Thiopurine Methyltransferase Genotype Research Articles

P798 Study of the concordance between the enzymatic activity of Thiopurine S-methyltransferase and polymorphisms in the TPMT gene in patients with Inflammatory Bowel Disease

Abstract Background Thiopurine therapy is associated with an increased risk of serious adverse events in patients with Inflammatory Bowel Disease (IBD). Thiopurine S-methyltransferase (TPMT) is the enzyme responsible for metabolism, and its determination helps identify patients at risk of toxicity. This study aims to explore the level of correspondence between the TPMT genotype and enzymatic activity (phenotype) with the objective of preventing adverse events prior to the commencement of thiopurine therapy. Methods Consecutive patients with IBD, Crohn’s Disease (CD) or Ulcerative Colitis (UC), were included. Genotype was conducted for the most prevalent TPMT variants (TPMT*2, TPMT*3B, TPMT*3C), TPMT enzymatic activity assessment, and quantification of 6-thioguanine nucleotide (6-TGN) levels. Adverse effects were documented, along with the administration of biologics, mesalazine, ACE inhibitors or allopurinol. The primary objective was to evaluate the concordance between TPMT genotype and phenotype. Secondary objectives included determining the prevalence of genotype and phenotype within our patient cohort, 6-TGN serum levels, the prevalence of adverse events and identifying potential pharmacological inducers of the phenotype. Results A total of 218 patients were included (131 with CD and 81 with UC). Thirteen patients did not initiate thiopurines, and 34.63% (71/205) had discontinued treatment. The most observed adverse events were myelotoxicity (15.12%), gastrointestinal toxicity (12.20%), and hepatic toxicity (5.37%). TPMT activity exhibited a trimodal distribution, with 87.61% (191/218) having normal activity, 11.47% having intermediate activity, and 1% having low activity. Genotypically, 90.83% (198/218) had a wild-type genotype, 8.72% (18/218) had a heterozygous genotype, and 0.5% (1/218) had a homozygous genotype. The concordance index between phenotype and genotype was calculated at 0.57. Significantly, myelotoxicity was associated with the presence of genotypic variants (p=0.004). 6-TGN levels did not correlate with TPMT activity and concurrent medications did not affect TPMT activity . Conclusion The proactive assessment of TPMT (phenotype or genotype) before initiating thiopurine therapy effectively mitigates the occurrence of myelotoxicity in IBD patients. Importantly, the level of concordance between TPMT activity and genotype is moderate, with genotyping serving as the more reliable approach for preselecting patients susceptible to myelotoxic reactions.

The Role of Pharmacogenetics in the Therapeutic Response to Thiopurines in the Treatment of Inflammatory Bowel Disease: A Systematic Review.

This study focuses on the use of thiopurines for treating inflammatory bowel diseases (IBD). These drugs undergo enzymatic changes within the body, resulting in active and inactive metabolites that influence their therapeutic effects. The research examines the role of genetic polymorphisms in the enzyme thiopurine S-methyltransferase (TPMT) in predicting the therapeutic response and adverse effects of thiopurine treatment. The TPMT genotype variations impact the individual responses to thiopurines. Patients with reduced TPMT activity are more susceptible to adverse reactions (AEs), such leukopenia, hepatotoxicity, pancreatitis, and nausea, which are common adverse effects of thiopurine therapy. The therapeutic monitoring of the metabolites 6-thioguanine nucleotides (6-TGN) and 6-methyl mercaptopurine (6-MMP) is proposed to optimize treatment and minimize AEs. Patients with higher 6-TGN levels tend to have better clinical responses, while elevated 6-MMP levels are linked to hepatotoxicity. Genotyping for TPMT before or during treatment initiation is suggested to tailor dosing strategies and enhance treatment efficacy while reducing the risk of myelosuppression. In conclusion, this study highlights the importance of considering genetic variations and metabolite levels in optimizing thiopurine therapy for IBD patients, focusing on balance therapeutic efficacy with the prevention of adverse effects and contributing to personalized treatment and better patient outcomes.

Azathioprine dose tailoring based on pharmacogenetic information: Insights of clinical implementation

Azathioprine is commonly used as an immunosuppressive antimetabolite in the treatment of acute lymphoblastic leukemia, autoimmune disorders (such as Crohn's disease and rheumatoid arthritis), and in patients receiving organ transplants. Thiopurine-S-methyltransferase (TPMT) is a cytoplasmic trans-methylase catalyzing the S-methylation of thiopurines. The active metabolites obtained from thiopurines are hydrolyzed into inactive forms by the Nudix hydrolase 15 (NUDT15). The TPMT*2 (defined by rs1800462), *3A (defined by rs1800460 and rs1142345), *3B (defined by rs1800460), *3C (defined by rs1142345), *6 (defined by rs75543815), and NUDT15 rs116855232 genetic variant have been associated, with the highest level of evidence, with the response to azathioprine, and, the approved drug label for azathioprine and main pharmacogenetic dosing guidelines recommend starting with reduced initial doses in TPMT intermediate metabolizer (IM) patients and considering an alternative treatment in TPMT poor metabolizer (PM) patients. This study aims to assess the clinical impact of azathioprine dose tailoring based on TPMT genotyping studying the azathioprine toxicity and efficacy, treatment starts, and dose adjustments during follow-up, comparing TPMT IM/PM and normal metabolizer (NM) patients. It also studied the association of NUDT15 rs116855232 with response to azathioprine in patients receiving a tailored treatment based on TPMT and characterized the TMPT and NUDT15 studied variants in our population. Results show that azathioprine dose reduction in TPMT IM patients (TPMT*1/*2, *1/*3A, or *1/*3C genotypes) is related to lower toxicity events compared to TPMT NM (TPMT *1/*1 genotype), and lower azathioprine dose adjustments during follow-up without showing differences in the efficacy. The results support the hypothesis of existing other genetic variants affecting azathioprine toxicity.

Implications of Tioguanine Dosing in IBD Patients with a TPMT Deficiency.

Tioguanine is metabolised by fewer enzymatic steps compared to azathioprine and mercaptopurine, without generating 6-methylmercaptopurine ribonucleotides. However, thiopurine S-methyl transferase (TPMT) plays a role in early toxicity in all thiopurines. We aimed to describe the hazards and opportunities of tioguanine use in inflammatory bowel disease (IBD) patients with aberrant TPMT metabolism and propose preventative measures to safely prescribe tioguanine in these patients. In this retrospective cohort study, all determined TPMT genotypes (2016-2021) were evaluated for aberrant metabolism (i.e., intermediate and poor TPMT metabolisers). Subsequently, all IBD patients on tioguanine with aberrant TPMT genotypes were evaluated for tioguanine dosages, adverse drug events, lab abnormalities, treatment duration and effectiveness. TPMT genotypes were determined in 485 patients, of whom, 50 (10.3%) and 4 patients (0.8%) were intermediate and poor metabolisers, respectively. Of these patients, 12 intermediate and 4 poor TPMT metabolisers had been prescribed tioguanine in varying doses. In one poor TPMT metaboliser, tioguanine 10 mg/day induced delayed pancytopenia. In general, reduced tioguanine dosages of 5 mg/day for intermediate TPMT metabolisers, and 10 mg two-weekly for poor TPMT metabolisers, resulted in a safe, long-term treatment strategy. Diminished or absent TPMT enzyme activity was related with a pharmacokinetic shift of tioguanine metabolism which is associated with relatively late-occurring myelotoxicity in patients on standard tioguanine dose. However, in strongly reduced dose regimens with strict therapeutic drug and safety monitoring, tioguanine treatment remained a safe and effective option in IBD patients with dysfunctional TPMT.

B-225 Assessment of the Genotype Frequency of Thiopurine Methyltransferase (TPMT) Deficiency in a Large Cohort of Patients With Immune Mediated Inflammatory Disease and Cancer

Abstract Background Thiopurine methyltransferase (TPMT) alleles (*1/*2/*3A/*3B/*3C) are routinely genotyped to determine the metabolizer status of patients that plan to undergo thiopurine therapies (6-Mercaptopurine and Azathioprine). Screening of these alleles allows for personalized patient dosing and reduces the risk of severe or even fatal myelosuppression. The objective of this study was to determine the genotype frequency of TPMT deficiency in a large clinical cohort of patients with immune mediated inflammatory diseases and cancer. Methods All genotyping was performed in a reference clinical laboratory. Patient specimens were tested using genomic DNA isolated from whole blood. Clinical patient diagnoses were physician provided (international classification of disease, ICD-10). The genomic DNA was tested for the presence of three single nucleotide polymorphisms (SNPs) in the TPMT gene coding region and known to associate with reduced TPMT activity (rs1800462-Exon 5, rs1800460-Exon 7 and rs1142345-Exon 10). These polymorphisms determine the presence of *2/*3A/*3B/*3C alleles. Testing occurred from 10/26/2000 to 9/9/2022. Three different testing technologies were used during the 22 years of testing (PCR-RFLP from 2000 to 2003, Taqman™ chemistries from 2004 to 2015, and Luminex™ xMAP™ bead-based since 2016). Results A total of 219 129 patients were tested (mean age: 38 yrs, 56% female). As presented in the Table the genotype frequency for the wild type *1/*1 was 90.8% (95%CI: 90.7–90.9%), it was 9.0% (95%CI: 8.8–9.1%) for the heterozygous and 0.22% (95%CI: 0.020–0.024%) for homozygous variant, with similar frequency across disease states. The TPMT *1/*3B genotype frequency was very rare (<0.1%, n = 5) and the *3B/*3C homozygous variant (which cannot be distinguished from the *1/*3A) was calculated to have a frequency of 1:7 706 230 (95%CI: 1:7 043 228–1:8 369 232) and lower than initial estimates. Conclusion We estimated the frequency of TPMT genotypes in the largest cohort reported to date. The occurrence of the *3B/*3C genotype may be rarer than initially estimated.

Key factors associated with 6-thioguanine and 6-methylmercaptopurine nucleotide concentrations in children treated by thiopurine for acute leukaemia and inflammatory bowel disease.

Azathioprine (AZA) and 6-mercaptopurine are prescribed in acute lymphoblastic leukaemia (ALL) and inflammatory bowel diseases (IBD). Metabolism to active 6-thioguanine (6TGN) and 6-methylmercaptopurine nucleotides (6MMPN) is variable but therapeutic drug monitoring (TDM) remains debatable. This study reports on factors impacting on red blood cell (RBC) metabolites concentrations in children to facilitate TDM interpretation. The first paediatric TDM samples received during year 2021 were analysed, whatever indication and thiopurine drug. Target concentration ranges were 200-500, <6000 pmol/8× 108 RBC for 6TGN and 6MMPN. Children (n= 492) had IBD (64.8%), ALL (22.6%) or another autoimmune disease (12.6%): mean ages at TDM were 7.5 in ALL and 13.7years in IBD (P <.0001). ALL received 6-mercaptopurine (mean dose 1.7mg/kg/d with methotrexate), IBD received AZA (1.9mg/kg/d with anti-inflammatory drugs and/or monoclonal antibodies). Median 6TGN and 6MMPN concentrations were 213.7 [interquartile range: 142.5; 309.6] and 1144.6 [419.4; 3574.3] pmol/8× 108 RBC, 38.8% of patients were in the recommended therapeutic range for both compounds. Aminotransferases and blood tests were abnormal in 57/260 patients: 8.1% patients had high alanine aminotransaminase, 3.4% of patients had abnormal blood count. Factors associated with increased 6TGN were age at TDM and thiopurine methyltransferase genotype in ALL and AZA dose in IBD. The impact of associated treatment in IBD patients was also significant. TDM allowed identification of children who do not reach target levels or remain over treated. Including TDM in follow-up may help physicians to adjust dosage with the aim of reducing adverse effects and improve treatment outcome.

Role of Pharmacogenomics in the Efficacy and Safety of Thiopurines in Inflammatory Bowel Disease: A Systematic Review and Meta-analysis.

Thiopurines' toxicity often leads to dose reduction or discontinuation. This systematic review aims to synthesize the evidence on the effect of genotype-based dosing of thiopurines on treatment efficacy and safety in inflammatory bowel disease (objective #1), and the association between genotype status and the efficacy and safety profile (objective #2). The Cochrane Library, MEDLINE, and EMBASE were searched in August 2021. A total of 80 studies (19,859 individuals) were included. Meta-analyses for mortality, different types of adverse events (AEs), withdrawal due to AE, change in disease activity and clinical remission were performed following mainly a fixed-effects model. PROSPERO registration: CRD42020148130. Genotype-based dosing was associated to a significantly lower incidence of hematologic AEs (risk ratio=0.71; 95% CI: 0.56-0.90; I2: 47%; 4 randomized controlled trials; moderate quality), which may be attributable to nudix hydrolase 15 (NUDT15) testing more than to thiopurine methyltransferase (TPMT) genotyping. No differences were found in other outcomes. Mutations in TPMT and NUDT15 genes were associated to a higher probability of serious AEs [odds ratio (OR) TPMT=4.98; OR NUDT15=11.44], hematologic AEs (OR TPMT=3.18), and serious hematologic AEs (OR TPMT=7.88; OR NUDT15=12.83). TPMT was also associated with a higher risk of withdrawals due to AEs (OR=3.38), and NUDT15 with gastrointestinal AEs (OR=2.04). Mutations in the ITPA gene did not lead to significant differences. Evidence of an association between other genes and clinical outcomes is still scarce. Mutations in TPMT and NUDT15 genes predispose patients to suffer thiopurine-induced toxicity, and genotype-guided treatment has been shown to contribute to the prevention of thiopurine-induced toxicity, especially in the case of NUDT15 in Asians.

Thiopurine S-Methyltransferase Polymorphisms Predict Hepatotoxicity in Azathioprine-Treated Patients with Autoimmune Diseases.

Thiopurine methyltransferase (TPMT) is the rate-limiting enzyme in Azathioprine (AZA) metabolization. Although studies have discussed the association between the TPMT polymorphisms and myelosuppression, the data about the relationship between TPMT genotypes and hepatoxicity in Asian patients remain limited. This study investigated the correlation between TPMT polymorphisms and AZA-related hepatotoxicity. This study enrolled the patients who had prior exposure to AZA from the Taichung Veterans General Hospital (TCVGH)-Taiwan Precision Medicine Initiative (TPMI) cohort. Genetic variants were determined using a single nucleotide polymorphism (SNP) array. Participants were accordingly categorized into normal metabolizer (NM) and non-normal metabolizer (non-NM) groups. From the TCVGH-TPMI cohort, we included 50 TPMT non-NM patients, including 1 poor metabolizer (PM), 49 intermediate metabolizers (IMs), and 1000 NM patients. The non-NM genotype was associated with hepatotoxicity compared with the NM genotype (hazard ratio (HR): 3.85, 95% confidence interval (CI): 1.83–8.10). In the non-NM group, the 3-year cumulative incidence of hepatotoxicity was higher than that in the NM group at 8.5% in the first year and 18.6% in the second and third years (p < 0.001). A TPMT non-NM genotype was associated with the occurrence of hepatotoxicity following AZA therapy. Preemptive testing helps individualize AZA therapy by minimizing the risk of hepatotoxicity.

Azathioprine‐induced pancytopenia: A case report

AbstractAzathioprine (AZA) is commonly used as immunosuppressive therapy for autoimmune diseases, including systemic lupus erythematosus (SLE). Myelosuppression is a common side effect of AZA. Here we report a case of severe myelosuppression following AZA therapy in a 15‐year‐old girl despite a normal thiopurine methyltransferase (TPMT) level. She had been receiving AZA for SLE and presented with neutropenic fever and pancytopenia. AZA was stopped. After stopping AZA, her blood counts steadily improved. When TPMT genotyping results were normal, AZA was reintroduced. Pancytopenia reappeared after starting AZA, despite normal TPMT genotype. AZA was replaced with mycophenolate mofetil which consequently resulted in improvement of blood counts. It is essential to understand the temporal relationship between AZA use and pancytopenia onset in patients with normal TPMT activity. This case illustrates that regular monitoring of blood cell counts should be routine practice after starting AZA regardless of TPMT activity.

Passive smoking and thiopurine methyl transferase genotyping in 6-mercaptopurine-induced myelosuppression in Egyptian children with acute lymphoblastic leukemia

6-mercaptopurine (6-MP) is a crucial component drug used to treat childhood acute lymphoblastic leukemia (ALL) in the maintenance phase. Thiopurine S-methyltransferase (TPMT) is an enzyme that metabolizes thiopurine drugs like 6-MP. In patients taking thiopurine medications, lack of TPMT activity is linked to an increased risk of myelosuppression. Patients with TPMT polymorphisms are more likely to develop mercaptopurine-induced myelosuppression. The goal of this study was to see how passive smoking and TPMT genetic variations affected 6-MP-induced myelosuppression in Egyptian ALL children. This study included 100 children: 50 with acute lymphoblastic leukemia in the maintenance phase of treatment who had a history of passive smoking and 50 age-matched healthy controls. 6-MP was given to patients in the maintenance phase, and myelosuppression was monitored at the end of this phase by comparing WBC counts to those obtained at the start of treatment. After six months of 6-MP administration, there was no significant association between cotinine levels and WBC count (as evidence of myelosuppression), although there was a significant correlation between TPMT genotypes and WBC count, with 75% of hetero genotype patients suffering from severe declines in WBC count and 91% of wild genotype people undergoing mild to moderate decreases in WBC count.

Genotype-Guided Prescription of Azathioprine Reduces the Incidence of Adverse Drug Reactions in TPMT Intermediate Metabolizers to a Similar Incidence as Normal Metabolizers.

IntroductionThiopurine drugs are purine nucleoside analogues used for treatment of different immune-related conditions. To date, different studies highlighted the importance of thiopurine methyltransferase (TPMT) genotyping in patients who initiate treatment with thiopurines to make an adequate dose adjustment. We aimed to investigate the influence of TPMT phenotype, concomitant treatments, and demographic characteristics on the incidence of adverse reactions (ADRs) in patients who start treatment with azathioprine (AZA).MethodsThis was an observational and retrospective study. The study population comprised 109 patients who started treatment with AZA following routine TPMT genotyping before June 2019 and who were routinely followed up at Hospital Universitario de La Princesa. The incidence of ADRs and treatment duration were evaluated according to TPMT phenotype.ResultsForty-five men and 64 women were recruited, with a mean age of 67.6 ± 18.5. The medical specialty with the most requests was dermatology (45.9%) and the most frequent disease for which genotyping was requested was bullous pemphigoid (27.5%). All patients were normal metabolizers (NM), except for eight intermediate metabolizers (IM) (7.3%); no poor metabolizers (PM) were found. The initial azathioprine dose was subtherapeutic in both groups (103.2 ± 45.4 mg in NMs and 75 ± 32.3 mg in IMs), increasing during the first months of treatment, especially in NMs (120.3 ± 41.3 vs. 78.6 ± 30.4 mg in IMs, p = 0.011). Most patients (73.4%) received corticosteroids to keep the disease under control; and for 41.2% of NMs, physicians were able to reduce the dose at 6 months post treatment. No IMs completed 6 months of treatment. Hepatotoxicity, gastric intolerance, and blood disorders were the most common ADRs. The incidence of ADRs in the sample was 28.4% (n = 31) with a similar trend between IMs (37.5%) and NMs (27.8%). Patients undergoing concomitant treatment with allopurinol were associated with a higher incidence of ADRs (n = 4, 100% vs. n = 105, 20%; p = 0.002).ConclusionTPMT genotyping before AZA prescription reduces ADR incidence in IMs to a similar level as NMs in the Spanish population. However, it is important to note no IMs completed 6 months of treatment, suggesting that there may be some differences in drug tolerability according to phenotype. In addition, most NMs are treated with subtherapeutic doses, are poorly followed up, and thus suffer avoidable ADRs. Finally, concomitant therapies that inhibit the xanthine oxidase enzyme (XDH), such as allopurinol, predispose to ADRs. Therefore, pharmacogenetic testing should be integrated as an additional clinical tool, in such a way that each patient receives personalized, precision treatment, where all factors influencing drug response are considered.

A Gastrointestinal Toxicity during Low-Dose Methotrexate Treatment in Two Pediatric Patients with Acute Lymphoblastic Leukemia

Low-dose Methotrexate (LD-MTX) and 6-mercaptopurine (6-MP) are used in the maintenance phase of treatment of childhood acute lymphoblastic leukaemia. It was reported that individuals with reduced activity of two of the enzymes involved in the pathway of purine metabolism, thiopurine S-methyltransferase (TPMT) and nudix hydrolase 15 (NUDT15), will be exposed to higher levels of active metabolites and will be at higher risk of side effects, such as myelosupression. Therefore, dosing recommendations for 6-MP based on TPMT and NUDT15 genotype have been approved by FDA and have been published by the Clinical Pharmacogenetics Implementation Consortium and the Ditch Working Group. Though several studies were conducted on the toxicity of LD and HD-MTX in childhood ALL patients, none of the genetic markers so far have been used in MTX therapy protocols due to the lack of a clear association with the response and/or toxicity. We describe two pediatric patients who suffered from gastrointestinal toxicity following peroral administration of LD-MTX during maintenance therapy of ALL that diminished after switching to parenteral administration of the drug.

Does Body Mass Index (BMI) during Maintenance Influence Relapse Risk in Children with Acute Lymphoblastic Leukemia (ALL)? Results from COG-AALL03N1

▪Introduction: Higher BMI at ALL diagnosis is associated with an increased risk of post-induction residual leukemia (Orgel, Blood 2014) and relapse (Butturini, JCO 2007). However, children may experience significant changes in BMI during the pre-maintenance phases of ALL treatment (Withycombe, Pediatr Blood Cancer 2009), necessitating an examination of the association between BMI during maintenance and relapse risk. We hypothesized that higher BMI during maintenance would be associated with a greater risk of relapse. We also explored the association between BMI and red cell thioguanine (TGN) levels to understand whether BMI-associated variations in TGN biodistribution explained the BMI-relapse association.Methods: We used data from COG-AALL03N1 (primary aim was 6MP adherence) to examine the association between BMI during maintenance and relapse risk. Eligibility for enrollment on AALL03N1 included age ≤21y at ALL diagnosis and receiving maintenance therapy in first remission. The current analysis was limited to patients with wild-type thiopurine methyltransferase genotype. BMI (exposure variable) was calculated as sex- and age-based percentile per CDC normative data, and operationalized as normal/underweight [<85%ile], overweight/obese [85-98%ile] and morbidly obese [≥99%ile]). Hazard of relapse (any site) was estimated using multivariable proportional subdistributional hazards regression after adjusting for age at study enrollment, sex, race/ethnicity, NCI risk group, cytogenetics, 6MP dose intensity (6MPDI), and time from initiation of maintenance. We compared fitted means of red cell TGN levels by BMI groups after adjusting for age at enrollment, sex, race/ethnicity, 6MPDI and time from initiation of maintenance using generalized estimated equations. The association between BMI and relapse risk, as well as between BMI and TGN levels, was also examined in a sub-cohort of patients with available 6MP adherence.Results: The sociodemographic and disease characteristics of the 676 study participants are summarized in the Table. Median BMI%ile was 88.5 (range, 0-100); normal/underweight: 43%, overweight/obese: 45%, and morbidly obese: 12%. As shown in the Figure, cumulative incidence of relapse at 2y from start of maintenance therapy was significantly greater among patients with morbid obesity (11.9±4.3%) when compared to those who were overweight/obese (5.4±1.6%) and underweight/normal weight (4.1±1.4%). After adjusting for the variables listed above, we found that patients with morbid obesity had a 3.2-fold greater hazard of relapse (95%CI=1.4-7.5, P=0.008) when compared to patients who were normal/underweight. After adjusting for age at enrollment, sex, race/ethnicity, 6MPDI, and time from initiation of maintenance, patients with morbid obesity had lower mean red cell TGN levels compared to normal/underweight patients (mean difference: -27.2±7.4 pmol/8 x 10 8 erythrocytes, P=0.0002). However, inclusion of TGN in the model did not alter the association between BMI and hazard of relapse (HR=3.8, 95%CI, 1.6-9.0, P=0.003). These findings did not change after adjusting for 6MP adherence in the sub-cohort with available adherence data (n=435).Conclusion: Morbid obesity during maintenance for childhood ALL is associated with relapse as well as lower systemic exposure to 6MP. However, lower TGN levels do not explain the relation between BMI and relapse risk. Therefore, there is a need to understand the mechanism of the relation between morbid obesity during maintenance and relapse risk in children with ALL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

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.

Understanding thiopurine methyltransferase polymorphisms for the targeted treatment of hematologic malignancies

ABSTRACT Introduction Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of thiopurines (mercaptopurine (MP) and tioguanine (TG)), chemotherapeutic agents used in the treatment of acute lymphoblastic leukemia (ALL). Polymorphisms in TPMT gene encode diminished activity enzyme, enhancing accumulation of active metabolites, and partially explaining the inter-individual differences in patients’ clinical response. Areas covered This review gives an overview on TPMT gene and function, and discusses the pharmacogenomic implications of TPMT variants in the prevention of severe thiopurine-induced hematological toxicities and the less known implication on TG-induced sinusoidal obstruction syndrome. Additional genetic and non-genetic factors impairing TPMT activity are considered. Literature search was done in PubMed for English articles published since1990, and on PharmGKB. Expert opinion To titrate thiopurines safely and effectively, achieve the right degree of lymphotoxic effect and avoid excessive myelosuppression, the optimal management will combine a preemptive TPMT genotyping to establish a safe initial dose with a close phenotypic monitoring of TPMT activity and/or of active metabolites during long-term treatment. Compared to current ALL protocols, replacement of TG by MP during reinduction phase in TPMT heterozygotes and novel individualized TG regimens in maintenance for TPMT wild-type subjects could be investigated to improve outcomes while avoiding risk of severe hepatotoxicity.

Cost-effectiveness analysis of genotype screening and therapeutic drug monitoring in patients with inflammatory bowel disease treated with azathioprine therapy: a Chinese healthcare perspective using real-world data.

BackgroundThis study aimed to analyze the cost-effectiveness of combining screening for thiopurine methyl transferase (TPMT) and nucleotide triphosphate diphosphatase (NUDT15) defective alleles with therapeutic drug monitoring (TDM) in Chinese patients with inflammatory bowel disease (IBD) treated with azathioprine (AZA).MethodsWe evaluated the cost-effectiveness of combining screening for NUDT15 and TPMT deficiency with TDM in patients receiving AZA treatment over a 1-year horizon by developing a decision tree model. Real-world data and published literature were used to derive model inputs. The model’s primary outcomes included quality-adjusted life-years (QALYs) and incremental cost-effectiveness ratios (ICERs). One-way and probabilistic sensitivity analyses were used to address uncertainty.ResultsCompared to NUDT15 genotyping, the combined TPMT/NUDT15 genotyping strategy cost an additional $13.83, yielding an ICER of $3,929.54/QALY, which was under the willingness-to-pay level of $30,425 per QALY in China. Compared to strategies with singular TPMT genotyping or no genotyping, the combined TPMT/NUDT15 genotyping strategy gained 0.00406 and 0.00782 QALYs and reduced the cost by $25.15 and $99.06, respectively. Additionally, incorporating TDM of AZA was more effective and less expensive than strategies without TDM. One-way sensitivity analysis revealed the expense attached to severe myelotoxicity to be the factor with the greatest influence in the present research. The application of the combined genotype screening strategy with TDM of AZA treatment was found to have a 91.7% chance of being cost-effective.ConclusionsFor Chinese patients with IBD who receive an AZA regimen, a strategy involving combined NUDT15/TPMT genotype screening prior to treatment initiation and incorporating TDM for treatment management is cost-effective compared to strategies involving genotyping of NUDT15 or TPMT alone or genotyping without TDM.

No association between relapse hazard and thiopurine methyltransferase geno- or phenotypes in non-high risk acute lymphoblastic leukemia: a NOPHO ALL2008 sub-study.

6-mercaptopurine(6MP)/methotrexate maintenance therapy is essential to reduce relapse of childhood acute lymphoblastic leukemia (ALL). Common germline variants in TPMT cause low activity of thiopurine methyltransferase (TPMT) and higher 6MP metabolite (TGN) levels. Higher levels of TGNs incorporated into DNA (DNA-TG) and low TPMT activity have previously been associated with a lower relapse risk. We explored if TPMT geno- or phenotype was associated with DNA-TG levels and relapse rate in NOPHO ALL2008. TPMT genotype, repeated phenotyping, and DNA-TG measurements were collected in 918 children with non-high risk ALL (NOPHO ALL2008 maintenance therapy study). Maintenance therapy started with 6MP at 50 and 75mg/m2 for TPMT heterozygous and wildtype patients and was adjusted to a target WBC of 1.5 - 3.0 × 109/L. Of 918 patients, 78 (8.5%) were TPMT heterozygous and 903 had at least one TPMT measurement (total 3063). Mean TPMT activities were higher with wildtype than heterozygous TPMT (N = 752, 16.6 versus 9.6 U/mL ery., p < 0.001). The 5-year cumulative incidence of relapse was 6.4% and 6.0% for TPMT heterozygous and wildtype patients, and there was no association between genotype and relapse rate (N = 918, hazard ratio = 1.01, 95% confidence interval [CI] 0.40 - 2.54, p = 0.98). Although TPMT heterozygous patients had higher DNA-TG (N = 548, median 760.9 [interquartile range (IQR) 568.7 - 890.3] versus 492.7 [IQR 382.1 - 634.6] fmol/µg, p < 0.001), TPMT activity was not associated with relapse rate (N = 813; hazard ratio = 0.98 per one U/mL ery. increase in TPMT activity, 95% CI 0.91 - 1.06, p = 0.67). TPMT geno- and phenotype were not associated with relapse in non-high risk NOPHO ALL2008.

Hepatic sinusoidal obstruction syndrome and short-term application of 6-thioguanine in pediatric acute lymphoblastic leukemia

Long-term treatment with 6-thioguanine (6-TG) for pediatric acute lymphoblastic leukemia (ALL) is associated with high rates of hepatic sinusoidal obstruction syndrome (SOS). Nevertheless, current treatment continues to use short-term applications of 6-TG with only sparse information on toxicity. 6-TG is metabolized by thiopurine methyltransferase (TPMT) which underlies clinically relevant genetic polymorphism. We analyzed the association between hepatic SOS reported as a serious adverse event (SAE) and short-term 6-TG application in 3983 pediatric ALL patients treated on trial AIEOP-BFM ALL 2000 (derivation cohort) and defined the role of TPMT genotype in this relationship. We identified 17 patients (0.43%) with hepatic SOS, 13 of which with short-term exposure to 6-TG (P < 0.0001). Eight of the 13 patients were heterozygous for low-activity TPMT variants, resulting in a 22.4-fold (95% confidence interval 7.1–70.7; P ≤ 0.0001) increased risk of hepatic SOS for heterozygotes in comparison to TPMT wild-type patients. Results were supported by independent replication analysis. All patients with hepatic SOS after short-term 6-TG recovered and did not demonstrate residual symptoms. Thus, hepatic SOS is associated with short-term exposure to 6-TG during treatment of pediatric ALL and SOS risk is increased for patients with low-activity TPMT genotypes.

TPMT Genetic Variability and Its Association with Hematotoxicity in Indonesian Children with Acute Lymphoblastic Leukemia in Maintenance Therapy.

PurposeHematotoxicity monitoring in children with acute lymphoblastic leukemia (ALL) is critical to preventing life-threatening infections and drug discontinuation. The primary drug that causes hematotoxicity in ALL children is 6-mercaptopurine (6-MP). Genetic variability of the drug-metabolizing enzymes of 6-MP, thiopurine S-methyltransferase (TPMT), is one factor that might increase the susceptibility of children to hematotoxicity. The present study aimed to determine the variability in TPMT genotypes and phenotypes and its association with the occurrence of hematotoxicity in ALL children in maintenance therapy.Patients and MethodsA cross-sectional study was conducted at Cipto Mangunkusumo and Dharmais National Cancer Hospital, Jakarta, Indonesia, from June 2017 to October 2018. We included ALL patients, 1–18 years, who were receiving at least one month of 6-MP during maintenance therapy according to the Indonesian protocol for ALL 2013. Direct sequencing was used to determine TPMT*3A, *3B, and *3C genotypes, and LC-MS/MS analysis was performed to measure the plasma concentrations of 6-MP and its metabolites. Association analysis between the TPMT genotype and hematotoxicity was evaluated using the unpaired t-test or Mann–Whitney’s test.ResultsThe prevalence of neutropenia, anemia, and thrombocytopenia in ALL children during maintenance therapy was 51.9%, 44.3%, and 6.6%, respectively. We found a low frequency of TPMT*3C, which is 0.95%. No association was found between hematotoxicity and TPMT genotypes or age, nutritional status, serum albumin levels, risk stratification, the daily dose of 6-MP, and cotrimoxazole co-administration. However, hematotoxicity was associated with 6-methylmercaptopurine (6-MeMP) plasma concentrations and the ratio 6-MeMP/6-thioguanine (6-TGN). We also found no association between TPMT genotypes and TPMT phenotypes.ConclusionThe 6-MeMP/6-TGN ratio is associated with hematotoxicity in ALL children during maintenance therapy but is not strong enough to predict hematotoxicity.

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.