Thiopurine Methyltransferase Status Research Articles

No Association between Relapse Risk and Thiopurine Methyltransferase Activity By Either Geno- or Phenotype in the NOPHO ALL-2008 Protocol

Background6-mercaptopurine/methotrexate (6MP/MTX) maintenance therapy is essential for high cure rates of childhood acute lymphoblastic leukemia (ALL). 6MP exerts its cytotoxicity through conversion into thioguanine nucleotides (TGN) that are substrates for DNA polymerase and compete with normal guanine for DNA incorporation. DNA-TGN occasionally mismatches to thymidine causing cell death due to futile attempts of repetitive mismatch repair. We recently showed that higher levels of DNA-incorporated TGN were associated with a reduced relapse risk in non-high risk ALL (Nielsen, Lancet Oncol 2017). Common germline variants in TPMT that codes for thiopurine methyltransferase (TPMT) lead to reduced TPMT activity and higher levels of cytosol TGN when measured in erythrocytes. Thus, low TPMT activity could theoretically lead to higher DNA-TGN and a lower relapse rate. Low TPMT activity (phenotype) has been associated with a lower relapse risk in the NOPHO ALL92 protocol (Schmiegelow, Leukemia 2009). We explored if TPMT status evaluated by either geno- or phenotype was related to DNA-TGN levels and relapse rate in the NOPHO ALL2008 protocol.MethodTPMTgenotype and repeated phenotyping (2141 measurements in total), and levels of DNA-TGN (10830 measurements in total) during maintenance therapy were determined for 918 children treated for non-high risk ALL (89% of all eligible patients). On a subset (N=204) TPMT phenotype was available both at diagnosis and during maintenance therapy. 6MP was started at 50 and 75 mg/m2 for TPMT heterozygous and wildtype patients, respectively, and subsequently adjusted to a target WBC of 1.5-3.0 x109/L. Cox analyses were adjusted by age, sex and WBC at diagnosis.Results78 of the 918 non-high risk patients were TPMT heterozygous (none homozygous deficient). TPMT activities were significantly higher during maintenance therapy than at diagnosis for both TPMT wildtype and heterozygous children (N=204; wildtype: 16.6 vs. 11.3 U/mL ery., p <0.001; heterozygous: 9.9 vs. 6.8 U/mL ery., p=0.047). Mean DNA-TGN levels were higher for TPMT heterozygous than wildtype children (N=523, children in remission at end of therapy with Maintenance II measurements, median 760.9 fmol/µg, range 125.7−1255.0 vs. 494.9, range 44.0−1559.0, p <0.001; Figure). The 5-year cumulative incidence of relapse was 4.2% (95% CI: 0-8.7%) for TPMT heterozygous and 5.7% (95% CI: 3.8-7.5%) forwildtype children (hazard ratio =0.87, 95% CI: 0.27−2.83, p=0.81). No association between mean TPMT activity during maintenance therapy (as a time-dependent covariate) and relapse-specific hazard was seen (N=812; hazard ratio =0.98 per one unit increase in TPMT activity (U/mL ery.), 95% CI: 0.89−1.08, p=0.63). In patients with positive MRD at end of induction (d29) results were similar (N=476; hazard ratio =1.04 per one unit increase in TPMT activity (U/mL ery.), 95% CI: 0.93−1.16, p=0.50).ConclusionAlthough mean DNA-TGN levels were higher in TPMT heterozygous than wildtype children, variability was high in both groups and DNA-TGN levels were largely overlapping, and no association with relapse hazard was seen for neither TPMT geno- nor phenotype. In accordance with previously published data TPMT phenotype determinations done in erythrocytes present at ALL diagnosis can lead to TPMT misclassification.FIGUREa) Mean DNA-TGN levels (N=523) during Maintenance II of children in clinical remission with completed therapy and at least one measurement.b) Cumulative incidence curves of relapse of children carrying TPMT wildtype vs. heterozygous. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Thiopurine methyltransferase activity in children with acute myeloid leukemia.

Activity of the enzyme thiopurine methyltransferase (TPMT) determines the anti-leukemic effect of thiopurines used in the chemotherapy of acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML). TPMT status and its effects on treatment outcome have been studied extensively in ALL and autoimmune disorders, but few data is available on TPMT in AML. The present study assessed the genetic polymorphisms and activity of TPMT in children with AML at different treatment stages, and compared the results with those obtained for children with ALL. The study included 33 children with AML (0.7-19.7 years) treated with 6-thioguanine (6-TG) according to the AML-BFM 2004 Protocol. Blood samples were collected at diagnosis, during and following maintenance chemotherapy from 8, 10 and 17 patients with AML (the assay was performed at two time points in 2 patients), respectively. Blood samples from 105 children with ALL were obtained at diagnosis, during the maintenance chemotherapy and following the cessation of the chemotherapy from 16, 55 and 34 children, respectively. The activity of TPMT in red blood cells lysates was measured using an enzymatic reaction based on the conversion of 6-mercaptopurine into 6-methylmercaptopurine, involving S-adenozyl-L-methionine as the methyl group donor. TPMT mutations were determined using a polymerase chain reaction/restriction fragment length polymorphism method. Median TPMT activity at diagnosis, during maintenance chemotherapy and following chemotherapy was 43.1, 47,3 and 41.7 nmol 6-mMP g-1 Hb h-1, respectively. All patients with AML exhibited the homozygous TPMT*1/*1 genotype, with the exception of 1, who was a heterozygote with the TPMT*1/*3C genotype and demonstrated a TPMT activity level at diagnosis of 42.5 nmol 6-mMP g-1 Hb h-1. At each chemotherapy stage, the median TPMT activities in children with AML were significantly increased compared with the median TPMT activities in children with ALL. The preliminary results suggest that the TPMT activity in AML may be increased compared with that in ALL. Comprehensive studies on the association between thiopurine metabolism and treatment outcome in AML are required, with regard to the cytogenetic and molecular factors currently used for AML risk stratification.

Usefulness of thiopurine methyltransferase polymorphism study and metabolites measurement for patients treated by azathioprine

Azathioprine is widely used in internal medicine and frequently implicated in occurrence of adverse events. Among these adverse events the bone marrow suppression, a dose-related one, is the most serious because of is potential morbidity and mortality. Severe myelosuppression, associated with abnormal AZA metabolism, is linked to the thiopurine methyltransferase (TPMT) genetic polymorphism that results in a high variability of its activity with 89% of patients with a normal activity, 11% with an intermediate activity, and 0.3% with very low activity leading to a very high risk of bonne marrow suppression. TPMT status can be assessed prior to AZA treatment by measuring enzyme activity or genotyping techniques to identify patients for which the standard dose is not advisable. Furthermore, azathioprine metabolites monitoring is helpful for the follow up of patients, especially in therapeutic failure, to distinguish non-compliant patients from under-dosed, "shunters" or resistant patients.

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.

Host thiopurine methyltransferase status affects mercaptopurine antileukemic effectiveness in a murine model

Thiopurines are used for many cancers, including acute lymphoblastic leukemia (ALL). Patients with an inherited host defect in thiopurine methyltransferase (TPMT) are at high risk for life-threatening toxicity if treated with conventional dosages, but the impact on antileukemic efficacy is less clear. We treated thiopurine-sensitive BCR-ABL+Arf-null Tpmt+/+ ALL in Tpmt+/+, +/-, or -/- recipient mice to test the impact of the host polymorphism on antileukemic efficacy. Median survival was similar in untreated mice of different Tpmt genotypes (16-18 days). However, in mice treated with low-dose mercaptopurine (such as tolerated by TPMT-/- patients), the difference in 30-day leukemia-free survival by Tpmt genotype was profound: 5% (±9%) for Tpmt+/+ mice, 47% (±26%) for Tpmt+/- mice, and 85% (±14%) for Tpmt-/- mice (P=5×10), indicating a substantial impact of host Tpmt status on thiopurine effectiveness. Among Tpmt+/+ recipient mice, leukemia-free survival improved with higher doses of mercaptopurine (similar to doses tolerated by wild-type patients) compared with lower doses, and at higher doses was comparable (P=0.6) to the survival of Tpmt-/- mice treated with the lower dose. These findings support the notion that germline polymorphisms in Tpmt affect not only host tissue toxicity but also antitumor effectiveness.

Implementation of TPMT testing

The activity of the enzyme thiopurine methyltransferase (TPMT) is regulated by a common genetic polymorphism. One in 300 individuals lack enzyme activity and 11% are heterozygous for a variant low activity allele and have an intermediate activity. The thiopurine drugs azathioprine, mercaptopurine and thioguanine are substrates for TPMT; these drugs exhibit well documented myelosuppressive effects on haematopoietic cells and have a track record of idiosyncratic drug reactions. The development of severe bone marrow toxicity, in patients taking standard doses of thiopurine drugs, is associated with TPMT deficiency whilst the TPMT heterozygote is at an increased risk of developing myelosuppression. Factors influencing TPMT enzyme activity, as measured in the surrogate red blood cell, are discussed in this review to enable an appreciation of why concordance between TPMT genotype and phenotype is not 100%. This is particularly important for lower/intermediate TPMT activities to avoid misclassification of TPMT status. TPMT testing is now widely available in routine service laboratories. The British National Formulary suggests TPMT testing before starting thiopurine drugs. Dermatologists were quick to adopt routine TPMT testing whilst gastroenterologists do not specifically recommend TPMT screening. TPMT testing is mandatory prior to the use of mercaptopurine in childhood leukaemia. Thiopurine drug dose and other treatment related influences on cell counts explain some of the differing recommendations between clinical specialities. TPMT testing is cost-effective and the major role is in the identification of the TPMT deficient individual prior to the start of thiopurine drugs.

How do we prepare ourselves for a new paradigm of medicine to advance the treatment of pediatric acute lymphoblastic leukemia?

The immunotherapeutic approaches that have been realized in clinical settings have begun a new era in the treatment of acute lymphoblastic leukemia (ALL). Gene-engineered autologous T-cell therapy, called living drug, has killed leukemic cells and had kept 88% of refractory adult patients with ALL alive in successful molecular remission [1]. We are fully armed with tailored chemotherapy, hematopoietic stem cell transplantation, and gene-engineered T-cell immunotherapy to fight ALL. Over the past several decades, there have been remarkable advances in childhood ALL. In recent clinical trials, which had better defined risk stratification using minimal residual disease, the 5-year survival rate was more than 85% in developed countries [2]. This improvement is the result of active chemotherapeutic agents being developed, a better understanding of prescribing doses and combining these agents, and the significant progress made in supportive care through multicenter clinical trials. However, we still have to fight a treatment failure of 10-15%. Continued research is required to identify and target specific molecular and genetic abnormalities within leukemic cells as well as to better understand and address individual differences in the pharmacogenetics of chemotherapeutic agents. Genomic sequencing and adequate analytic platforms, such as gene expression profiling, single-nucleotide polymorphism arrays, and next-generation sequencing, have become available in the treatment of childhood ALL. Molecular biology techniques have revealed many different childhood ALL subtypes that bear features such as iAMP21, CRLF2 rearrangements, IKZF1 alteration, JAK1/2 mutations, BCR-ABL1-like signatures, and early T-cell precursors that are associated with poorer outcomes [3]. In addition, recent analysis has identified new genomic alteration-associated prognosis; therefore, these genomics can be translated into a better risk stratification that will make tailored therapy available in the near future. The increasing number of genomic alterations that have been discovered recently gives us important information for designing future studies. The first is whether these genotypes can be used to improve patient stratification; the second concerns the role of new discoveries in genetics and molecular biology in determining specific treatment modalities. Pharmacogenomics in cancer treatment hold great promise for yielding genetic polymorphisms that could be used to individualize antileukemic agent dosages. Thus far, the only well-established clinical effect refers to mercaptopurine and the genetic polymorphism status of the thiopurine methyltransferase (TPMT ) gene. Indeed, tailoring the dosages of methotrexate and mercaptopurine to the limits of tolerance has been associated with a better outcome [4]. Therefore, customizing the dosage of mercaptopurine based on preemptive testing for TPMT status will likely decrease the risk of mercaptopurine-induced toxicities associated with an inherited TPMT deficiency. This, in turn, might reduce the likelihood of acute myelosuppression (without compromising disease control) and the risk for developing mercaptopurine-induced myeloid malignancy. TPMT also has a significant impact on thioguanine pharmacokinetics because patients with this enzyme deficiency are at an increased risk for developing hepatic venoocclusive disease. It is possible that, in the near future, genetic guidance might ensure a better use of standard drugs, improved efficacy, and reduced toxicity and long-term side effects. TPMT mutations in the Asian populations are rare (about 2-3%) compared with that in the Western populations (about 10%) [5]. However, this cannot account for the reduced tolerance to mercaptopurine among the Asian populations. Further study is needed to ascertain whether polymorphisms of genes encoding enzymes involved in folate metabolism, or perhaps other genes, affect the response to antimetabolites in the Asian populations. Korea has many centers with enhanced resources that can adapt new technologies to clinical practices [6]. However, in Korea, where the national health insurance system controls the application of new laboratory tests or treatments to clinical practice, national health insurance policy limitations hinder the addition of these advances to standard practice guidelines, which would be unlikely in the United States or Europe [7]. In the past, our management guidelines in pediatric ALL have been based mainly on treatment experiences in the United States or Europe, and those guidelines are often different in each institution. However, several years ago, we started multicenter clinical trials for high-risk pediatric ALL patients and have finished patient enrollment. Currently, we are developing new protocols for newly diagnosed high-risk and relapsed ALL patients. In these trials, risk assignment, bone marrow response, and minimal residual disease measurements will be performed on day 7 or day 15 by flow cytometry and reverse transcription PCR (HemaVision). TPMT studies will be carried out for pharmacogenetics. Because defining the complete genetic repertoire of ALL and the progressive availability of new targeted therapy will make patient subgroups smaller, we will be asked to participate in international collaborative trials. For this to become a reality, we have to keep up with global standards. Now is the time to come together.

Association of thiopurine methyltransferase status with azathioprine side effects in Chinese patients with systemic lupus erythematosus

Azathioprine (AZA) is indicated for the treatment of systemic lupus erythematosus (SLE). Thiopurine methyltransferase (TPMT) is the rate-limiting enzyme in the steps of AZA metabolization. Heritable deficiency of TPMT enzyme activity and polymorphisms may lead to leukopenia. This study aims to detect TPMT polymorphisms and TPMT enzyme activity in Chinese SLE patients and to describe the association between TPMT genotypes and adverse effects of AZA. One hundred and twenty-six SLE patients with present or previous thiopurine therapy were identified from a local database. Adverse effects were documented. No TPMT*2, TPMT*3A, or TPMT*3B mutant alleles were detected. TPMT*3C was detected in four patients (3.17%). The heterozygotes had significantly lower mean TPMT activity as compared to the homozygotes (2.38 ± 1.24 vs. 12.56 ± 7.02U/mL, P < 0.001). Twenty-seven cases (21.42%) exhibited adverse effects. All of the heterozygotes (4/4, 100%) developed severe leukopenia, and three cases (3/4, 75%) of whom exhibited alopecia simultaneously. The specificity of TPMT*3C for predicting leukopenia and alopecia was 100 and 99.17%, respectively, and the sensitivity was 28.57 and 60.00%, respectively. The mean value of TPMT activity with leukopenia (4.67 ± 3.01 vs. 13.2 ± 6.94U/mL RBC, P < 0.001) or alopecia (2.31 ± 1.16 vs. 12.65 ± 6.98U/mL RBC, P < 0.001) was significantly lower than those without. TPMT*3C was the most common mutant polymorphism found in the study group. TPMT activity is reduced in TPMT*3C mutant. AZA-induced leukopenia and alopecia were partly correlated to TPMT*3C heterozygotes and low TPMT activity. The results of this study suggest that the value of TPMT genotyping before AZA therapy was limited in Chinese SLE patients, considering the low sensitivity. Routine monitoring of TPMT activity before prescribing and continuous hematological monitoring dose were recommended.

Thiopurine methyltransferase genotype–phenotype discordance and thiopurine active metabolite formation in childhood acute lymphoblastic leukaemia

In children with acute lymphoblastic leukaemia (ALL) bone marrow activity can influence red blood cell (RBC) kinetics, the surrogate tissue for thiopurine methyltransferase (TPMT) measurements. The aim of this study was to investigate TPMT phenotype-genotype concordance in ALL, and the influence of TPMT on thiopurine metabolite formation. We measured TPMT (activity, as units ml(-1) packed RBCs and genotype) at diagnosis (n = 1150) and TPMT and thioguanine nucleotide (TGN) and methylmercaptopurine nucleotide (MeMPN) metabolites (pmol/8 × 10(8) RBCs) during chemotherapy (n = 1131) in children randomized to thioguanine or mercaptopurine on the United Kingdom trial ALL97. Median TPMT activity at diagnosis (8.5 units) was significantly lower than during chemotherapy (13.8 units, median difference 5.1 units, 95% confidence interval (CI) 4.8, 5.4, P < 0.0001). At diagnosis genotype-phenotype was discordant. During chemotherapy the overall concordance was 92%, but this fell to 55% in the intermediate activity cohort (45% had wild-type genotypes). For both thiopurines TGN concentrations differed by TPMT status. For mercaptopurine, median TGNs were higher in TPMT heterozygous genotype (754 pmol) than wild-type (360 pmol) patients (median difference 406 pmol, 95% CI 332, 478, P < 0.0001), whilst median MeMPNs, products of the TPMT reaction, were higher in wild-type (10 650 pmol) than heterozygous patients (3868 pmol) (P < 0.0001). In TPMT intermediate activity patients with a wild-type genotype, TGN (median 366 pmol) and MeMPN (median 8590 pmol) concentrations were similar to those in wild-type, high activity patients. In childhood ALL, TPMT activity should not be used to predict heterozygosity particularly in blood samples obtained at disease diagnosis. Genotype is a better predictor of TGN accumulation during chemotherapy.

Nomenclature for alleles of the thiopurine methyltransferase gene

The drug-metabolizing enzyme thiopurine methyltransferase (TPMT) has become one of the best examples of pharmacogenomics to be translated into routine clinical practice. TPMT metabolizes the thiopurines 6-mercaptopurine, 6-thioguanine, and azathioprine, drugs that are widely used for treatment of acute leukemias, inflammatory bowel diseases, and other disorders of immune regulation. Since the discovery of genetic polymorphisms in the TPMT gene, many sequence variants that cause a decreased enzyme activity have been identified and characterized. Increasingly, to optimize dose, pretreatment determination of TPMT status before commencing thiopurine therapy is now routine in many countries. Novel TPMT sequence variants are currently numbered sequentially using PubMed as a source of information; however, this has caused some problems as exemplified by two instances in which authors' articles appeared on PubMed at the same time, resulting in the same allele numbers given to different polymorphisms. Hence, there is an urgent need to establish an order and consensus to the numbering of known and novel TPMT sequence variants. To address this problem, a TPMT nomenclature committee was formed in 2010, to define the nomenclature and numbering of novel variants for the TPMT gene. A website (http://www.imh.liu.se/tpmtalleles) serves as a platform for this work. Researchers are encouraged to submit novel TPMT alleles to the committee for designation and reservation of unique allele numbers. The committee has decided to renumber two alleles: nucleotide position 106 (G>A) from TPMT*24 to TPMT*30 and position 611 (T>C, rs79901429) from TPMT*28 to TPMT*31. Nomenclature for all other known alleles remains unchanged.

Improved Outcomes in a Quality Improvement Collaborative for Pediatric Inflammatory Bowel Disease

Unintended variation in the care of patients with Crohn disease (CD) and ulcerative colitis (UC) may prevent achievement of optimal outcomes. We sought to improve chronic care delivery and outcomes for children with inflammatory bowel disease by using network-based quality improvement methods. By using a modified Breakthrough Series collaborative structure, 6 ImproveCareNow Network care centers tested changes in chronic illness care and collected data monthly. We used an interrupted time series design to evaluate the impact of these changes. Data were available for 843 children with CD and 345 with UC. Changes in care delivery were associated with an increase in the proportion of visits with complete disease classification, measurement of thiopurine methyltransferase (TPMT) before initiation of thiopurines, and patients receiving an initial thiopurine dose appropriate to their TPMT status. These were significant in both populations for all process variables (P < .01) except for measurement of TPMT in CD patients (P = .12). There were significant increases in the proportion of CD (55%-68%) and UC (61%-72%) patients with inactive disease. There was also a significant increase in the proportion of CD patients not taking prednisone (86%-90%). Participating centers varied in the success of achieving these changes. Improvements in the outcomes of patients with CD and UC were associated with improvements in the process of chronic illness care. Variation in the success of implementing changes suggests the importance of overcoming organizational factors related to quality improvement success.

The value of tpmt measurement in the routine clinical management of patients with IBD

IntroductionThe measurement of thiopurine methyl transferase (TPMT) can be used to identify patients at risk of marrow suppression from azathioprine. Across the medical specialities, there is no clear consensus or...

Characterisation and utility of thiopurine methyltransferase and thiopurine metabolite measurements in autoimmune hepatitis

Corticosteroids alone or in conjunction with azathioprine (AZA) is the standard treatment in autoimmune hepatitis (AiH). Individual variations in thiopurine (TP) metabolism may affect both drug efficacy and toxicity. Our aim was to investigate the utility of thiopurine methyltransferase (TPMT) as well as thioguanine nucleotide (TGN) and methylthioinosine monophosphate (meTIMP) metabolite measurements with regard to clinical outcome. Two hundred thirty-eight patients with AiH were included in this cross-sectional study. TPMT status was assessed in all patients, while TGN and meTIMP were measured in patients with ongoing TP medication. Clinical outcome was evaluated by liver tests and the ability to withdraw steroids. TPMT genotyping (n=229) revealed 207 (90.4%) wild-type and 22 heterozygous patients. One hundred forty-three patients had ongoing TP therapy with AZA (n=134) or mercaptopurine (MP; n=9); response was judged as complete response (CR) in 113 patients and partial response (PR) in 30 patients. Both TP dose (1.64 vs 1.19 mg/kg; p=0.012) and TPMT activity (14.3 vs 13.5; p=0.05) were higher in PR, resulting in similar TGN levels (PR: 121 pmol/8 x 10(8) red blood cells [RBC]; CR: 113 pmol/8 x 10(8) RBC; p=0.33) but higher meTIMP levels in PR (1350 vs 400 pmol/8 x 10(8) RBC; p=0.004). Patients able to withdraw steroids or who were using 5 mg prednisolone daily were treated with lower TP doses than patients on higher steroid doses (1.15 vs 1.18 vs 1.82 mg/kg; p<0.001). TP metabolite measurements are of clinical value in AiH patients who do not respond to standard TP treatment and for the identification of a shifted metabolism, which may demand an alternative treatment strategy.

Thiopurinemethyltransferase (TPMT) genotype and TPMT activity in patients with anti-neutrophil cytoplasmic antibody-associated vasculitis: relation to azathioprine maintenance treatment and adverse effects

Azathioprine is used in patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) to maintain remission. Azathioprine is inactivated and converted into hepatotoxic metabolites by the enzyme thiopurine methyltransferase (TPMT). Polymorphisms...

Thiopurine Methyltransferase Gene Polymorphisms in Chinese Patients with Inflammatory Bowel Disease

Background: The thiopurine drugs azathioprine and 6-mercaptopurine are well established in the treatment of inflammatory bowel disease (IBD). However, great interpatient variability exists in the efficacy and toxicity of the drugs, and this results from thiopurine methyltransferase (TPMT) gene polymorphisms. The aim of this study was to identify the TPMT gene polymorphisms in Chinese IBD patients and to study the relationship between TPMT status and thiopurine-related toxicity in these patients. Materials and Methods: A total of 189 IBD patients, 87 with Crohn’s disease and 102 with ulcerative colitis, and 273 healthy controls were enrolled. All subjects were from the Han Chinese ethnic group. Polymorphisms in TPMT*2, *3A, *3B and *3C were analyzed using allele-specific polymerase chain reaction and polymerase chain reaction-restriction fragment length polymorphism. Direct sequencing was used to confirm the mutation results. Exons of the TPMT gene from patients who suffered from azathioprine-induced toxicity were amplified and sequenced to detect TPMT mutations. Results: No TPMT*2, *3A or *3B mutant alleles were detected. The allele frequency of TPMT*3C in the IBD group was 1.59%, which was similar to that of the healthy control group (1.59 vs. 1.47%, p = 1.000). Forty-three patients were treated with azathioprine therapy, 4 experienced myelotoxicity, and 1 experienced hepatotoxicity, so the incidence of drug toxicity was 11.7% (5/43). No TPMT*2, *3A, *3B or *3C polymorphisms were detected in these 5 patients. After directly sequencing the exons of the TPMT gene in these 5 patients, a synonymous single-nucleotide polymorphism (TPMT*1S), which does not alter the encoded amino acid, was found in 3 patients. Conclusion: TPMT*3C seemed to be a unique variant allele in this Han Chinese population. The overall frequencies of variant TPMT alleles in this population were lower than those in Caucasians, but thiopurinetoxicity in Han Chinese IBD patients is not low. Factors other than TPMT polymorphisms may be responsible for the development of toxicity.

The effect of thiopurine drugs on DNA methylation in relation to TPMT expression

The thiopurine drugs 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are well-established agents for the treatment of leukaemia but their main modes of action are controversial. Thiopurine methyltransferase (TPMT) metabolises thiopurine drugs and influences their cytotoxic activity. TPMT, like DNA methyltransferases (DNMTs), transfers methyl groups from S-adenosylmethionine (SAM) and generates S-adenosylhomocysteine (SAH). Since SAM levels are dependent on de novo purine synthesis (DNPS) and the metabolic products of 6-TG and 6-MP differ in their ability to inhibit DNPS, we postulated that 6-TG compared to 6-MP would have differential effects on changes in SAM and SAH levels and global DNA methylation, depending on TPMT status. To test this hypothesis, we used a human embryonic kidney cell line with inducible TPMT. Although changes in SAM and SAH levels occurred with each drug, decrease in global DNA methylation more closely reflected a decrease in DNMT activity. Inhibition was influenced by TPMT for 6-TG, but not 6-MP. The decrease in global methylation and DNMT activity with 6-MP, or with 6-TG when TPMT expression was low, were comparable to 5-aza-2′-deoxycytidine. However, this was not reflected in changes in methylation at the level of an individual marker gene ( MAGE1A). The results suggest that a non-TPMT metabolised metabolite of 6-MP and 6-TG and the TPMT-metabolised 6-MP metabolite 6-methylthioguanosine 5’-monophosphate, contribute to a decrease in DNMT levels and global DNA methylation. As demethylating agents have shown promise in leukaemia treatment, inhibition of DNA methylation by the thiopurine drugs may contribute to their cytotoxic affects.

Current use of pharmacogenetic testing: a national survey of thiopurine methyltransferase testing prior to azathioprine prescription

Azathioprine is an immunosuppressant prescribed for the treatment of inflammatory conditions and after organ transplantation. Risk of neutropaenia has limited the effective use of azathioprine (AZA) and driven requirements for careful monitoring and blood tests. Thiopurine methyltransferase (TPMT) is a genetically moderated key enzyme involved in the metabolism of AZA that can be used to stratify individuals into different levels of risk of developing neutropaenia. Two techniques can be used to measure TPMT status: enzyme-level testing (phenotype testing) and DNA based testing (genotype testing). To identify the current uptake of TPMT enzyme-level testing, TPMT genotype testing, and, the role of guidelines; to inform the prescribing and monitoring of AZA. A survey was mailed to a consultant dermatologist, gastroenterologist, and rheumatologist at every NHS Hospital Trust in England. The survey comprised mainly closed questions exploring: use of AZA and monitoring; use of TPMT enzyme-level testing and genotype testing; and, the role of guidelines to guide prescribing practice. A 70% (n=287) response rate was obtained. The majority of respondents reported prescribing AZA (99%, n=283). Prescribing and monitoring patterns differed between individual respondents and between the three disciplines. TPMT enzyme-level testing was reportedly used by 67% (n=189) of respondents, but this differed by discipline (dermatologists 94%, gastroenterologists 60%, rheumatologists 47%). In 91% of cases enzyme-level testing was carried out prior to prescribing AZA. Genotype testing is not typically available to NHS clinicians but 15 clinicians (six dermatologists, six gastroenterologists, three rheumatologists) reported using it. Most consultants (82%) reported using guidelines to inform their AZA prescribing and monitoring (dermatologists 81%, gastroenterologists 75%, rheumatologists 94%). Two-thirds of the consultants surveyed in England are using TPMT enzyme-level testing, prior to AZA treatment. Uptake differs between specialities. High uptake of TPMT enzyme-level testing by dermatologists, compared with gastroenterologists and rheumatologists, may reflect national guidelines advocating its use prior to AZA. Uptake of enzyme-level testing may alter in other specialties as other guidelines are developed.

Assessment of thiopurine methyltransferase enzyme activity is superior to genotype in predicting myelosuppression following azathioprine therapy in patients with inflammatory bowel disease

Myelosuppression occurs in 2-7% of inflammatory bowel disease (IBD) patients treated with azathioprine, and can be associated with reduced activity of thiopurine methyltransferase (TPMT) in some patients. It has been proposed that pretreatment assessment of TPMT status reduces the incidence of toxicity and is cost-effective. To determine if screening for TPMT status predicts side-effects to azathioprine in patients with IBD and to ascertain whether screening by TPMT enzyme activity or genotype is superior. Sequential IBD patients were identified and azathioprine tolerance recorded. Blood was collected for measurement of TPMT activity and TPMT*3C, TPMT*3A and TPMT*2 genotypes. Of 130 patients, 25% stopped azathioprine because of toxicity. Four patients experienced severe myelosuppression (WCC < 2). Eleven of 17 patients with reduced TPMT activity were heterozygotes, including one patient with marked TPMT deficiency who experienced severe myelosuppression. There was no association between intermediate TPMT deficiency and any side-effect. Moderate reduction of TPMT activity in heterozygotes was not associated with toxicity, but very low TPMT activity caused severe myelosuppression in one patient. This would have been predicted by measuring TPMT activity but not by genotyping. Measurement of TPMT activity may therefore be superior to genotype in predicting severe myelosuppression.

Thiopurines in Inflammatory Bowel Disease - The Role of Pharmacogenetics and Therapeutic Drug Monitoring

Pharmacogenetics represents the study of variability in drug response due to genetic variations. Inflammatory bowel disease (IBD, i.e. primarily Crohn's disease and ulcerative colitis) is characterized by a chronic or relapsing inflammation of the digestive tract. The thiopurines 6-mercaptopurine (6-MP) and azathioprine (AZA), an imidazol derivative and pro-drug of 6-MP, are widely used in IBD, particularly in Crohn's disease. The metabolism of thiopurines is complex and individually variable. Thiopurine methyltransferase (TPMT) is a key enzyme in this metabolism and exhibits a genetic variability due to a number of variant alleles coding for a defective enzyme. The formation of biologically active thioguanine nucleotides (TGN) and methylated metabolites may vary considerably due to the TPMT activity. Patients with decreased TPMT activity are at increased risk of developing severe side effects if treated with conventional thiopurine doses, due to the accumulation of toxic metabolites. Determination of the TPMT phenotype or genotype is often used to identify individuals with increased risk for adverse events. Twenty-one variant TPMT alleles have been described, of which three are more common than the others. An association between inosine triphosphate pyrophosphatase polymorphisms and adverse events during thiopurine treatment has also been proposed. In this review, the clinical value of TPMT status determination and pharmacological monitoring of thiopurine metabolites are discussed as well as the increased interest in the use of 6-thioguanine, a thiopurine with a less complex metabolism, as an alternative for patients who do not tolerate AZA or 6-MP. It can be concluded that TPMT determination before start of thiopurine therapy is of value to identify individuals with increased risk for adverse reactions due to genetic enzyme deficiency. However, large prospective studies are still needed to evaluate the true benefit of monitoring thiopurine metabolites during thiopurine treatment. © 2006 Bentham Science Publishers Ltd.

Risk of Second Malignant Neoplasm Is Related to TPMT Activity and Duration of MTX/6MP Maintenance Therapy of ALL.

Risk of SMN has been linked to irradiation, alkylating agents, and topo-II inhibitors with less attention to factors that may interfere with repair of treatment induced DNA damage. Due to mutations, 10% of children with ALL have low thiopurine methyl transferase (TPMT) activity with low 6-mercaptopurine (6MP) and -metabolite methylation and increased intracellular levels of cytotoxic 6-thioguanine nucleotides, which could affect DNA-repair. 1703 Nordic children 1.0–14.9y were diagnosed with ALL 1/92–10/01. 1617 were treated by the NOPHO 92 protocol, obtained CR1 after induction therapy, and were then treated by risk group based on WBC and age (standard risk (SR): 2.0–9.9y and WBC <10; intermediate risk (IR): 1.0–1.9y or ≥10.0y and/or WBC 10–49.9) and presence of high (HR) or very high risk (VHR) features: WBC ≥50.0, T-ALL, CNS or testicular disease, t(9;22), t(4;11), M3 d15 or M2/M3 d29. VHR included pts with certain HR features and age >5y (due to RTx). During CR1, 121 non-SCT pts received CNS RTx and 53 received a SCT. After multidrug induction/consolidation therapy (Gustafsson Leukemia 2000) oral 6MP (75mg/m2)/MTX (20mg/m2/w) maintenance therapy was given to SR, IR and HR pts from weeks 13, 32, and 63. VHR pts received LSA2L2 maintenance from week 48. TPMT status was available for 603 patients. 528 were classified as wild type (WT), 72 as heterozygous, 3 as deficient. The pEFS10y of the 1617 pts was 0.75. Thirty-four pts died in CR1. 330 relapsed. Eighteen patients developed SMN (3 after SCT in CR1) 10–98 months (median 40) from Dx, and 11 of these died. Eleven of the 15 SMNs in the 1564 non-SCT pts occurred in the SR-group. In multivariate analysis, the risk of SMN was significantly related to the duration of maintenance therapy (highest in SR-ALL, p=0.01) and to low TPMT-activity (p=0.003), but not to sex, age or WBC at Dx, immunophenotype (B- vs T-lineage), or CNS RTx. Four of 75 TPMT low activity pts developed SMN vs 3 of 528 TPMT wild type pts (5% vs 1% risk, p<0.001). In contrast, the 10y risk of relapse was significantly higher for the TPMT wild type pts (18% vs 8% at 10y, p<0.05). The excessive risk of SMN in pts who received the least chemo-/radio-intensive induction and consolidation therapy (SR) indicates that their longer duration of MTX/6MP maintenance therapy may have increased their risk for SMN through DNA-damage or interference with DNA-repair, not least for the pts that carry TPMT-mutations.Treatment and occurrence of SMNSRIRHRVHRNo of patients554586292167Anthracycline (mg/m.sq.)120240240360Cyclophosphamide (mg/m.sq.)0300030006000Prophylactic CNS RTxNoNoNoYesTotal duration of Tx2.5y2.0y2.0y2.0yDuration of MTX/6MP (weeks)11772410AML/MDS/other6/5/01/1/12*/0/1*0/0/1*TPMT (WT/low)248/27221/3653/126/0SMN TPMT (WT/low)3/20/10/10/NA*each represent a patient with an SMN after SCT in CR1; NA=not applicable