Pharmacogenetics of thiopurine S-methyltransferase and thiopurine therapy

Most medications exhibit wide interpatient variability in their efficacy and toxicity. For many medications, these interindividual differences result in part from polymorphisms in genes encoding drug-metabolizing enzymes, drug transporters, and/or drug targets (eg, receptors, enzymes). Pharmacogenomics is a burgeoning field aimed at elucidating the genetic basis of differences in drug efficacy and toxicity, using genome-wide approaches to identify the network of genes that govern an individual's response to drug therapy. For some genetic polymorphisms, such as thiopurine S-methyltransferase (TPMT), monogenic traits have a marked effect on the pharmacokinetics of medications, such that individuals who inherit an enzyme deficiency must be treated with markedly different doses of the affected medications (eg, 5-10% of the standard thiopurine dose). This review uses the TPMT polymorphism and thiopurine therapy (eg, azathioprine, mercaptopurine) to illustrate the potential of pharmacogenomics to elucidate genetic determinants of drug response, and optimize the selection of drug therapy for individual patients.     

Determinants of mercaptopurine toxicity in paediatric acute lymphoblastic leukemia maintenance therapy

AIMS

6-mercaptopurine (6-MP) is used in the treatment of childhood acute lymphoblastic leukaemia (ALL). Its red blood cell (RBC) metabolite concentrations (6-thioguanine [6-TGN] and 6-methylmercaptopurine nucleotides [6-MMPN]) are related to drug response. We investigated the impact of non-genetic covariates and pharmacogenetic polymorphisms affecting thiopurine methyltransferase (TPMT) and inosine triphosphate pyrophosphatase (ITPA) on 6-MP metabolism and response.

METHODS

Sixty-six children with ALL treated according to EORTC 58951 protocol were included in this study. Six patients had a heterozygous genotype for the most common TPMT polymorphisms, nine for ITPA 94 C > A and 17 for ITPA IVS2+21 A > C. 6-MP metabolites concentrations were analyzed by mixed model analysis.

RESULTS

During maintenance, steady-state RBC 6-TGN concentrations were lower in patients aged 6 years or younger (493 pmol/8 × 10⁸RBC) than in older children (600 pmol/8 × 10⁸RBC). 6-MMPN concentrations were low in patients with TPMT variant/wild-type ITPA (1862 pmol/8 × 10⁸RBC), intermediate in wild-type patients and high (16468 pmol/8 × 10⁸RBC) in patients wild-type TPMT/variant ITPA. A 6-MMPN threshold of 5000 pmol/8 × 10⁸RBC was associated with an increased risk of hepatotoxicity.

CONCLUSION

In this study, age and both TPMT and ITPA genotypes influenced 6-MP metabolism. High 6-MMPN was associated with hepatotoxicity. These pharmacological tools should be used to monitor ALL treatment in children.     

Novel therapy for childhood acute lymphoblastic leukemia

Introduction: During recent decades, the prognosis of childhood acute lymphoblastic leukemia (ALL) has improved dramatically, nowadays, reaching a cure rate of almost 90%. These results are due to a better management and combination of old therapies, refined risk-group stratification and emergence of minimal residual disease (MRD) combined with treatment’s intensification for high-risk subgroups. However, the subgroup of patients with refractory/relapsed ALL still presents a dismal prognosis indicating necessity for innovative therapeutic approaches.

Areas covered: We performed an exhaustive review of current first-line therapies for childhood ALL in the worldwide main consortia, summarized the major advances for front-line and relapse treatment and highlighted recent and promising innovative therapies with an overview of the most promising ongoing clinical trials.

Expert opinion: Two major avenues marked the beginning of 21^st century. First, is the introduction of tyrosine-kinase inhibitor coupled to chemotherapy for treatment of Philadelphia positive ALL opening new treatment possibilities for the recently identified subgroup of Ph-like ALL. Second, is the breakthrough of immunotherapy, notably CAR T-cell and specific antibody-based therapy, with remarkable success observed in initial studies.

This review gives an insight on current knowledge in these innovative therapeutic directions, summarizes currently ongoing clinical trials and addresses challenges these approaches are faced with.     

Thiopurine S-methyltransferase phenotype-genotype correlation in children with acute lymphoblastic leukemia

Thiopurine S-methyltransferase (TPMT) is an enzyme that catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine, 6-thioguanine, and azathioprine. TPMT activity exhibits an interindividual variability mainly as a result of genetic polymorphism. Patients with intermediate or deficient TPMT activity are at risk for toxicity after receiving standard doses of thiopurine drugs. The aim of this study was to determine the TPMT genotype and phenotype (activity) and investigate the correlation between TPMT genotype and enzyme activity in 43 Polish children receiving 6-MP during maintenance therapy in course of acute lymphoblastic leukemia (ALL), in 16 children with ALL at diagnosis and 39 healthy controls. TPMT activity was measured in RBC by HPLC method. Patients were genotyped for TPMT *2, *3A and *3C variant allelesusing PCR-RFLP and allele-specific PCR methods. In the group of children with ALL during maintenance therapy, median TPMT activity (29.3 nmol 6-mMP g(-1) Hb h(-1)) was significantly higher compared to the group of children with ALL at diagnosis (20.6 nmol 6-mMP g(-1) Hb h(-1), p = 0.0028), as well as to the control group (22.8 nmol 6-mMP g(-1) Hb h(-1), p = 0.0002). Percentages of individuals heterozygous for TPMT variant allele in respective groups were: 9.3, 6.2 and 15.5% (p > 0.05). In all the study groups heterozygous patients manifested a significantly lower TPMT activity as compared to the wild type homozygotes (16.7 +/- 2.1 vs. 31.2 +/- 6.8 nmol 6-mMP g(-1) Hb h(-1), p = 0.002, in children during maintenance therapy, 11.9 +/- 2.7 vs. 24.6 +/- 9.5, p = 0.0003, in the combined group of children with ALL at diagnosis and controls). The results present that commencement of the thiopurine therapy caused an increase in the TPMT activity in RBCs by approximately 20%. All patients heterozygous for the TPMT variant allele revealed decreased TPMT activity compared to TPMT wild-type patients. Since decreased TPMT activity is associated with higher risk for toxicity after receiving standard doses of thiopurine drugs, pretreatment determination of TPMT status, with phenotypic or genetic assay, should be performed routinely, also in Poland.     

Pharmacogenetic determinants of mercaptopurine disposition in children with acute lymphoblastic leukemia

BACKGROUND: The backbone of drug therapy used in acute lymphoblastic leukemia (ALL) in children includes 6-mercaptopurine (6-MP). Intracellular metabolism of this prodrug is a key component of the therapeutic response. Many metabolizing enzymes are involved in 6-MP disposition and active 6-MP metabolites are represented by 6-thioguanine nucleotides (6-TGN) and methylated metabolites primarily methylated by the thiopurine S-methyltransferase enzyme (TPMT). The genetic polymorphism affecting TPMT activity displays an important inter-subject variability in metabolites pharmacokinetics and influences the balance between 6-MP efficacy and toxicity: patients with high 6-TGN levels are at risk of myelosuppression while patients with high levels of methylated derivates are at hepatotoxic risk. However, the genetic TPMT polymorphism does not explain all 6-MP adverse events and some severe toxicities leading to life-threatening conditions remain unexplained. Additional single nucleotide polymorphisms (SNPs) in genes encoding enzymes involved in 6-MP metabolism and 6-MP transporters may also be responsible for this inter-individual 6-MP response variability. AIM: This review presents the pharmacogenetic aspects of 6-MP metabolism in great detail. We have focused on published data on ALL treatment supporting the great potential of 6-MP pharmacogenetics to improve efficacy, tolerance, and event-free survival rates in children with ALL.     

Frequency distribution of thiopurine S-methyltransferase alleles in a polish population

Thiopurine S-methyltransferase (TPMT) is an enzyme that catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine, 6-thioguanine, and azathioprine. TPMT activity exhibits an interindividual variability mainly a result of genetic polymorphism. Patients with intermediate or deficient TPMT activity are at risk for toxicity after receiving standard doses of thiopurine drugs. It has previously been reported that 3 variant alleles:TPMT*2, *3A, and *3C are responsible for over 95% cases of lower enzyme activity. The purpose of this study was to determine the frequency of TPMT variant alleles in a Polish population. DNA samples were obtained from 358 unrelated healthy Polish subjects of white origin, and TPMT genetic polymorphism was determined using PCR-RFLP and allele-specific PCR methods. The results showed that allelic frequencies were 0.4% for TPMT*2, 2.7% for TPMT*3A, and 0.1% for TPMT*3C, respectively. A TPMT*3B allele was not found in the studied population. The general pattern of TPMT allele disposition in the Polish population is similar to those determined for other white populations, but the frequency of total variant alleles is lower than in other European populations studied to date.     

Genetic polymorphism of thiopurine S-methyltransferase: molecular mechanisms and clinical importance

The activity of thiopurine S-methyltransferase (TPMT) is inherited as an autosomal co-dominant trait. In most large world populations studied to date, approximately 10% of the population have intermediate activity due to heterozygosity at the TPMT locus, and about 0.33% is TPMT deficient. TPMT is now one of the most well characterized genetic polymorphisms of drug metabolism, with the genetic basis having been well defined in most populations, providing molecular strategies for studying this genetic polymorphism in human and experimental models. Three mutant alleles, TPMT(*)2, TPMT(*)3A and TPMT(*)3C, account for the great majority of mutant alleles in all human populations studied to date. Significant ethnic differences occur in the frequencies of these mutant alleles. Progress in DNA analysis has made it practical to use genotyping techniques for the molecular diagnosis of TPMT deficiency and heterozygosity, thereby avoiding adverse effects that are more prevalent in TPMT-deficient and heterozygous patients prescribed thiopurine medications.     

Genetic polymorphism of inosine-triphosphate-pyrophosphatase influences mercaptopurine metabolism and toxicity during treatment of acute lymphoblastic leukemia individualized for thiopurine-S-methyl-transferase status

Importance of the field: Although genetic polymorphisms in the gene encoding human thiopurine methyltransferase (TPMT) are known to have a marked effect on mercaptopurine metabolism and toxicity, there are many patients with wild-type TPMT who develop toxicity. Furthermore, when mercaptopurine dosages are adjusted in patients who are heterozygous at the TPMT locus, there are still some patients who develop toxicity for reasons that are not fully understood. Therefore, we recently studied the effects of a common polymorphism in another gene encoding an enzyme involved in mercaptopurine metabolism (SNP rs1127354 in inosine-triphospate-pyrophosphatase, ITPA), showing that genetic polymorphism of ITPA is a significant determinant of mercaptopurine metabolism and of febrile neutropenia following combination chemotherapy of acute lymphoblastic leukemia (ALL) in which mercaptopurine doses are individualized based on TPMT genotype.

Area covered in this review: In this review, we summarize the knowledge available about the effect and clinical relevance of TPMT and ITPA on mercaptopurine pharmacogenomics, with a particular focus on the use of this medication in pediatric patients with ALL.

What the reader will gain: Reader will gain insights into: i) the effects of pharmacogenomic traits on mercaptopurine toxicity and efficacy for the treatment of ALL and ii) individualization strategies that can be used to mitigate toxicity without compromising efficacy in pediatric patients with ALL.

Take home message: Mercaptopurine dose can be adjusted on the basis of TPMT genotype to mitigate toxicity in pediatric patients with ALL. As treatment is individualized in this way for the most relevant genetic determinant of drug response (i.e., for mercaptopurine, TPMT), the importance of other genetic polymorphisms emerges (e.g., ITPA).     

Thiopurine S-methyltransferase genetic polymorphism in the Thai population

AIMS: To determine the incidence of the thiopurine S-methyltransferase (TPMT) genetic polymorphism in the Thai population. METHODS: Genomic DNAs were isolated from peripheral blood leucocytes of 200 healthy Thais. The frequencies of five allelic variants of the TPMT gene, TPMT*2, *3A, *3B, *3C and *6 were determined using allele specific polymerase chain reaction (PCR) or PCR-Restriction fragment length polymorphism technique. RESULTS: Of the 200 Thai subjects participating in this study, 181 subjects (90.5%) were homozygous for TPMT*1, 18 subjects (9.0%) were heterozygous for TPMT*1/*3C. Only one subject (0.5%) was homozygous for TPMT*3C. The frequency of TPMT*3C mutant allele was 0.050. CONCLUSIONS: Although the TPMT*3C is the most prevalent mutant allele in Asian populations, the frequency of this defective allele is significantly higher in Thais than has been reported in other Asian populations.     

Azathioprine therapy and adverse drug reactions in patients with inflammatory bowel disease: impact of thiopurine S-methyltransferase polymorphism

The efficacy of the immunosuppressants azathioprine and 6-mercaptopurine has been well established in the therapy of inflammatory bowel diseases (IBD). However, its use has been complicated by a high incidence of serious adverse drug reactions such as hematotoxicity, hepatotoxicity, pancreatitis and gastrointestinal disturbances. Whereas azathioprine-related pancytopenia has been clearly linked to thiopurine S-methyltransferase (TPMT) polymorphism limited data are available to explain gastrointestinal side effects. In a retrospective analysis of 93 adults with IBD and azathioprine therapy both phenotyping and genotyping was used to explore systematically the relationship between TPMT and azathioprine-related adverse reactions. At time of inclusion, 69 patients were still receiving azathioprine therapy and had never experienced side effects. Azathioprine had been withdrawn in 10 patients for non-medical reasons or lack of response and 14 patients (15%) had stopped medication or were on reduced dose due to severe azathioprine-related side effects. Nine of these 14 patients had developed gastrointestinal side effects (hepatotoxicity, n = 3; pancreatitis, n = 3; others, n = 3), but their normal red blood cell TPMT activities were in accordance to TPMT wild-type. TPMT deficiency in one patient had led to pancytopenia whereas only two of the remaining four patients with hematotoxicity displayed an intermediate phenotype of TPMT. This study demonstrates that azathioprine-related gastrointestinal side effects are independent of the TPMT polymorphism. Nevertheless pharmacogenetic testing for TPMT prior to commencing thiopurine therapy should become routine practice in order to avoid severe hematotoxicity in TPMT deficient patients and lowering the incidence of hematological side effects in individuals heterozygous for TPMT.     

Antileukemic drug effects in childhood acute lymphoblastic leukemia

Cancer is the second most common cause of death in children between 1 and 15 years of age in industrialized countries. Although acute lymphoblastic leukemia (ALL) is still among the leading causes of death in childhood cancer, remarkable progress in treatment results has been made with this disease. With current antileukemic therapy, more than 80% of children with ALL can be cured. Antileukemic medications, however, are generally toxic and can cause significant adverse drug reactions. Moreover, some patients have leukemia cell clones that are resistant to conventional antileukemic treatment. Overall, a fifth of children with ALL will not be cured despite intensified therapy, including allogeneic stem cell transplantation. Pharmacogenomics aims to elucidate functionally relevant genomic determinants for drug disposition and response in order to establish valid pharmacogenomic models that can help to select medications and their dose based on a patient’s genetic make-up. This review focuses on investigations that provide important basic insights into ALL pharmacogenomics and/or hold great promise in providing clinically valuable pharmacogenomic models that can help to improve childhood ALL treatment by facilitating appropriate dose individualization, optimal treatment selection and drug discovery.     

硫嘌呤甲基转移酶遗传多态性检测在6-MP个体化治疗中的意义

目的：研究硫嘌呤甲基转移酶活性和基因型检测对6-巯基嘌呤（6-MP）的个体化治疗的意义。方法-94例口服6-MP维持治疗的白血病患者，19例口服6-Mp后出现血液系统危象的为观察组，同时服用6-MP没有出现血液系统损害的75患者为对照组，采用高效液相色谱法测定硫嘌呤甲基转移酶（TPMT）活性，等位基因特异性PCR和限制性片段长度多态性（RFLP）的方法检测TPMT＊2、TPMT＊3A、TPMT＊3B和TPMT＊3C的等位基因频率。通过临床白细胞记数和骨髓象检查及临床表现判定巯嘌呤的疗效和不良反应。结果：观察组TPMT活性是（6．1±2．1）U·mL^-1 pRBCs显著低于对照组（15．3±2．3）U·mL^-1 pRBCs，而观察组的基因突变率10．7％高于对照组1．2％。结论：TPMT活性低下和／或TPMT基因突变型个体，在服用标准剂量的巯嘌呤后，发生不良反应的危险较高，应及时发现此类患者并积极调整剂量实现安全有效的治疗。     

培门冬酶治疗儿童急性淋巴细胞白血病临床观察

目的探讨培门冬酶在儿童急性淋巴细胞白血病(ALL)中应用的疗效及安全性。方法 62例初发ALL患儿,均按ALL-XH-99方案进行治疗。对照组34例采用VDLP方案(长春新碱+柔红霉素+左旋门冬酰胺酶+泼尼松)进行诱导缓解治疗和巩固强化治疗;试验组28例将前方案中左旋门冬酰胺酶改为培门冬酶,余不变。比较2组临床疗效和安全性。结果试验组的完全缓解率为93%,对照组为91%,无显著差异(P>0.05)。试验组2年无进展生存率为75%,总生存率为89%;对照组2年无进展生存率为71%,总生存率为85%,差异均无显著意义(P>0.05)。试验组肝功能损害发生率(17%)低于对照组(56%),差异有显著意义(P<0.05),且试验组未出现严重过敏反应发生。结论培门冬酶在治疗儿童ALL中具有与左旋门冬酰胺酶相当的疗效和较低的不良反应。     

中国新疆维吾尔族硫嘌呤甲基转移酶基因突变研究

目的 研究硫嘌呤甲基转移酶(thiopurine S-methyltransferase，TPMT)在新疆维吾尔族中的基因突变频率。方法 用等位基因特异性的PCR方法和限制性片断长度多态性的方法检测4种常见的导致酶活性降低的突变类型：TPMT*2、TPMT*3A、TPMT*3B和TPMT*3C。结果 在160名维吾尔族中发现了1例TPMT*3A(A719G／G460A)杂合子、5例TPMT*3C(A719G)杂合子，TPMT*3A和TPMT*3C的等位基因频率分别是0．3％和1．6％。结论 维吾尔族总的TPMT突变等位基因频率(1．9％)同中国其他民族相近；TPMT*3C是维吾尔族最主要的突变类型。     

Relationships between thiopurine S-methyltransferase polymorphism and azathioprine-related adverse drug reactions in Chinese renal transplant recipients

Objective  To systematically investigate the relationships between thiopurine S-methyltransferase (TPMT) polymorphisms and azathioprine-related adverse drug reactions in patients with kidney transplantation. Methods  Erythrocyte TPMT activity of 150 patients with kidney transplantation and AZA therapy was determined by HPLC. The frequency of four common TPMT mutant alleles, TPMT*2, *3A, *3B, and *3C was determined by allele-specific PCR and PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. Results  Thirty cases (20%) had stopped azathioprine medication or were on reduced dose due to azathioprine-related side effects. The TPMT activity range of cases who never experienced side effects was 16.63-68.25 U, the mean of the controls was 38.43 ± 11.59 U. The mean value of 12 cases with hematotoxicity was 23.50 ± 10.33 U, much lower than the control mean (P < 0.05). No significant difference between the mean value of 18 cases with hepatotoxicity and the control mean (P > 0.05) was seen. No case with TPMT deficiency was found in all patients studied, and TPMT*2, *3A, and *3B were not detected in any of them. TPMT*3C heterozygous alleles were found in 4.7% (seven cases) of these patients, all seven cases had intermediate TPMT activity, and the mean was 16.75 ± 2.09 U, much lower than other TPMT wild-type patients (P < 0.05). In the seven TPMT*3C patients, four cases experienced side effects (hematotoxicity, n = 2; hepatotoxicity, n = 2). Conclusions  This study demonstrates that TPMT activity is reduced in patients with TPMT*3C mutation. AZA-induced hematotoxicity is related to the reduced TPMT activity.     

Methotrexate binds to recombinant thiopurine S-methyltransferase and inhibits enzyme activity after high-dose infusions in childhood leukaemia

Purpose

Important drugs in the treatment of childhood acute lymphoblastic leukaemia (ALL) are 6-mercaptopurine (6-MP) and methotrexate (MTX). Thiopurine methyltransferase (TPMT) is a polymorphic enzyme causing variability in 6-MP response and toxicity. The aim of this study was to investigate the fluctuation in TPMT enzyme activity over time and the effect of high-dose MTX infusions on TPMT enzyme activity and 6-MP metabolites in paediatric ALL patients.

Methods

Fifty-three children with ALL treated according to the NOPHO-ALL 2000 protocol were included in the study. TPMT enzyme activity was measured at six different times starting from diagnosis until after the end of maintenance treatment. TPMT and 6-MP metabolites were measured before the initiation of high-dose MTX (HD-MTX) infusions and at 66 h post-infusion. The interaction between MTX and TPMT was investigated in vitro using recombinant TPMT protein and a leukaemic cell line.

Results

Forty percent of TPMT wild-type individuals had deceptively low TPMT enzyme activity according to genotype at the time of diagnosis. TPMT activity had decreased significantly 66 h after the start of HD-MTX infusions (?9.2 %; p?=?0.013). MTX bound to recombinant TPMT protein severely inhibiting TPMT enzyme activity (remaining activity 16 %).

Conclusions

Our results show that TPMT genotyping should be performed in children with ALL, since 40 % of the children in our study who carried the wild-type TPMT gene were at risk of initial underdosing of 6-MP in cases where only TPMT enzyme activity was determined. MTX inhibits the TPMT enzyme activity after HD-MTX infusions due to protein binding.