TPMT phenotyping

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]. 

Deficiency of thiopurine S-methyl transferase (TPMT) is another phenotype that exhibits inter-ethnic differences in frequency. TPMT is an enzyme that catalyzes methylation of therapeutic agents used in the treatment of acute lymphoblastic leukemia, rheumatoid arthritis, and autoimmune/inflammatory diseases, as well as in organ transplantation. Patients who have TPMT deficiency experience less efficient methylation and are at greater risk of fatal toxicity when treated with standard doses of fhiopurines. TPMT phenotype is defined by erythrocyte 6-mercapto-purine methylation. African American populations exhibit a 20% lower erythrocyte TPMT than Caucasian Americans, and persons of Chinese descent tend to exhibit greater activity than either of these other American subpopulations. 

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. 

The most comprehensive analysis of TPMT phenotype versus genotype published to date was conducted by Schaeffeler et al. (140). In their study, RBC TPMT activity and genotype TPMT 2 and 3 alleles) were analyzed in 1214 healthy Caucasian blood donors. Discordant cases between phenotype and genotype were systematically sequenced. The frequencies of the mutant alleles were 4.4% for TPMT 3A, 0.4% for TPMT 3C, and 0.2% for TPMT 2. All seven TPMT-deficient subjects identified by Schaeffeler and colleagues were homozygous or compound heterozygous carriers for these alleles. 

Most studies in leukemia patients have used RBC as a surrogate tissue for phenotyping TPMT activity, and several of them have shown that childhood leukemia patients with TPMT /TPMT " phenotypes are at high risk of developing severe hematologic toxicity after treatment with standard doses of thiopurines (196,197,198). Thus, dose adjustment... 

Schaeffeler E, Fischer C, Brockmeier D, et al. (2004). Comprehensive analysis of thiopurine S-methyltransferase (TPMT) phenotype-genotype correlation in a large population of German-Caucasians and identification of novelTPMT variants. Pharmacogen. 14 407-417. 

Spire-Vayron de la Moureyre C, De-buysere H, Mastain B et al. Genotypic and phenotypic analysis of the polymorphic thiopurine S-methyltransferase gene (TPMT) in a European population. Br J Pharmacol 1998 125 879-887. 

Based on the population genotype-phenotype studies performed to date, assays for the molecular diagnosis of TPMT deficiency have focussed on alleles TPMT 2, TPMT 3A and TPMT 3C, as these represent 80-95% of all mutant alleles of this gene in Caucasians [46, 50]. However, the frequency and pattern of mutant alleles of this gene is different among various ethnic populations. For example, Southwest Asians (Indian, Pakistani) have a lower frequency of mutant TPMT alleles and all mutant alleles identified to date are TPMT 3A (Table 24.1) [52]. This is in contrast to Kenyans and Ghanaians where the frequency of mutant alleles is similar to Caucasians, and all mutant alleles are TPMT 3C (Table 24.1) [53, 54]. Among African Americans, TPMT 3C is the most prevalent allele, but TPMT 2... 

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... 

Another approach to optimize pharmacotherapy of leukemia with thiopurines is phenotyping and/or genotyping of TPMT. TPMT is a ubiquitously expressed cytosolic methyltransferase that catalyzes the S -methylation of aromatic and heterocyclic sulfhydryl compounds (130, 131). Neither the biological function of TPMT nor its endogenous substrates are known. In thiopurine-treated patients, the activity of TPMT determines the balance between 6-TGN and 6-MMPR. 

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). 

With regard to long-term toxicity, some reports in the literature have suggested a relationship of secondary malignancies after treatment of childhood ALL with TPMT /TPMT and TPMTVTPMT phenotypes. In a study at St. Jude Children s Research Hospital, SJCRH Total XlllHR, patients with lower TPMT activity showed a trend towards a higher incidence of AML associated with application of the topoiso-merase II inhibitor etoposide (203). 

Of major concern is a report of an increased incidence of secondary brain tumors after radiotherapy in children with decreased TPMT activity phenotypes and/or high concentrations of thioguanine nucleotides in blood... 

TPMT) or the liver (for UGT and CYP2C9). Certain mutations in these isoforms, or gene variants, produce different phenotypes but most important to drug dosing is the poor metabolizer phenotype that results in heightened exposure to either the parent drug or a major metabolite, or reduced exposure to an active metabolite (e.g., morphine from codeine administration). 

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