TPMT activity

Reduced Tolerance to 6-MP in Patients with Genetic Impairment of TPMT Activity... 

TMPT activity in human erythrocytes is transmitted as an autosomic codominant trait [15] and is trimodally distributed, with 89-94% of the individuals having high, 6-11% intermediate, and 0.3% low activity [7, 15-17] (Figure 14.2). The measurement of TPMT activity in erythrocytes closely reflects the ability of bone marrow to inactivate 6-MP. TPMT activity is inversely related to erythrocyte 6-TGN levels [7, 13, 18, 19], and children with low TPMT activity and very high 6-TGN levels experienced profound myelotoxicity [20, 21]. Moreover, TPMT phenotype in erythrocyte reflects that in leukemic blasts [22]. Patients with intermediate TPMT activity had a 5-fold greater cumulative incidence of dose reductions than subjects with high activity [13], and TPMT activity has been inversely related to the time of treatment withdrawal due to cytopenia [21]. 

Interethnic variability in RBC TPMT activity has been reported in several populations. RBC TPMT was 29% higher in Saami subjects in Northern Norway compared to white subjects from the same geographic region [41]. African American subjects have 17-33% lower RBC TPMT activity than American white subjects [34, 42]. The TPMT activity in African and white Americans was substantially lower than that reported in 119 Chinese subjects [43]. 

The association between low TPMT activity and excessive hematological toxicity has been recognized [31, 35, 37]. Molecular analysis of the TPMT genotype is able to identify patients at risk for acute toxicity from thiopurines. A recent study involving 180 children identified that the TPMT genotype plays an important role in a patients tolerance to 6-MP therapy [51]. Two of the patients, who were TPMT-de-... 

Tai HI, Krynetski EY, Schuetz EG, Ya-shinevski Y, Evans WE. Enhanced proteolysis of thiopurine S-methyltransferase (TPMT) encoded by mutant alleles in human (TPMT 3A, TPMT 2) mechanism for the genetic polymorphism of TPMT activity. Proc Natl Acad Sci USA 1997 94 6444-6449. 

The activity of TPMT is influenced by genetic polymorphisms that can alter the rate of 6-MP metabolism by TPMT. The enzyme activity of TPMT varies among patients 86.6% of the Caucasian population has high TPMT activity, 11.1% has intermediate activity, and 0.3% are deficient in TPMT (42-44). The TPMT found in patients with normal TPMT activity is classified as the wild-type TPMT 1. 

Eight TPMT alleles have been identified. Three of these alleles, TPMT 2 (45), 3A, and 3C (G460A), account for 80-95% of intermediate or low enzyme activity cases. High concentrations of variant TPMT (TPMT 3A, 3B, and 3C) are found in patients with decreased TPMT activity Patients with TMPT-3A have a complete loss of TPMT catalytic activity, patients with TPMT 3B have a 9-fold reduction, and those with TPMT 3C have a 1.4-fold reduction (43,46). The frequency of loss of TPMT activity (TPMT 3A) appears to vary with ethnicity but not with gender or age. In addition, haplotyping methods have been developed to discriminate the genotypes TPMT 1/ 3A (intermediate metabolizer) and TPMT 3B/ 3C (poor metabolizer) (47). 

Tests for the TPMT genotype and phenotype are commercially available. Attention should be paid for those patients who test negative for TPMT status. Patients with poor or intermediate TPMT activity may tolerate only 1/10 to 1/2 of the average 6-MP dose. A pharmacoeconomic model has been developed to analyze the potential cost of screening to prevent azathioprine toxicity. In this model, it was assumed that TPMT deficiency is present in 0.3% of the population, that intermediate activity is present in 11%, and that both groups have an increased risk of developing myelosuppression. Under these circumstances, the model predicted that the costs per Caucasian patient for the first 6 mo of therapy with screening are lower... 

Factors that affect drug metabolism will influence the development of serious adverse effects. TPMT, one of the three enzymes that metabolize 6-MP, is encoded by a gene on chromosome 6 that contains functional polymorphisms. Multiple variants have been described ( 2, 3A, 3B, 3C, 3D, 4, 5, 6, 7, 10 31,32) that result in lower TPMT activity, leading to preferential metabolism of 6-MP by the HGPRT enzyme. This results in an increase in the amount of cytotoxic thioguanine nucleotides and greater myelotoxicity. There appears to be an allele dose-dependent... 

Other stndies have examined the association between the activity of TPMT and other enzymes in the pnrine pathway and AZA toxicity. In one stndy, TPMT, HPRT, 5 -nncleotidase, and pnrine nncleoside phosphorylase activity in the RBCs of 33 RA patients on AZA (dose of approximately 2mg/kg/d) and 66 controls was meas-nred. Compared to patients with normal TPMT activity, 14 RA patients with low (p = 0.004) and 7 patients with intermediate TPMT activity (RR 3.1) developed AZA toxicity(4d). None of the patients were genotyped. Another study measured TPMT activity in 3 RA patients who had experienced AZA-induced hematologic toxicity and 16 RA patients withont AZA toxicity. In this study, 2 patients with AZA-indnced hematologic toxicity were TPMT deficient, one partial and the other complete (50). Patients were not genotyped in either of these studies. 

Thus, both TPMT genotyping and measnrement of TPMT activity in RBCs may be usefnl in predicting and preventing AZA toxicity. Clearly, large, prospective studies are needed to validate the observations from the smaller stndies described (see Table 14.2). Of note, TPMT genotyping is available to clinicians currently to... 

Thiopurine methyltransferase methylates 6-mercaptopurine, a commonly used treatment for childhood acute lymphocytic leukemia, reducing its conversion to the active form of the drug. Approximately 10% of patients have intermediate enzyme activity, and 0.3% are deficient for TPMT activity. Intermediate activity patients have a greater incidence of thiopurine toxicity, whereas TPMT-deficient patients have severe or fatal hematological toxicity from 6-mercaptopurine therapy. In one study, patients deficient for TPMT tolerated only 7% of a 2.5-yr mercaptopurine treatment regimen. Patients with intermediate TPMT activity tolerated 65% of total weeks of therapy and patients with normal TPMT activity tolerated 84% of total weeks of therapy (3). 

There are several known polymorphisms in TPMT (4). Alleles TPMT 2, TPMT 3A, and TPMT 3C account for up to 95% of reduced TPMT activity. Patients heterozygous for these alleles have intermediate TPMT levels (5), and patients homozygous for the variant TPMT alleles are at high risk for severe, sometimes life-threatening, toxicity requiring significant reductions in mercaptopurine doses (5). 

Thiouric acid is formed by XO and is excreted renally. Whereas the cytosolic enzyme TPMT is expressed ubiquitously in humans [e.g., in the intestine, liver, red blood cells (RBC) and white blood cells], XO is not expressed in hematopoietic tissue (94). Therefore, TPMT-dependent methylation is critical in white blood cells, leading to an enhanced cytotoxic effect in patients with low TPMT activity. 

The half-lives of 6-MP and 6-TG are 21 min and 90 min, respectively, and their plasma areas under the curve are highly variable and not strongly predictive of therapeutic response or side effects (107,108,109,110, 111). It has been suggested that the major cytotoxic effect of thiopurine drugs is dependent on the intracellular accumulation of 6-TGN and 6-MMPR where on average a steady-state concentration is reached within 1-4 weeks after initiation of treatment (112). In individuals deficient of TPMT activity, 6-TGN accumulate more rapidly in RBC (113) with an 8- to 15-fold increase of 6-TGN concentration compared with wild-type patients, subsequently leading to an exaggerated cytotoxic effect (114). 

Altogether, no consensus recommendations on therapeutic ranges for 6-MP and 6-TG metabolite monitoring in treatment of childhood ALL are currently available. Moreover, 6-TGN levels may be biased in individuals who have received repeated RBC transfusions due to high variability in TPMT activity of transfused donor RBC. 

This distribution of TPMT activity in Caucasian populations has been subsequently confirmed by other studies (134,135). However, TPMT activity was also shown to differ between different ethnic populations (136). For example, African Americans were demonstrated to have lower enzyme activity in comparison with Americans of Caucasian descent. Other factors that have been variably associated with TPMT activity include age (higher in children, especially neonates), gender (slightly higher in men), smoking (higher in smokers), and thiopurine treatment (137,138,139,140). 

TPMT activity can be analyzed by different phenotyping assays including the radiochemical method developed by Weinshilboum and more recent non-radioactive HPLC methods using either 6-MP or 6-TG as substrates ( 145, 146, 147, 148, 149,150,151,152,153). The radiochemical and HPLC assays have been shown to lead to comparable results with 6-MP as a substrate (146,147,148,149,150). However, when using 6-TG, TPMT activity was measured at 30% higher levels (152). 

TlPMr genotype can be used as a surrogate marker of TPMT activity. In several independent studies, rPMT genotype showed excellent concordance with TPMT phenotype. For example, Yates and colleagues analyzed the TPMT phenotype in 282 unrelated Caucasian Americans (170). Subsequently, all individuals phenotypically deficient or with intermediate TPMT activity and a randomly selected sample of individuals with high TPMT activity were rPMT genotyped TPMT 2 and 3A, 3B, 3C). 

In this study, 21 patients had a heterozygous TPMT phenotype. With a frequency of 85%, TPMT 3A was the most prevalent variant allele, followed by TPMT 2 and TPMT 3C with about 5% each. All 6 patients who phenotypically displayed TPMT deficiency had two mutant alleles 20 of the 21 patients with intermediate TPMT activity had one variant allele and all of the selected 21 patients with high activity did not carry one of the tested JPMT variant alleles. Thus, the major inactivating TPMT variants can be detected reliably by a PCR-based method and demonstrated an excellent concordance with TPMT phenotype. 

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