Thiopurine Methyltransferase (TPMT; EC 2.1.1.67) is a cytoplasmic enzyme that catalyzes the S-methylation of aromatic and heterocyclic sulfhydryl compounds (Ames et al., 1986; Weinshilboum, 1989; Deininger et al., 1994). S-adenosylmethionine (SAM) is the methyl donor in this Mtase catalyzed reaction, and S-adenosylhomocysteine (SAH) and 6-methylmercaptopurine are released as reaction products.
TPMT is the enzyme responsible for metabolizing several anti-cancer thiopurine drugs, including thioguanine, azathioprine, and mercaptopurine, through the addition of a methyl group to the sulfhydryl group of these drugs. Methylation prevents the incorporation of the drugs into extending nucleic acid polymers, limiting the drugs' potency. Consequently, the dosage of the drug delivered to a patient must exceed the patient's ability to inactivate the drug. As there are numerous polymorphisms of TPMT possessing disparate levels of activity, it is important to tailor the drug dosage to the patient.
TPMT activity exhibits genetic polymorphism in the human population. About ˜89% of Caucasians and African Americans have high TPMT activity (wild type), ˜11% show intermediate activity (presumed heterozygotes), and ˜1 in 300 (homozygotes) display complete TPMT deficiency, which is inherited as an autosomal recessive trait (Weinshilboum, 1980; Weinshilboum et al, 1999). Such frequent mutations which occur in over 1% of the population are formally termed polymorphisms (Meyer et al, 1990; Iyer and Ratain, 1998). As a result of these genetic polymorphisms, a significant fraction of the population cannot metabolize certain commonly employed therapeutic drugs. For example, in TPMT-deficient patients, 6-mercaptopurine cannot be methylated to 6-methylmercaptopurine. 
The methylated reaction product, 6-methylmercaptopurine (6-MMP), is normally metabolized in the liver. However, unmethylated 6-mercaptopurine builds up to toxic levels in the bloodstream in TPMT-deficient individuals. As a part of multiagent chemotherapy for the treatment of acute lymphoblastic leukemia, 6-mercaptopurine (or 6-thioguanine) is typically used in large doses over an extended time period. Mercaptopurine is a prodrug with no intrinsic anticancer activity, requiring intracellular conversion to 6-thioguanine nucleotides. These metabolites, in turn, are incorporated into DNA, as one mechanism of its antiproliferative effects (Lennard et al, 1987; Elion, 1989).
Patients with low levels of TPMT activity accumulate significantly higher levels of thioguanine nucleotides when treated with standard mercaptopurine dosages, leading to severe hematopoietic toxicity. On the other hand, “standard” doses of these drugs may undertreat patients with high levels of enzyme activity (Lennard et al, 1990). Due to TPMT deficiency, about one in ten childhood leukemia patients cannot tolerate the high doses of thiopurines which are used in the normal chemotherapeutic regimen (Lennard et al, 1987; Lennard et al, 1993). Similar problems are encountered in certain dermatology patients (Jackson et al, 1997), patients with Crohn's Disease (Sandborn et al, 1995), or organ transplant recipients (Schutz et al, 1996) when they are treated with thiopurine drugs.
The most common strategy for thiopurine administration is to give all patients low doses of 6-mercaptopurine or azathioprine; and to then gradually increase the dose (Weinshilboum, 1984; Evans et al, 1991). At certain dosages, one in ten patients will become ill. Lower doses of thiopurines and/or alternative therapy are then given to TPMT-intolerant patients. Obviously, this course of action is less than ideal since 90% of patients receive unaggressive treatment; and the remaining 10% are poisoned by their ‘therapy’ (Lennard and Lilleyman, 1987; Lennard et al, 1993).
TPMT-deficient patients suffer greatly when given thiopurine drugs. Not only do children with leukemia suffer from a life-threatening cancer, but the consequences of their chemotherapy can actually be worse than their disease (Lennard et al, 1987). Some TPMT-deficient patients die from acute thiopurine toxicity (Schutz et al, 1993).
A more rational therapeutic approach for thiopurine therapy strategy would be to first measure TPMT enzyme activity in patients who are candidates for thiopurine drugs, and then adjust the thiopurine dosage on an individual basis (Lennard et al., 1987; Lennard and Lilleyman, 1996; Jackson et al., 1997; Krynetski and Evans, 1998; Lennard, 1998; Lennard, 1999).
TPMT activity is typically measured from lysed red blood cells. Enzyme activity in red blood cell lysates corresponds to the level of TPMT in human liver, kidney, and normal lymphocytes. Currently used methods for measuring erythrocyte TPMT activity rely on the transfer of methyl groups from 14C-methyl-SAM to 6-mercaptopurine, extraction of the radiolabeled 14C-methylated reaction product into 20% isoamyl alcohol: 80% toluene, followed by liquid scintillation counting (Weinshilboum et al., 1978; University of Rochester, 1999). The total time needed to carry out this 8-step TPMT assay procedure is >3 hours, requires the use of 14C-radiolabeled SAM, involves the disposal of toxic organic waste, and costs over $100/assay (University of Rochester, 1999). At the present time, radioenzymatic TPMT assays are necessarily carried out in laboratories which are licensed and equipped for the handling of radioisotopes, such as hospital nuclear medicine laboratories and university medical genetics departments.
Non-isotopic TPMT assays based upon HPLC chromatography have been described (Boulieu and Lenoir, 1995; Kroplin et al., 1996; Micheli et al., 1997). However, these labor-intensive assays require extensive liquid handling and sample processing. Further, such heterogeneous Mtase assays are not suitable for routine point-of-care diagnosis.
Alternatively, DNA-based tests have been developed for common TPMT allelic variants (Krynetski et al., 1995; Szumlanski et al., 1996; Yates et al., 1997; Evans and Krynetski, 1999; Krynetski et al., 1999). Unfortunately, such genotypic tests have three major limitations: (i) DNA-based tests cannot detect any new or uncharacterized allelic variants; (ii) genotypic tests do not allow the clinician to determine what dose of thiopurine drugs, if any, should be given to a particular patient; and (iii) rapid DNA-based tests are relatively expensive. For example, the DNA Chip-based technology for TPMT detection developed by Nanogen (San Diego, Calif.) relies upon an instrument which costs over $175,000; and each DNA Chip costs approximately $200 (Heller, 2000). While elegant in principle, such high cost devices and reagents are generally not affordable. Even the designers of DNA-based tests for TPMT deficiency admit that a phenotypic test for blood or tissue enzyme activity would be preferable (Krynetski et al., 1995; Spire-Vayron de la Moureyre et al., 1998).
The development of rapid homogenous assays for TPMT would be technically advantageous, especially if the assays could be carried out without the need for radioisotopes. The cost and environmental hazard of radioenzymatic assays would be reduced, while the speed and technical simplicity of Mtase assays would be improved.
It is therefore a primary objective of the present invention to provide a method and means of providing an improved phenotypic assay to detect DNA sequence alterations in the human TPMT gene.
It is a further objective of the present invention to provide a method and means of detecting TPMT enzyme levels that is less labor intensive, time-consuming, and expensive than conventional assays.
It is a further objective of the present invention to provide a method and means of measuring TPMT that is both qualitative and quantitative.
It is yet a further objective of the present invention to provide an immunochemical method for detecting the methylated product of TPMT-catalyzed reactions.
It is still a further objective of the present invention to provide a method and means of detecting TPMT enzyme activity that allows the clinician to determine what dose of thiopurine drugs, if any, should be given to a particular patient.
These and other objectives will become clear from the following detailed description of the invention.