J. Kevin Hicks, PharmD, PhD

The thiopurines azathioprine, mercaptopurine, and thioguanine are prodrugs that are converted to active thioguanine nucleotide metabolites or methylated by thiopurine methyltransferase (TPMT) to compounds with less pharmacologic activity. In the absence of TPMT activity, patients are likely to have higher concentrations of thioguanine nucleotides, which can pose an increased risk of severe life-threatening myelosuppression. Determining TPMT activity, either directly by phenotyping or indirectly by determining the specific genetic allele (different alleles have different enzymatic activity), can help identify patients at greater risk of severe myelosuppression. Therefore, we recommend that TPMT testing be strongly considered before initiating therapy with a thiopurine.

THIOPURINES AND TPMT

Azathioprine, mercaptopurine, and thioguanine are used for treating autoimmune and inflammatory diseases^1–3 and certain types of cancer such as leukemias and lymphomas.^1,4–6 Typically, azathioprine is used to treat nonmalignant conditions, thioguanine is used to treat malignancies, and mercaptopurine can be used to treat both malignant and nonmalignant conditions.

Although the exact mechanism of action of these drugs has not been completely elucidated, the active thioguanine nucleotide metabolites are thought to be incorporated into the DNA of leukocytes, resulting in DNA damage that subsequently leads to cell death and myelosuppression.^7–9

Variants of the TPMT gene may alter the activity of the TPMT enzyme, resulting in individual variability in thiopurine metabolism. Compared with people with normal (high) TPMT activity, those with intermediate or low TPMT activity metabolize the drugs more slowly, and are likely to have higher thioguanine nucleotide concentrations and therefore an increased risk of myelosuppression.

One of the earliest correlations between TPMT activity and thiopurine-induced myelosuppression was described in a pediatric patient with acute lymphocytic leukemia.¹⁰ After being prescribed a conventional mercaptopurine dosage (75 mg/m² daily), the patient developed severe myelosuppression and was observed to have a thioguanine nucleotide metabolite concentration seven times the observed population median. TPMT phenotyping demonstrated that the patient had low TPMT activity. Reducing the mercaptopurine dose by approximately 90% resulted in normalization of thioguanine nucleotide metabolite concentrations, and the myelosuppression subsequently resolved.

Approximately 10% of the population has intermediate TPMT activity and 0.3% has low or absent TPMT activity, though these percentages vary depending on ancestry.¹ Research has demonstrated that approximately 30% to 60% of those with intermediate TPMT activity cannot tolerate a full thiopurine dose (eg, azathioprine 2–3 mg/kg/day or mercaptopurine 1.5 mg/kg/day).¹ Almost all patients with low TPMT activity will develop life-threating myelosuppression if prescribed a full thiopurine dose.¹

SHOULD TPMT ACTIVITY BE DETERMINED FOR EVERY PATIENT PRESCRIBED A THIOPURINE?

Although determining TPMT activity in thiopurine-naïve patients will assist clinicians in selecting a thiopurine starting dose or in deciding if an alternative agent is warranted, there are instances when a clinician may elect to not perform a TPMT genotype or phenotype test. For example, determining TPMT activity is not recommended for patients who previously tolerated thiopurine therapy at full steady-state doses.

The required starting dose of a thiopurine can influence the decision on whether or not to test for TPMT activity. TPMT genotyping or phenotyping may be of most benefit for patients requiring immediate full doses of a thiopurine.¹¹ Ideally, TPMT activity should be determined before prescribing immediate full doses of a thiopurine. This could be achieved by preemptively ordering a TPMT test in patients likely to require immunosuppression—for example, in patients diagnosed with inflammatory or autoimmune diseases. If therapy cannot be delayed and TPMT activity is unknown, ordering a TPMT test at the time of prescribing a full thiopurine dose is still of benefit. Depending on the clinical laboratory utilized for testing, TPMT phenotype results are usually reported in 3 to 5 days, and TPMT genotype results are usually reported in 5 to 7 days. Because most patients will not reach steady-state concentrations for 2 to 6 weeks, clinicians could initiate immediate full doses of a thiopurine and modify therapy based on TPMT test results before accumulation of thioguanine nucleotide metabolites occurs. Caution should be used with this approach, particularly in situations where the clinical laboratory may not return results in a timely manner.

For patients who are candidates for an initial low dose of a thiopurine, clinicians may choose to slowly titrate doses based on response and tolerability instead of determining TPMT activity.¹¹ Depending on the starting dose and how slowly titration occurs, initiating a thiopurine at a low dose and titrating based on response can be a feasible approach for patients with intermediate TPMT activity. Because drastic thiopurine dose reductions of approximately 10-fold are required for patients with low TPMT activity, which is a much smaller dosage than most clinicians will initially prescribe, the starting dosage will likely not be low enough to prevent myelosuppression in patients with low TPMT activity.^1,10

Determining TPMT activity can help clinicians establish an appropriate titration schedule. Patients with normal TPMT activity will usually reach thiopurine steady-state concentrations in 2 weeks, and the dosage can be titrated based on response.¹ Alterations in TPMT activity influence the pharmacokinetic parameters of thiopurines, and the time to reach steady-state is extended to 4 or 6 weeks for those with intermediate or low TPMT activity.¹ Increasing the thiopurine dosage before reaching steady state can lead to the prescribing of doses that will not be tolerated, resulting in myelosuppression.

Factors to consider when deciding if TPMT activity should be assessed include the disease state being treated and corresponding starting dose, the need for immediate full doses, and previous documented tolerance of thiopurines at steady-state doses. As with many aspects of medicine that have multiple options, coupled with an increase in patient access to healthcare information, the decision to test for TPMT activity may include shared decision-making between patients and providers. Although TPMT genotyping or phenotyping can help identify those at greatest risk of severe myelosuppression, such assays do not replace routine monitoring for myelosuppression, hepatotoxicity, or pancreatitis that may be caused by thiopurines.

WHAT TESTS ARE AVAILABLE TO DETERMINE TPMT ACTIVITY?

Patients with intermediate or low TPMT activity can be identified by either genotyping or phenotyping. There are considerations, though, that clinicians should be aware of before selecting a particular test.

TPMT genotyping

Four TPMT alleles, TPMT*2, *3A, *3B, and *3C, account for over 90% of inactivating polymorphisms.¹² Therefore, most reference laboratories only analyze for those genetic variants. Based on the reported test result, a predicted phenotype (eg, normal, intermediate, or low TPMT activity) can be assigned. Table 1 lists the predicted phenotypes for select genotyping results.

TPMT phenotyping

Phenotyping quantitates TPMT enzyme activity in erythrocytes, and based on the result, patients are classified as having normal, intermediate, or low TPMT activity. Because internal standards and other testing conditions may differ between reference laboratories, test results must be interpreted in the context of the laboratory that performed the assay.

Which test is right for my patient?

In most cases, either the genotype or the phenotype test provides sufficient information to guide thiopurine therapy. There are certain circumstances, though, in which the genotype or phenotype test is less informative.

TPMT genotyping, when performed using a blood specimen, is not recommended in those with a history of allogeneic bone marrow transplantation, as the result would reflect the donor’s genotype, not the patient’s. In such instances, monitoring of white blood cell counts and thiopurine metabolites may be more beneficial.

TPMT phenotyping may be inaccurate if performed within 30 to 90 days of an erythrocyte transfusion, as the test result may be influenced by donor erythrocytes. If a patient is receiving erythrocyte transfusions, TPMT genotyping is preferable to phenotyping.

Test cost may also be a consideration when determining if the genotype or phenotype test is best for your patient. Costs vary by laboratory, but phenotyping is generally less expensive than genotyping. The cost of genotyping, though, continues to decrease.¹³ The approximate commercial cost is $200 for phenotyping and $450 for genotyping, but laboratory fees may be substantially higher. Several insurance plans, including Medicare, cover TPMT testing, but reimbursement and copayments vary, depending on the patient’s specific plan.

There are conflicting data as to whether determining TPMT status is^11,14–18 or is not¹⁹ cost-effective. Multiple studies suggest that the cost of genotyping a sufficient number of patients to identify a single individual at high risk of myelosuppression is cheaper than the costs associated with treating an adverse event. Additional cost-benefit studies are needed, particularly studies that consider how bundled payments and outcomes-based reimbursement influence cost-effectiveness.

MODIFYING THIOPURINE THERAPY BASED ON TPMT ACTIVITY

There is a strong correlation between TPMT activity and tolerated thiopurine doses, with those having intermediate or low TPMT activity requiring lower doses.^10,20–23 Adjusting mercaptopurine doses based on TPMT activity to prevent hematopoietic toxicity has been successfully demonstrated in pediatric patients with acute lymphoblastic leukemia.²⁴ Furthermore, reducing initial thiopurine doses to avoid myelosuppression and titrating based on response has been shown to not compromise outcomes.^1,25,26 The Clinical Pharmacogenetic Implementation Consortium (CPIC) has developed an evidence-based guideline on how to adjust thiopurine doses based on TPMT activity,¹ summarized in Table 2. These dosing recommendations are classified as “strong.”

Patients with normal TPMT activity should be prescribed the usual thiopurine starting dose as indicated by disease-specific guidelines.

For those with intermediate TPMT activity, the CPIC guideline recommends reducing the initial targeted full dose of azathioprine and mercaptopurine by 30% to 70% and reducing the targeted full dose of thioguanine by 30% to 50%. The percentage of dose reduction depends on the targeted full dose. Siegel and Sands²⁷ suggested that for those who are diagnosed with inflammatory bowel disease and have intermediate TPMT activity, azathioprine should be initiated at a low dose and titrated to 1.25 mg/kg and mercaptopurine should be initiated at a low dose and titrated to 0.75 mg/kg. Based on these titration goals, if the targeted full dose for mercaptopurine is 1 mg/kg, then a dose reduction of approximately 30% would be more appropriate. If the targeted full dose is 1.5 mg/kg, a dose reduction of approximately 50% would be more appropriate. Thiopurine doses should be titrated based on response and disease-specific guidelines, allowing 2 to 4 weeks to reach steady state before dose titration.

For those with low TPMT activity, alternative therapy should be considered for nonmalignant conditions because of the risk of severe myelosuppression. For malignancy, or if a thiopurine is warranted for a nonmalignant condition, consider a 90% dose reduction and give the drug three times per week instead of daily. For example, acute lymphoblastic leukemia patients with low TPMT activity can be started on mercaptopurine 10 mg/m² three times per week instead of the usual starting dose.¹⁰ Thiopurine doses should be titrated based on response and disease-specific guidelines, allowing 4 to 6 weeks to reach steady state before dose titration.

RECOMMENDATIONS

Individuals with intermediate or low TPMT activity have an increased risk of myelosuppression. Because of the elevated risk for morbidity and death, especially for patients with low TPMT activity, multiple guidelines and regulatory agencies recommend TPMT genotyping or phenotyping if a thiopurine is prescribed.^25,28–32 Although additional cost-benefit analysis studies are needed, evidence suggests testing for TPMT activity may be cheaper than the costs associated with treating myelosuppression.

In view of treatment guidelines, the recommendations of regulatory agencies, cost-benefit analyses, and the availability of gene-based dosing recommendations, we consider the benefits of testing for TPMT activity to greatly outweigh any associated risks. Therefore, we recommend that TPMT testing be strongly considered before initiating therapy with a thiopurine.

The thiopurines azathioprine, mercaptopurine, and thioguanine are prodrugs that are converted to active thioguanine nucleotide metabolites or methylated by thiopurine methyltransferase (TPMT) to compounds with less pharmacologic activity. In the absence of TPMT activity, patients are likely to have higher concentrations of thioguanine nucleotides, which can pose an increased risk of severe life-threatening myelosuppression. Determining TPMT activity, either directly by phenotyping or indirectly by determining the specific genetic allele (different alleles have different enzymatic activity), can help identify patients at greater risk of severe myelosuppression. Therefore, we recommend that TPMT testing be strongly considered before initiating therapy with a thiopurine.

THIOPURINES AND TPMT

Azathioprine, mercaptopurine, and thioguanine are used for treating autoimmune and inflammatory diseases^1–3 and certain types of cancer such as leukemias and lymphomas.^1,4–6 Typically, azathioprine is used to treat nonmalignant conditions, thioguanine is used to treat malignancies, and mercaptopurine can be used to treat both malignant and nonmalignant conditions.

Although the exact mechanism of action of these drugs has not been completely elucidated, the active thioguanine nucleotide metabolites are thought to be incorporated into the DNA of leukocytes, resulting in DNA damage that subsequently leads to cell death and myelosuppression.^7–9

Variants of the TPMT gene may alter the activity of the TPMT enzyme, resulting in individual variability in thiopurine metabolism. Compared with people with normal (high) TPMT activity, those with intermediate or low TPMT activity metabolize the drugs more slowly, and are likely to have higher thioguanine nucleotide concentrations and therefore an increased risk of myelosuppression.

One of the earliest correlations between TPMT activity and thiopurine-induced myelosuppression was described in a pediatric patient with acute lymphocytic leukemia.¹⁰ After being prescribed a conventional mercaptopurine dosage (75 mg/m² daily), the patient developed severe myelosuppression and was observed to have a thioguanine nucleotide metabolite concentration seven times the observed population median. TPMT phenotyping demonstrated that the patient had low TPMT activity. Reducing the mercaptopurine dose by approximately 90% resulted in normalization of thioguanine nucleotide metabolite concentrations, and the myelosuppression subsequently resolved.

Approximately 10% of the population has intermediate TPMT activity and 0.3% has low or absent TPMT activity, though these percentages vary depending on ancestry.¹ Research has demonstrated that approximately 30% to 60% of those with intermediate TPMT activity cannot tolerate a full thiopurine dose (eg, azathioprine 2–3 mg/kg/day or mercaptopurine 1.5 mg/kg/day).¹ Almost all patients with low TPMT activity will develop life-threating myelosuppression if prescribed a full thiopurine dose.¹

SHOULD TPMT ACTIVITY BE DETERMINED FOR EVERY PATIENT PRESCRIBED A THIOPURINE?

Although determining TPMT activity in thiopurine-naïve patients will assist clinicians in selecting a thiopurine starting dose or in deciding if an alternative agent is warranted, there are instances when a clinician may elect to not perform a TPMT genotype or phenotype test. For example, determining TPMT activity is not recommended for patients who previously tolerated thiopurine therapy at full steady-state doses.

The required starting dose of a thiopurine can influence the decision on whether or not to test for TPMT activity. TPMT genotyping or phenotyping may be of most benefit for patients requiring immediate full doses of a thiopurine.¹¹ Ideally, TPMT activity should be determined before prescribing immediate full doses of a thiopurine. This could be achieved by preemptively ordering a TPMT test in patients likely to require immunosuppression—for example, in patients diagnosed with inflammatory or autoimmune diseases. If therapy cannot be delayed and TPMT activity is unknown, ordering a TPMT test at the time of prescribing a full thiopurine dose is still of benefit. Depending on the clinical laboratory utilized for testing, TPMT phenotype results are usually reported in 3 to 5 days, and TPMT genotype results are usually reported in 5 to 7 days. Because most patients will not reach steady-state concentrations for 2 to 6 weeks, clinicians could initiate immediate full doses of a thiopurine and modify therapy based on TPMT test results before accumulation of thioguanine nucleotide metabolites occurs. Caution should be used with this approach, particularly in situations where the clinical laboratory may not return results in a timely manner.

For patients who are candidates for an initial low dose of a thiopurine, clinicians may choose to slowly titrate doses based on response and tolerability instead of determining TPMT activity.¹¹ Depending on the starting dose and how slowly titration occurs, initiating a thiopurine at a low dose and titrating based on response can be a feasible approach for patients with intermediate TPMT activity. Because drastic thiopurine dose reductions of approximately 10-fold are required for patients with low TPMT activity, which is a much smaller dosage than most clinicians will initially prescribe, the starting dosage will likely not be low enough to prevent myelosuppression in patients with low TPMT activity.^1,10

Determining TPMT activity can help clinicians establish an appropriate titration schedule. Patients with normal TPMT activity will usually reach thiopurine steady-state concentrations in 2 weeks, and the dosage can be titrated based on response.¹ Alterations in TPMT activity influence the pharmacokinetic parameters of thiopurines, and the time to reach steady-state is extended to 4 or 6 weeks for those with intermediate or low TPMT activity.¹ Increasing the thiopurine dosage before reaching steady state can lead to the prescribing of doses that will not be tolerated, resulting in myelosuppression.

Factors to consider when deciding if TPMT activity should be assessed include the disease state being treated and corresponding starting dose, the need for immediate full doses, and previous documented tolerance of thiopurines at steady-state doses. As with many aspects of medicine that have multiple options, coupled with an increase in patient access to healthcare information, the decision to test for TPMT activity may include shared decision-making between patients and providers. Although TPMT genotyping or phenotyping can help identify those at greatest risk of severe myelosuppression, such assays do not replace routine monitoring for myelosuppression, hepatotoxicity, or pancreatitis that may be caused by thiopurines.

WHAT TESTS ARE AVAILABLE TO DETERMINE TPMT ACTIVITY?

Patients with intermediate or low TPMT activity can be identified by either genotyping or phenotyping. There are considerations, though, that clinicians should be aware of before selecting a particular test.

TPMT genotyping

Four TPMT alleles, TPMT*2, *3A, *3B, and *3C, account for over 90% of inactivating polymorphisms.¹² Therefore, most reference laboratories only analyze for those genetic variants. Based on the reported test result, a predicted phenotype (eg, normal, intermediate, or low TPMT activity) can be assigned. Table 1 lists the predicted phenotypes for select genotyping results.

TPMT phenotyping

Phenotyping quantitates TPMT enzyme activity in erythrocytes, and based on the result, patients are classified as having normal, intermediate, or low TPMT activity. Because internal standards and other testing conditions may differ between reference laboratories, test results must be interpreted in the context of the laboratory that performed the assay.

Which test is right for my patient?

In most cases, either the genotype or the phenotype test provides sufficient information to guide thiopurine therapy. There are certain circumstances, though, in which the genotype or phenotype test is less informative.

TPMT genotyping, when performed using a blood specimen, is not recommended in those with a history of allogeneic bone marrow transplantation, as the result would reflect the donor’s genotype, not the patient’s. In such instances, monitoring of white blood cell counts and thiopurine metabolites may be more beneficial.

TPMT phenotyping may be inaccurate if performed within 30 to 90 days of an erythrocyte transfusion, as the test result may be influenced by donor erythrocytes. If a patient is receiving erythrocyte transfusions, TPMT genotyping is preferable to phenotyping.

Test cost may also be a consideration when determining if the genotype or phenotype test is best for your patient. Costs vary by laboratory, but phenotyping is generally less expensive than genotyping. The cost of genotyping, though, continues to decrease.¹³ The approximate commercial cost is $200 for phenotyping and $450 for genotyping, but laboratory fees may be substantially higher. Several insurance plans, including Medicare, cover TPMT testing, but reimbursement and copayments vary, depending on the patient’s specific plan.

There are conflicting data as to whether determining TPMT status is^11,14–18 or is not¹⁹ cost-effective. Multiple studies suggest that the cost of genotyping a sufficient number of patients to identify a single individual at high risk of myelosuppression is cheaper than the costs associated with treating an adverse event. Additional cost-benefit studies are needed, particularly studies that consider how bundled payments and outcomes-based reimbursement influence cost-effectiveness.

MODIFYING THIOPURINE THERAPY BASED ON TPMT ACTIVITY

There is a strong correlation between TPMT activity and tolerated thiopurine doses, with those having intermediate or low TPMT activity requiring lower doses.^10,20–23 Adjusting mercaptopurine doses based on TPMT activity to prevent hematopoietic toxicity has been successfully demonstrated in pediatric patients with acute lymphoblastic leukemia.²⁴ Furthermore, reducing initial thiopurine doses to avoid myelosuppression and titrating based on response has been shown to not compromise outcomes.^1,25,26 The Clinical Pharmacogenetic Implementation Consortium (CPIC) has developed an evidence-based guideline on how to adjust thiopurine doses based on TPMT activity,¹ summarized in Table 2. These dosing recommendations are classified as “strong.”

Patients with normal TPMT activity should be prescribed the usual thiopurine starting dose as indicated by disease-specific guidelines.

For those with intermediate TPMT activity, the CPIC guideline recommends reducing the initial targeted full dose of azathioprine and mercaptopurine by 30% to 70% and reducing the targeted full dose of thioguanine by 30% to 50%. The percentage of dose reduction depends on the targeted full dose. Siegel and Sands²⁷ suggested that for those who are diagnosed with inflammatory bowel disease and have intermediate TPMT activity, azathioprine should be initiated at a low dose and titrated to 1.25 mg/kg and mercaptopurine should be initiated at a low dose and titrated to 0.75 mg/kg. Based on these titration goals, if the targeted full dose for mercaptopurine is 1 mg/kg, then a dose reduction of approximately 30% would be more appropriate. If the targeted full dose is 1.5 mg/kg, a dose reduction of approximately 50% would be more appropriate. Thiopurine doses should be titrated based on response and disease-specific guidelines, allowing 2 to 4 weeks to reach steady state before dose titration.

For those with low TPMT activity, alternative therapy should be considered for nonmalignant conditions because of the risk of severe myelosuppression. For malignancy, or if a thiopurine is warranted for a nonmalignant condition, consider a 90% dose reduction and give the drug three times per week instead of daily. For example, acute lymphoblastic leukemia patients with low TPMT activity can be started on mercaptopurine 10 mg/m² three times per week instead of the usual starting dose.¹⁰ Thiopurine doses should be titrated based on response and disease-specific guidelines, allowing 4 to 6 weeks to reach steady state before dose titration.

RECOMMENDATIONS

Individuals with intermediate or low TPMT activity have an increased risk of myelosuppression. Because of the elevated risk for morbidity and death, especially for patients with low TPMT activity, multiple guidelines and regulatory agencies recommend TPMT genotyping or phenotyping if a thiopurine is prescribed.^25,28–32 Although additional cost-benefit analysis studies are needed, evidence suggests testing for TPMT activity may be cheaper than the costs associated with treating myelosuppression.

In view of treatment guidelines, the recommendations of regulatory agencies, cost-benefit analyses, and the availability of gene-based dosing recommendations, we consider the benefits of testing for TPMT activity to greatly outweigh any associated risks. Therefore, we recommend that TPMT testing be strongly considered before initiating therapy with a thiopurine.

The thiopurines azathioprine, mercaptopurine, and thioguanine are prodrugs that are converted to active thioguanine nucleotide metabolites or methylated by thiopurine methyltransferase (TPMT) to compounds with less pharmacologic activity. In the absence of TPMT activity, patients are likely to have higher concentrations of thioguanine nucleotides, which can pose an increased risk of severe life-threatening myelosuppression. Determining TPMT activity, either directly by phenotyping or indirectly by determining the specific genetic allele (different alleles have different enzymatic activity), can help identify patients at greater risk of severe myelosuppression. Therefore, we recommend that TPMT testing be strongly considered before initiating therapy with a thiopurine.

THIOPURINES AND TPMT

Azathioprine, mercaptopurine, and thioguanine are used for treating autoimmune and inflammatory diseases^1–3 and certain types of cancer such as leukemias and lymphomas.^1,4–6 Typically, azathioprine is used to treat nonmalignant conditions, thioguanine is used to treat malignancies, and mercaptopurine can be used to treat both malignant and nonmalignant conditions.

Although the exact mechanism of action of these drugs has not been completely elucidated, the active thioguanine nucleotide metabolites are thought to be incorporated into the DNA of leukocytes, resulting in DNA damage that subsequently leads to cell death and myelosuppression.^7–9

Variants of the TPMT gene may alter the activity of the TPMT enzyme, resulting in individual variability in thiopurine metabolism. Compared with people with normal (high) TPMT activity, those with intermediate or low TPMT activity metabolize the drugs more slowly, and are likely to have higher thioguanine nucleotide concentrations and therefore an increased risk of myelosuppression.

One of the earliest correlations between TPMT activity and thiopurine-induced myelosuppression was described in a pediatric patient with acute lymphocytic leukemia.¹⁰ After being prescribed a conventional mercaptopurine dosage (75 mg/m² daily), the patient developed severe myelosuppression and was observed to have a thioguanine nucleotide metabolite concentration seven times the observed population median. TPMT phenotyping demonstrated that the patient had low TPMT activity. Reducing the mercaptopurine dose by approximately 90% resulted in normalization of thioguanine nucleotide metabolite concentrations, and the myelosuppression subsequently resolved.

Approximately 10% of the population has intermediate TPMT activity and 0.3% has low or absent TPMT activity, though these percentages vary depending on ancestry.¹ Research has demonstrated that approximately 30% to 60% of those with intermediate TPMT activity cannot tolerate a full thiopurine dose (eg, azathioprine 2–3 mg/kg/day or mercaptopurine 1.5 mg/kg/day).¹ Almost all patients with low TPMT activity will develop life-threating myelosuppression if prescribed a full thiopurine dose.¹

SHOULD TPMT ACTIVITY BE DETERMINED FOR EVERY PATIENT PRESCRIBED A THIOPURINE?

Although determining TPMT activity in thiopurine-naïve patients will assist clinicians in selecting a thiopurine starting dose or in deciding if an alternative agent is warranted, there are instances when a clinician may elect to not perform a TPMT genotype or phenotype test. For example, determining TPMT activity is not recommended for patients who previously tolerated thiopurine therapy at full steady-state doses.

The required starting dose of a thiopurine can influence the decision on whether or not to test for TPMT activity. TPMT genotyping or phenotyping may be of most benefit for patients requiring immediate full doses of a thiopurine.¹¹ Ideally, TPMT activity should be determined before prescribing immediate full doses of a thiopurine. This could be achieved by preemptively ordering a TPMT test in patients likely to require immunosuppression—for example, in patients diagnosed with inflammatory or autoimmune diseases. If therapy cannot be delayed and TPMT activity is unknown, ordering a TPMT test at the time of prescribing a full thiopurine dose is still of benefit. Depending on the clinical laboratory utilized for testing, TPMT phenotype results are usually reported in 3 to 5 days, and TPMT genotype results are usually reported in 5 to 7 days. Because most patients will not reach steady-state concentrations for 2 to 6 weeks, clinicians could initiate immediate full doses of a thiopurine and modify therapy based on TPMT test results before accumulation of thioguanine nucleotide metabolites occurs. Caution should be used with this approach, particularly in situations where the clinical laboratory may not return results in a timely manner.

For patients who are candidates for an initial low dose of a thiopurine, clinicians may choose to slowly titrate doses based on response and tolerability instead of determining TPMT activity.¹¹ Depending on the starting dose and how slowly titration occurs, initiating a thiopurine at a low dose and titrating based on response can be a feasible approach for patients with intermediate TPMT activity. Because drastic thiopurine dose reductions of approximately 10-fold are required for patients with low TPMT activity, which is a much smaller dosage than most clinicians will initially prescribe, the starting dosage will likely not be low enough to prevent myelosuppression in patients with low TPMT activity.^1,10

Determining TPMT activity can help clinicians establish an appropriate titration schedule. Patients with normal TPMT activity will usually reach thiopurine steady-state concentrations in 2 weeks, and the dosage can be titrated based on response.¹ Alterations in TPMT activity influence the pharmacokinetic parameters of thiopurines, and the time to reach steady-state is extended to 4 or 6 weeks for those with intermediate or low TPMT activity.¹ Increasing the thiopurine dosage before reaching steady state can lead to the prescribing of doses that will not be tolerated, resulting in myelosuppression.

Factors to consider when deciding if TPMT activity should be assessed include the disease state being treated and corresponding starting dose, the need for immediate full doses, and previous documented tolerance of thiopurines at steady-state doses. As with many aspects of medicine that have multiple options, coupled with an increase in patient access to healthcare information, the decision to test for TPMT activity may include shared decision-making between patients and providers. Although TPMT genotyping or phenotyping can help identify those at greatest risk of severe myelosuppression, such assays do not replace routine monitoring for myelosuppression, hepatotoxicity, or pancreatitis that may be caused by thiopurines.

WHAT TESTS ARE AVAILABLE TO DETERMINE TPMT ACTIVITY?

Patients with intermediate or low TPMT activity can be identified by either genotyping or phenotyping. There are considerations, though, that clinicians should be aware of before selecting a particular test.

TPMT genotyping

Four TPMT alleles, TPMT*2, *3A, *3B, and *3C, account for over 90% of inactivating polymorphisms.¹² Therefore, most reference laboratories only analyze for those genetic variants. Based on the reported test result, a predicted phenotype (eg, normal, intermediate, or low TPMT activity) can be assigned. Table 1 lists the predicted phenotypes for select genotyping results.

TPMT phenotyping

Phenotyping quantitates TPMT enzyme activity in erythrocytes, and based on the result, patients are classified as having normal, intermediate, or low TPMT activity. Because internal standards and other testing conditions may differ between reference laboratories, test results must be interpreted in the context of the laboratory that performed the assay.

Which test is right for my patient?

In most cases, either the genotype or the phenotype test provides sufficient information to guide thiopurine therapy. There are certain circumstances, though, in which the genotype or phenotype test is less informative.

TPMT genotyping, when performed using a blood specimen, is not recommended in those with a history of allogeneic bone marrow transplantation, as the result would reflect the donor’s genotype, not the patient’s. In such instances, monitoring of white blood cell counts and thiopurine metabolites may be more beneficial.

TPMT phenotyping may be inaccurate if performed within 30 to 90 days of an erythrocyte transfusion, as the test result may be influenced by donor erythrocytes. If a patient is receiving erythrocyte transfusions, TPMT genotyping is preferable to phenotyping.

Test cost may also be a consideration when determining if the genotype or phenotype test is best for your patient. Costs vary by laboratory, but phenotyping is generally less expensive than genotyping. The cost of genotyping, though, continues to decrease.¹³ The approximate commercial cost is $200 for phenotyping and $450 for genotyping, but laboratory fees may be substantially higher. Several insurance plans, including Medicare, cover TPMT testing, but reimbursement and copayments vary, depending on the patient’s specific plan.

There are conflicting data as to whether determining TPMT status is^11,14–18 or is not¹⁹ cost-effective. Multiple studies suggest that the cost of genotyping a sufficient number of patients to identify a single individual at high risk of myelosuppression is cheaper than the costs associated with treating an adverse event. Additional cost-benefit studies are needed, particularly studies that consider how bundled payments and outcomes-based reimbursement influence cost-effectiveness.

MODIFYING THIOPURINE THERAPY BASED ON TPMT ACTIVITY

There is a strong correlation between TPMT activity and tolerated thiopurine doses, with those having intermediate or low TPMT activity requiring lower doses.^10,20–23 Adjusting mercaptopurine doses based on TPMT activity to prevent hematopoietic toxicity has been successfully demonstrated in pediatric patients with acute lymphoblastic leukemia.²⁴ Furthermore, reducing initial thiopurine doses to avoid myelosuppression and titrating based on response has been shown to not compromise outcomes.^1,25,26 The Clinical Pharmacogenetic Implementation Consortium (CPIC) has developed an evidence-based guideline on how to adjust thiopurine doses based on TPMT activity,¹ summarized in Table 2. These dosing recommendations are classified as “strong.”

Patients with normal TPMT activity should be prescribed the usual thiopurine starting dose as indicated by disease-specific guidelines.

For those with intermediate TPMT activity, the CPIC guideline recommends reducing the initial targeted full dose of azathioprine and mercaptopurine by 30% to 70% and reducing the targeted full dose of thioguanine by 30% to 50%. The percentage of dose reduction depends on the targeted full dose. Siegel and Sands²⁷ suggested that for those who are diagnosed with inflammatory bowel disease and have intermediate TPMT activity, azathioprine should be initiated at a low dose and titrated to 1.25 mg/kg and mercaptopurine should be initiated at a low dose and titrated to 0.75 mg/kg. Based on these titration goals, if the targeted full dose for mercaptopurine is 1 mg/kg, then a dose reduction of approximately 30% would be more appropriate. If the targeted full dose is 1.5 mg/kg, a dose reduction of approximately 50% would be more appropriate. Thiopurine doses should be titrated based on response and disease-specific guidelines, allowing 2 to 4 weeks to reach steady state before dose titration.

For those with low TPMT activity, alternative therapy should be considered for nonmalignant conditions because of the risk of severe myelosuppression. For malignancy, or if a thiopurine is warranted for a nonmalignant condition, consider a 90% dose reduction and give the drug three times per week instead of daily. For example, acute lymphoblastic leukemia patients with low TPMT activity can be started on mercaptopurine 10 mg/m² three times per week instead of the usual starting dose.¹⁰ Thiopurine doses should be titrated based on response and disease-specific guidelines, allowing 4 to 6 weeks to reach steady state before dose titration.

RECOMMENDATIONS

Individuals with intermediate or low TPMT activity have an increased risk of myelosuppression. Because of the elevated risk for morbidity and death, especially for patients with low TPMT activity, multiple guidelines and regulatory agencies recommend TPMT genotyping or phenotyping if a thiopurine is prescribed.^25,28–32 Although additional cost-benefit analysis studies are needed, evidence suggests testing for TPMT activity may be cheaper than the costs associated with treating myelosuppression.

In view of treatment guidelines, the recommendations of regulatory agencies, cost-benefit analyses, and the availability of gene-based dosing recommendations, we consider the benefits of testing for TPMT activity to greatly outweigh any associated risks. Therefore, we recommend that TPMT testing be strongly considered before initiating therapy with a thiopurine.

To the Editor: We previously addressed whether VKORC1 and CYP2C9 pharmacogenomic testing should be considered when prescribing warfarin.¹ Our recommendation, based on available evidence at that time, was that physicians should consider pharmacogenomic testing for any patient who is started on warfarin therapy.

Since the publication of this recommendation, two major trials, COAG (Clarification of Optimal Anticoagulation Through Genetics)² and EU-PACT (European Pharmacogenetics of Anticoagulant Therapy-Warfarin),³ were published along with commentaries debating the clinical utility of warfarin pharmacogenomics.^4–15 Based on these publications, we would like to update our recommendations for pharmacogenomic testing for warfarin therapy.

COAG compared the efficacy of a clinical algorithm or a clinical algorithm plus VKORC1 and CYP2C9 genotyping to guide warfarin dosage. At the end of 4 weeks, the mean percentage of time within the therapeutic international normalized ratio (INR) range was 45.4% for those in the clinical algorithm arm and 45.2% for those in the genotyping arm (95% confidence interval [CI] –3.4 to 3.1, P = .91). For both treatment groups, clinical data that included body surface area, age, target INR, concomitantly prescribed drugs, and smoking status were used to predict warfarin dose, with the genotyping arm including VKORC1 and CYP2C9. Although VKORC1 and CYP2C9 genotyping offered no additional benefit, caution should be used when extrapolating this conclusion to clinical settings in which warfarin therapy is initiated using a standardized starting dose (eg, 5 mg daily) instead of a clinical dosing algorithm.

Of interest, in the COAG trial, among black patients, the mean percentage of time in the therapeutic INR range was significantly less for those in the genotype-guided arm than for those in the clinically guided arm—ie, 35.2% vs 43.5% (95% CI –15.0 to –2.0, P = .01). The percentage of time with therapeutic INR has been identified as a surrogate marker for poor outcomes such as death, stroke, or major hemorrhage, with those with a lower percentage of time in therapeutic INR being at greater risk of an adverse event.¹⁶ Wan et al¹⁷ demonstrated that a 6.9% improvement of time in therapeutic INR decreased the risk of major hemorrhage by one event per 100 patient-years.¹⁷ Therefore, black patients in the COAG genotyping arm may have been at greater risk for an adverse event because of a lower observed percentage of time within the therapeutic INR range.

In the COAG trial, genotyping was done for only one VKORC1 variant and for two CYP2C9 alleles (CYP2C9*2, and CYP2C9*3). Other genetic variants are of clinical importance for warfarin dosing in black patients, and the lack of genotyping for these additional variants may explain why black patients in the genotyping arm performed worse.^5,7,11 In particular, CYP2C9*8 may be an important predictor of warfarin dose in black patients.¹⁸

EU-PACT compared the efficacy of standardized warfarin dosing and that of a clinical algorithm.³ Patients in the standardized dosing arm were prescribed warfarin 10 mg on the first day of treatment (5 mg for those over age 75), and 5 mg on days 2 and 3, with subsequent dosing adjustments based on INR. Patients in the genotyping arm were prescribed warfarin based on an algorithm that incorporated clinical data that included body surface area, age, and concomitantly prescribed drugs, as well as VKORC1 and CYP2C9 genotypes. At the end of 12 weeks, the mean percentage of time in the therapeutic INR range was 60.3% for those in the standardized-dosing arm and 67.4% for those in the genotyping arm (95% CI 3.3 to 10.6, P < .001).² The approximate 7% improvement in percentage of time in the therapeutic INR range may predict a lower risk of hemorrhage for those in the genotyping arm.¹⁷ Although patients in the genotyping arm had a higher percentage of time in the therapeutic INR range, it is unclear whether genotyping alone is superior to standardized dosing because the dosing algorithm used both clinical data and genotype data.

There are substantial differences between the COAG and EU-PACT trials, including dosing schemes, racial diversity, and trial length, and these differences could have contributed to the conflicting results. Based on these two trials, a possible conclusion is that genotype-guided warfarin dosing may be superior to standardized dosing, but may be no better than utilizing a clinical algorithm in white patients. For black patients, additional studies are needed to determine which genetic variants are of importance for guiding warfarin dosing.

We would like to update the recommendations we made in our previously published article,¹ to state that genotyping for CYP2C9 and VKORC1 may be of clinical utility in white patients depending on whether standardized dosing or a clinical algorithm is used to initiate warfarin therapy. Routine genotyping in black patients is not recommended until further studies clarify which genetic variants are of importance for guiding warfarin dosing.

The ongoing Genetics Informatics Trial of Warfarin to Prevent Venous Thrombosis may bring much needed clarity to the clinical utility of warfarin pharmacogenomics. We hope to publish a more detailed update of our 2013 article after completion of that trial.

To the Editor: We previously addressed whether VKORC1 and CYP2C9 pharmacogenomic testing should be considered when prescribing warfarin.¹ Our recommendation, based on available evidence at that time, was that physicians should consider pharmacogenomic testing for any patient who is started on warfarin therapy.

Since the publication of this recommendation, two major trials, COAG (Clarification of Optimal Anticoagulation Through Genetics)² and EU-PACT (European Pharmacogenetics of Anticoagulant Therapy-Warfarin),³ were published along with commentaries debating the clinical utility of warfarin pharmacogenomics.^4–15 Based on these publications, we would like to update our recommendations for pharmacogenomic testing for warfarin therapy.

COAG compared the efficacy of a clinical algorithm or a clinical algorithm plus VKORC1 and CYP2C9 genotyping to guide warfarin dosage. At the end of 4 weeks, the mean percentage of time within the therapeutic international normalized ratio (INR) range was 45.4% for those in the clinical algorithm arm and 45.2% for those in the genotyping arm (95% confidence interval [CI] –3.4 to 3.1, P = .91). For both treatment groups, clinical data that included body surface area, age, target INR, concomitantly prescribed drugs, and smoking status were used to predict warfarin dose, with the genotyping arm including VKORC1 and CYP2C9. Although VKORC1 and CYP2C9 genotyping offered no additional benefit, caution should be used when extrapolating this conclusion to clinical settings in which warfarin therapy is initiated using a standardized starting dose (eg, 5 mg daily) instead of a clinical dosing algorithm.

Of interest, in the COAG trial, among black patients, the mean percentage of time in the therapeutic INR range was significantly less for those in the genotype-guided arm than for those in the clinically guided arm—ie, 35.2% vs 43.5% (95% CI –15.0 to –2.0, P = .01). The percentage of time with therapeutic INR has been identified as a surrogate marker for poor outcomes such as death, stroke, or major hemorrhage, with those with a lower percentage of time in therapeutic INR being at greater risk of an adverse event.¹⁶ Wan et al¹⁷ demonstrated that a 6.9% improvement of time in therapeutic INR decreased the risk of major hemorrhage by one event per 100 patient-years.¹⁷ Therefore, black patients in the COAG genotyping arm may have been at greater risk for an adverse event because of a lower observed percentage of time within the therapeutic INR range.

In the COAG trial, genotyping was done for only one VKORC1 variant and for two CYP2C9 alleles (CYP2C9*2, and CYP2C9*3). Other genetic variants are of clinical importance for warfarin dosing in black patients, and the lack of genotyping for these additional variants may explain why black patients in the genotyping arm performed worse.^5,7,11 In particular, CYP2C9*8 may be an important predictor of warfarin dose in black patients.¹⁸

EU-PACT compared the efficacy of standardized warfarin dosing and that of a clinical algorithm.³ Patients in the standardized dosing arm were prescribed warfarin 10 mg on the first day of treatment (5 mg for those over age 75), and 5 mg on days 2 and 3, with subsequent dosing adjustments based on INR. Patients in the genotyping arm were prescribed warfarin based on an algorithm that incorporated clinical data that included body surface area, age, and concomitantly prescribed drugs, as well as VKORC1 and CYP2C9 genotypes. At the end of 12 weeks, the mean percentage of time in the therapeutic INR range was 60.3% for those in the standardized-dosing arm and 67.4% for those in the genotyping arm (95% CI 3.3 to 10.6, P < .001).² The approximate 7% improvement in percentage of time in the therapeutic INR range may predict a lower risk of hemorrhage for those in the genotyping arm.¹⁷ Although patients in the genotyping arm had a higher percentage of time in the therapeutic INR range, it is unclear whether genotyping alone is superior to standardized dosing because the dosing algorithm used both clinical data and genotype data.

There are substantial differences between the COAG and EU-PACT trials, including dosing schemes, racial diversity, and trial length, and these differences could have contributed to the conflicting results. Based on these two trials, a possible conclusion is that genotype-guided warfarin dosing may be superior to standardized dosing, but may be no better than utilizing a clinical algorithm in white patients. For black patients, additional studies are needed to determine which genetic variants are of importance for guiding warfarin dosing.

We would like to update the recommendations we made in our previously published article,¹ to state that genotyping for CYP2C9 and VKORC1 may be of clinical utility in white patients depending on whether standardized dosing or a clinical algorithm is used to initiate warfarin therapy. Routine genotyping in black patients is not recommended until further studies clarify which genetic variants are of importance for guiding warfarin dosing.

The ongoing Genetics Informatics Trial of Warfarin to Prevent Venous Thrombosis may bring much needed clarity to the clinical utility of warfarin pharmacogenomics. We hope to publish a more detailed update of our 2013 article after completion of that trial.

To the Editor: We previously addressed whether VKORC1 and CYP2C9 pharmacogenomic testing should be considered when prescribing warfarin.¹ Our recommendation, based on available evidence at that time, was that physicians should consider pharmacogenomic testing for any patient who is started on warfarin therapy.

Since the publication of this recommendation, two major trials, COAG (Clarification of Optimal Anticoagulation Through Genetics)² and EU-PACT (European Pharmacogenetics of Anticoagulant Therapy-Warfarin),³ were published along with commentaries debating the clinical utility of warfarin pharmacogenomics.^4–15 Based on these publications, we would like to update our recommendations for pharmacogenomic testing for warfarin therapy.

COAG compared the efficacy of a clinical algorithm or a clinical algorithm plus VKORC1 and CYP2C9 genotyping to guide warfarin dosage. At the end of 4 weeks, the mean percentage of time within the therapeutic international normalized ratio (INR) range was 45.4% for those in the clinical algorithm arm and 45.2% for those in the genotyping arm (95% confidence interval [CI] –3.4 to 3.1, P = .91). For both treatment groups, clinical data that included body surface area, age, target INR, concomitantly prescribed drugs, and smoking status were used to predict warfarin dose, with the genotyping arm including VKORC1 and CYP2C9. Although VKORC1 and CYP2C9 genotyping offered no additional benefit, caution should be used when extrapolating this conclusion to clinical settings in which warfarin therapy is initiated using a standardized starting dose (eg, 5 mg daily) instead of a clinical dosing algorithm.

Of interest, in the COAG trial, among black patients, the mean percentage of time in the therapeutic INR range was significantly less for those in the genotype-guided arm than for those in the clinically guided arm—ie, 35.2% vs 43.5% (95% CI –15.0 to –2.0, P = .01). The percentage of time with therapeutic INR has been identified as a surrogate marker for poor outcomes such as death, stroke, or major hemorrhage, with those with a lower percentage of time in therapeutic INR being at greater risk of an adverse event.¹⁶ Wan et al¹⁷ demonstrated that a 6.9% improvement of time in therapeutic INR decreased the risk of major hemorrhage by one event per 100 patient-years.¹⁷ Therefore, black patients in the COAG genotyping arm may have been at greater risk for an adverse event because of a lower observed percentage of time within the therapeutic INR range.

In the COAG trial, genotyping was done for only one VKORC1 variant and for two CYP2C9 alleles (CYP2C9*2, and CYP2C9*3). Other genetic variants are of clinical importance for warfarin dosing in black patients, and the lack of genotyping for these additional variants may explain why black patients in the genotyping arm performed worse.^5,7,11 In particular, CYP2C9*8 may be an important predictor of warfarin dose in black patients.¹⁸

EU-PACT compared the efficacy of standardized warfarin dosing and that of a clinical algorithm.³ Patients in the standardized dosing arm were prescribed warfarin 10 mg on the first day of treatment (5 mg for those over age 75), and 5 mg on days 2 and 3, with subsequent dosing adjustments based on INR. Patients in the genotyping arm were prescribed warfarin based on an algorithm that incorporated clinical data that included body surface area, age, and concomitantly prescribed drugs, as well as VKORC1 and CYP2C9 genotypes. At the end of 12 weeks, the mean percentage of time in the therapeutic INR range was 60.3% for those in the standardized-dosing arm and 67.4% for those in the genotyping arm (95% CI 3.3 to 10.6, P < .001).² The approximate 7% improvement in percentage of time in the therapeutic INR range may predict a lower risk of hemorrhage for those in the genotyping arm.¹⁷ Although patients in the genotyping arm had a higher percentage of time in the therapeutic INR range, it is unclear whether genotyping alone is superior to standardized dosing because the dosing algorithm used both clinical data and genotype data.

There are substantial differences between the COAG and EU-PACT trials, including dosing schemes, racial diversity, and trial length, and these differences could have contributed to the conflicting results. Based on these two trials, a possible conclusion is that genotype-guided warfarin dosing may be superior to standardized dosing, but may be no better than utilizing a clinical algorithm in white patients. For black patients, additional studies are needed to determine which genetic variants are of importance for guiding warfarin dosing.

We would like to update the recommendations we made in our previously published article,¹ to state that genotyping for CYP2C9 and VKORC1 may be of clinical utility in white patients depending on whether standardized dosing or a clinical algorithm is used to initiate warfarin therapy. Routine genotyping in black patients is not recommended until further studies clarify which genetic variants are of importance for guiding warfarin dosing.

The ongoing Genetics Informatics Trial of Warfarin to Prevent Venous Thrombosis may bring much needed clarity to the clinical utility of warfarin pharmacogenomics. We hope to publish a more detailed update of our 2013 article after completion of that trial.