TPMT activity returned to usual reference activity (43 nmol/g/h). This remarkable change in enzymatic activity of TPMT may be explained by differences in the age of red blood cells, as younger erythrocytes have a higher TPMT activity. Determination of a patient's TPMT status by phenotyping should therefore not be performed just after bone marrow depression or in cases of activated erythropoieses.

Impracticalities of thiopurine S‐methyltransferase determination in daily inflammatory bowel disease practice

Sirs, We greatly enjoyed the discussion concerning the cost-effectiveness of prior-to-treat genotyping of thiopurine S-methyltransferase (TPMT) in inflammatory bowel disease patients (IBD).1, 2 Thiopurines (azathioprine and mercaptopurine) are frequently used immunosuppressive agents in the treatment of Crohn's disease and ulcerative colitis. The metabolism of thiopurines is complex as multiple enzymatic steps are needed to ultimately generate the pharmacologically active 6-thioguanine nucleotides (6-TGN).3 The activity of the methylating enzyme TPMT plays a key role in the bioavailability of 6-TGN. Less efficient methylation by TPMT leads to relative high levels of 6-TGN, which are considered to be a causative factor in the development of myelotoxicity.4 High levels of methylated thiopurine metabolites [e.g. 6-methylmercaptopurine (6-MMP)] will be generated as a consequence of high-TPMT activity. Elevated 6-MMP levels have been associated with the development of hepatotoxicity, but also with refractoriness to thiopurine therapy.5 For this reason, prior-to-treat determination of the TPMT status by genotyping (or phenotyping) has been proposed to avoid toxicity by adjusting the dosage to the individual TPMT status. Winter et al.1 conclude that pre-treatment screening for TPMT polymorphisms in IBD patients commencing thiopurine therapy represents good value for money. In order to evaluate whether prior-to-treat determination of the TPMT status is currently utilized in daily IBD practice, a questionnaire regarding this topic was sent to all Dutch registered gastroenterologists (n = 250) per e-mail. A total of 133 questionnaires (53%) was returned. The results of the questions concerning the currently used and preferred toxicity screening techniques are depicted in Table 1. Five percent of gastroenterologists currently determined the TPMT status and 36% wants to determine the TPMT status prior to initiation of thiopurine therapy (χ2-test, P = 0.001). The aforementioned discrepancy may be explained by the lack of proper laboratory facilities in the Netherlands. Therefore, it is likely that gastroenterologists will expand their toxicity screening by determining the TPMT status when facilities become generally available. Nevertheless, the greater part of physicians (64%) does not consider TPMT determination as an additional or valuable toxicity screening tool. Possible explanations, as stated by several respondents, are that knowledge of the TPMT status may result in a decreased possibility of induction of toxicity because of thiopurine treatment; however, frequent determination of laboratory parameters remains mandatory for optimal care.6 Knowledge of the patients’ TPMT status seems, for that reason, not needed. In addition, genotyping of TPMT alleles is no guarantee for not developing a leucopenia as the majority of reported cases is reported to be in IBD patients without one or two mutant TPMT alleles.3, 4 Finally, a logistic problem arises when adapting to this novel pharmacogenetic strategy as the determination of TPMT status may take some time and patients in need of thiopurines have to await the results before thiopurine therapy can be initiated. We conclude that in spite of possible clinical and pharmaco-economic advantages, TPMT determination in daily Dutch IBD practice is considered to be of limited value and surplus to requirements. Its implementation seems hampered by logistical difficulties and by the lack of convincing evidence.

Introducing a fast and simple PCR‐RFLP analysis for the detection of mutant thiopurine S‐methyltransferase alleles TPMT*3A and TPMT*3C

Azathioprine, in combination with corticosteroids, is the first-line therapy of severe forms of pemphigus vulgaris. Patients with an impaired thiopurine S-methyltransferase (TPMT) activity are at risk of developing severe myelo-suppression upon treatment with thiopurines such as azathioprine. Analysis of the TPMT status prior to drug administration is therefore highly recommended. However, because of the limited availability of TPMT testing outside of specialized centres, pre-emptive TPMT testing is not widespread. To avoid laborious biochemical and sequencing assays, we evaluated a new restriction fragment length polymorphism (RFLP) analysis. We designed a rapid genetic polymerase chain reaction (PCR)-RFLP screen for the most prevalent mutant TPMT*3A and TPMT*3C alleles that are known to result in reduced TPMT enzyme activity. Validating our fast system on 871 Caucasian DNA samples, we observed that 8.61% of our probands carried the TPMT*3A allele and 0.23% were heterozygous for the TPMT*3C allele, which is in concordance with previously reported allele frequencies. This simple and low-cost PCR-RFLP TPMT polymorphism testing approach can be performed in a standard laboratory. It should be applied to all patients prior to receiving thiopurine drug therapy to avoid the severe, but predictable, haematopoietic side-effects of thiopurine drug administration.

Whose TPMT activity is it anyway?

Determination of thiopurine S-methyltransferase (TPMT) activity prior to starting azathioprine therapy is used to identify individuals with low or deficient TPMT activities who are at risk of severe complications and even death This case describes a patient treated with azathioprine without prior knowledge of TPMT status. Pancytopaenia developed over several months and at this point TPMT activity was determined and found to be low at 16 nmol 6-methyl thioguanine (6-MTG)/g Hb/h, which is within the reference interval associated with heterozygosity for TPMT mutant alleles. On repeating the TPMT measurement 3 months later, the TPMT activity was 2 nmol 6-MTG/g Hb/h, consistent with deficient TPMT activity (homozygosity for TPMT mutant alleles), suggesting the patient is at high risk of myelosuppression if treated with thiopurine drugs. Retrospectively, it was found that the patient had received transfusions of red blood cell and platelets 6 days before TPMT activity was first measured. This case underlines the importance of determining TPMT activity status prior to azathioprine treatment, rather than taking a dose incrementation approach. It also highlights the caution that must be taken in interpreting TPMT activity in patients who have recently been transfused.