Thiopurine Methyltransferase Activity

Indications for Testing

Consider TPMT testing in patients who will receive thiopurine drugs

Laboratory Testing

TPMT activity in red blood cells (RBC)
- If enzymatic level is reported as
  - Normal (25-65 U/mL) – low risk of bone marrow toxicity as a consequence of standard thiopurine therapy; no dose adjustment necessary
  - Abnormal/low (<25 U/mL) – high risk of bone marrow toxicity as a consequence of standard thiopurine dosing; reduction of drug dose by 80-90% recommended
  - Intermediate – reduction of drug dose by 20-50% may be necessary
  - High (>65 U/mL) – risk of therapeutic failure due to excessive inactivation of thiopurine drugs; may require higher drug dosing
Patient should not be taking certain drugs when tested (xanthine oxidase inhibitors) because levels may appear falsely low
- If RBC level is abnormal, consider TPMT genotyping

Therapeutic monitoring (thiopurine metabolite concentrations) may be appropriate for individuals with deficient or high TPMT activity

Thiopurine drugs such as azathioprine (AZA), 6-mercaptopurine (6-MP), and 6-thioguanine (6-TG) are widely used in the treatment of acute lymphoblastic leukemia (ALL), autoimmune diseases, inflammatory bowel disease, and post-transplant organ rejection. Patients with abnormal thiopurine methyltransferase (TPMT) enzyme activity have an increased risk of toxicity when given thiopurines.

Epidemiology

Prevalence – 1/300 Caucasians are TPMT deficient
- 11% of Caucasians are heterozygous for TPMT mutations and may have intermediate TPMT activity

Genetics

Autosomal recessive inheritance
- Homozygous state – associated with deficient enzymatic activity
- Heterozygous state – may be associated with intermediate enzymatic activity
>20 mutational TPMT alleles identified to date; *2, *3A and *3C alleles account for 80-95% of low-to-intermediate activity states

Pathophysiology

AZA, 6-MP, and 6-TG are inactive prodrugs used to treat a variety of different disease states and are metabolized by three different enzymes into three different 6-thioguanine nucleotides for activity
- AZA is metabolized to 6-MP (one of most commonly prescribed)
  - 6-MP is converted into two pharmacologically inactive metabolites
    - 6-thiouric acid by the enzyme xanthine oxidase (XO)
    - 6-methylmercaptopurine (6-MMP) by thiopurine S-methyltransferase (TPMT)
      - Primary metabolic route for inactivation of nucleotides is catalyzed by TPMT
  - 6-MP is also converted into active thioguanine nucleotides by the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT) to exert therapeutic cytotoxic effects
  - When 6-MP is converted to inactive metabolites, the amount of 6-TG nucleotides reduces, which balances the amount of 6-TG nucleotides needed to achieve required cytotoxicity for therapeutic treatment
  - When TPMT enzyme activity is low, proportionately more 6-MP is converted into cytotoxic 6-TG nucleotides, which increases toxicity
- Accumulation of excessive nucleotides inhibit purine synthesis, most dramatically noted in the bone marrow, causing toxicity with myelosuppression
- Reduced drug dosing may prevent myelosuppression in patients with intermediate and low TPMT activity

Tests generally appear in the order most useful for common clinical situations

Test name: Thiopurine Methyltransferase, RBC

ARUP #: 0092066

Methodology: Enzymatic/Quantitative Liquid Chromatography-Tandem Mass Spectrometry

Use:

Measures enzymatic levels to determine risk of toxicity of thiopurine therapy

Limitations:

Does not measure concentration of parent drug (6-TG, AZA, or 6-MP) or metabolites

Does not replace need for clinical monitoring of patients treated with thiopurine drugs

Genotype for TPMT cannot be inferred from TPMT activity in red blood cells

Phenotype testing should not be requested for patients currently treated with thiopurine drugs because results will be falsely low

Current TPMT phenotype may not reflect future TPMT phenotype, particularly in patients who received blood transfusion within 30-60 days of testing

TPMT enzyme activity can be inhibited by naproxen (Aleve^®), ibuprofen (Advil^®, Motrin^®), ketoprofen (Orudis^®), furosemide (Lasix^®), sulfasalazine (Azulfidine^®), mesalamine (Asacol^®), olsalazine (Dipentum^®), mefenamic acid (Ponstel^®), thiazide diuretics, and benzoic acid inhibitors

Patients should abstain from these drugs for at least 48 hours prior to TPMT testing

Test name: TPMT Genotype

ARUP #: 2002573

Methodology: Qualitative Polymerase Chain Reaction

Use:

Consider genotyping if RBC level is abnormal

Test name: Thiopurine Metabolites

ARUP #: 2002575

Methodology: Qualitative High Pressure Liquid Chromatography
(High Performance Liquid Chromatography)

Use:

Use to monitor patients with deficient or high TPMT activity