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Research Article
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Are patients with intermediate TPMT activity at
increased risk of myelosuppression when taking
thiopurine medications?
Thiopurine S-methyltransferase (TPMT) metabolizes thiopurine medications, including azathioprine and
6-mercaptopurine. Absent TPMT activity (i.e., in individuals homozygous for a variant TPMT allele) is
associated with an increased risk of myelosuppression in patients taking thiopurine drugs. However, it is
not clear if there is also an increased risk for patients with intermediate TPMT activity (i.e., in individuals
heterozygous for a variant TPMT allele). Aims: To quantify the increased risk of myelosuppression for
patients with intermediate TPMT activity. Materials & methods: A systematic review identified published
studies, up to 29 September 2008, that explored the relationship between TMPT and hematological adverse
drug reactions to thiopurines. Following a critical appraisal of the quality of published studies, a metaana­lysis calculated the odds ratio of myelosuppression for patients with intermediate TPMT activity
compared with wild-type. Results: A total of 67 studies were identified, the majority retrospective cohort
in design. Patients with two TPMT variant alleles who are TPMT deficient have a substantial increase in
their risk of myelotoxicity (86% of deficient patients developed myelosuppression). The increase in odds
ratio of developing leukopenia for patients with intermediate TPMT activity or one TPMT variant allele
compared with wild-type was 4.19 (95% CI: 3.20–5.48). Conclusion: This meta-ana­lysis suggests that
individuals with both intermediate and absent TPMT activity have an increased risk of developing
thiopurine-induced myelosuppression, compared with individuals with normal activity. However, there is
significant variability in the quality of the reported studies and large prospective studies to clarify the size
of the effect of TPMT variant alleles on the risk of myelosuppression should be conducted. Accurate risk
assessments will provide important data to inform clinical guidelines.
KEYWORDS: adverse drug reaction n azathioprine n myelosuppression
n pharmacogenetics n pharmacogenomics n thiopurine n TPMT
Deficiency of the drug-metabolism enzyme thiopurine S-methyltransferase (TPMT) was first
described nearly 30 years ago [1] . Approximately
one in 300 individuals were found to be deficient
in the enzyme, with a further one in ten having
intermediate activity. Subsequent studies established a close correlation between red blood cell
TPMT activity and 6-thioguanine nucleotide
levels in patients treated with thiopurine medications [2] . Nearly 10 years after its initial description, a relationship between absent TPMT activity and thiopurine-induced myelotoxicity was
established [3] . Numerous case reports supported
the association between profound neutropenia
and TPMT deficiency in individuals treated
with standard doses of thiopurine drugs [4–6] .
Although these were only isolated reports, the
use of azathioprine and 6-mercaptopurine to
treat a growing number of conditions, including inflammatory bowel disease, rheumatoid
arthritis, systemic lupus, immunobullous disease, generalized eczematous disorders and
acute lymphoblastic leukemia, meant that not
identifying TPMT deficiency could have wide
implications. TPMT deficiency is generally
associated with early and more severe myelo­
toxicity [7] . Conversely, high TPMT activity
may be associated with a poor response to treatment, with patients requiring higher doses to
achieve a therapeutic effect [8] .
Thiopurine S-methyltransferase activity can
be analyzed either through an enzyme assay
(conversion of 6-mercaptopurine to radiolabeled
6-methyl mercaptopurine in red cell lysate) or
by ana­lysis of common genetic polymorphisms
(e.g., TPMT*2, TPMT*3A or TPMT*3C ).
Measurement of TPMT activity using either
the phenotypic or genetic test was adopted by
some clinicians prior to azathioprine use [9,10] .
Uptake of TPMT testing in the UK and across
Europe was initially slow, as reservations
regarding sensitivity and specificity, cost, test
availability, turnaround time and results interpretation outweighed the evidence of clinical
utility [11,12] . TPMT deficiency only accounts
for a quarter of all cases of myelosuppression on
thiopurine treatment [13] , and a number of other
adverse reactions associated with thiopurine use,
10.2217/PGS.09.155 © 2010 Future Medicine Ltd
Pharmacogenomics (2010) 11(2), 177–188
Jenny E Higgs1,
Katherine Payne2,
Chris Roberts2 &
William G Newman1,3†
Author for correspondence:
Central Manchester
& Manchester Children’s
University Hospitals NHS Trust,
Manchester Royal Infirmary,
Manchester, UK
2
Health Methodology Research
Group, The University of
Manchester, Manchester, UK
3
Department of Medical
Genetics, St Mary’s Hospital,
Manchester Academic Health
Sciences Centre (MAHSC),
The University of Manchester,
Manchester, M13 9WL, UK
Tel.: +44 161 276 6264
Fax: 44 161 276 6145
william.newman@
manchester.ac.uk
†
1
part of
ISSN 1462-2416
177
Research Article
Higgs, Payne, Roberts & Newman
including allergic reactions, hepato­toxicity,
pancreatitis, nausea and vomiting, cannot be
predicted by TPMT testing.
A review has been carried out defining the risk
of myelosuppression for patients on thiopurine
medication for inflammatory bowel disease [7] .
This review does not address whether there is
an increased risk of myelosuppression associated
with intermediate TPMT enzyme activity. An
early study by Black et al. identified that five
out of six individuals who stopped azathioprine
owing to leukopenia were heterozygous for a
TPMT variant [14] . Other studies have also indicated an increased risk in individuals with intermediate TPMT activity [15,16] , which is counterbalanced by increased efficacy in this subset [15] .
Guidelines drawn up by the British Society of
Gastroenterology regarding the use of azathioprine
and the related monitoring of patients have not
necessarily advocated TPMT testing [17] . However,
recent guidelines in rheumato­logy and dermatology have been more forthright in recommending TPMT testing as part of the management of
patients who are due to commence azathioprine
therapy [18,19] . This has mirrored an exponential
increase in the uptake of TPMT testing in the
UK [12] . These recommendations were made
based on “evidence obtained from expert committee reports or opinions, and/or clinical experience of respected authorities… [with] an absence
of directly applicable studies of good quality” [18] .
Although numerous small studies have been
carried out testing TPMT activity and/or genotype, the effect of intermediate TPMT activity on
thiopurine-induced myelo­suppression is uncertain. There is an unmet need for a systematic
review of the risks of myelosuppression associated
with intermediate TPMT activity, and we present
a meta-ana­lysis of the published literature.
Materials & methods
The primary aim of this systematic review was to
identify all studies that observed hematological
adverse reactions to a thiopurine medication and
tested for TPMT activity or genotype. This was
to allow assessment of the quality of the studies and their contribution to the evidence base
for TPMT testing. The secondary aim was to
quantify the level of any increased risk of myelosuppression as a result of intermediate TPMT
activity. The systematic review was conducted by
following the guidelines described by Cochrane
to identify published studies [101] . The focus of
the review was to explore the relationship between
TPMT and hematological adverse drug reactions
to azathioprine in published literature. For the
178
Pharmacogenomics (2010) 11(2)
purpose of this review, the definitions shown
in Table 1 were adopted for the key adverse drug
reactions and descriptors of the level of TPMT
activity. It should be noted that the studies used
different methods for testing TPMT activity and,
therefore, reported their results in different units.
„„
Search strategy
Searches strategies were designed for OVID
MEDLINE, EMBASE, Cumulative Index to
Nursing and Allied Health Literature (CINAHL),
The Cochrane library, Web of Knowledge, and
PsycINFO using appropriate MeSH headings
(Online Supplementary Appendix 1; www.futuremedicine.
com/doi/suppl/10.2217/pgs.09.155/suppl_file/
suppl_material.xls). The search was run from
1978 to 29 September 2008. This start year was
selected because this was the year that the TPMT
enzyme was identified [19] . The key words in the
search strategy included terms relevant to TPMT
and thiopurines, for example, ‘azathioprine’ or
‘6-mercaptopurine’, and hematological events.
In addition, references in review articles and the
bibliographies of identified studies were examined
for further potentially relevant studies. Although
reviews were not included in the systematic review,
their references were checked for relevant studies.
„„
Study selection
Abstracts were checked for relevance to the topic
of the review against predefined inclusion and
exclusion criteria independently by two reviewers (JE Higgs and WG Newman). Disagreements
were resolved by discussion with a third party, if
necessary. Full text articles of the relevant abstracts
were then obtained and assessed for relevance by
two reviewers (JE Higgs and WG Newman).
„„
Inclusion criteria
The following inclusion criteria were used for this
review: all primary studies of patients on a thiopurine medication that tested for TPMT, either
the genotype or phenotype, and reported cases
of hematological adverse reactions. The search
was not limited to a specific disease or condition.
Expert opinion supported by a preliminary literature review indicated that there was likely to be
very few randomized, controlled trials (RCTs) on
this topic; therefore, any study design was included
in the review.
A second set of inclusion criteria were then used
to assess whether the identified studies would be
included in the subsequent meta-ana­lysis. Two criteria were used: availability of data on the number of cases of myelosuppression for each level of
TPMT activity; and adequate study quality.
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TPMT activity & myelosuppression when taking thiopurine medications
Research Article
Table 1. Hematological definitions.
Hematological term
Definition
Myelosuppression/myelotoxicity/
bone marrow suppression
Myelotoxicity*
Severe myelotoxicity*
A decrease in bone marrow activity
Leukopenia
Neutropenia‡
Thrombocytopenia§
TPMT activity definitions¶
High/normal TPMT activity
Intermediate TPMT activity
Low TPMT activity
Mild leukopenia: leukocyte count of between 3 and 4 × 109 /l
Severe leukopenia: leukocyte count of <2.5–2 × 109 /l or
leukopenia requiring hospitalization
A decrease in the number of white blood cells (leukocytes)
Definitions in the literature reviewed ranged from a leukocyte
count of <1x109 /l to <4 × 109 /l
A decrease in the number of neutrophils, defined in the literature
reviewed as a neutrophil count of <1 × 109 /l to <2.5 × 109 /l
A decrease in the number of platelets, defined as <100 × 109 /l
>13.7 units/mlRBC
Between 5 and 13.7 units/mlRBC
<5 units/mlRBC
*Definitions from [7].
‡
Reported by eight studies [24,25,27,29,30,73,74,75].
§
Reported by two studies [25,76].
¶
There was variation in the units used. The most commonly used range is shown [26,29,31,32,77,78].
RBC: Red blood cell; TMPT: Thiopurine S-methyltransferase.
„„
Exclusion criteria
Studies were excluded if they did not test TPMT
genotype or phenotype (e.g., TPMT metabolite
monitoring) or did not report hematological
adverse reactions. This study focused on primary
studies and excluded all reviews. Case studies were
excluded as they provided limited new data. Studies
published in only abstract form were excluded as
they did not provide enough information regarding results and study quality. Non‑English and
nonhuman articles were excluded.
A study was excluded from the meta-ana­lysis if
the sample included only patients that had been
selected for TPMT testing because they had experienced a hematological adverse drug reaction.
This is because it is not possible to calculate an
odds ratio of myelosuppression using data from
these studies.
„„
Quality-assessment strategy
All studies were quality assessed using published guidelines specifically designed to assess
the quality of pharmacogenetic studies ( Online
Supplementary Table 1; www.futuremedicine.com/
doi/suppl/10.2217/pgs.09.155/suppl_file/suppl_
material.xls) [20] .
„„
Data-extraction strategy
The key purpose of the data-extraction strategy
was to summarize the studies in terms of their
design. To conduct the meta-ana­lysis and calculate the odds ratio for the risk of myelo­suppression
in TPMT heterozygotes or intermediate activity, the following data were extracted; number
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of patients that were wild-type for TPMT with
and without a hematological event; and TPMT
heterozygous or intermediate activity with and
without a hematological event. If this information was not reported explicitly in the study, it
was excluded from the meta-ana­lysis.
„„
Data ana­lysis
The data from the systematic review and the
assessment of study quality were reported as a
narrative review. The number of patients with
and without TPMT heterozygosity and/or intermediate activity and with and without myelosuppression was tabulated (Online Supplementary Table 2;
www.futuremedicine.com/doi/suppl/10.2217/
pgs.09.155/suppl_file/suppl_material.xls). In
some categories, there were no patients, and these
were labeled ‘zero-cells’. A meta-ana­lysis using the
Mantel Haenszel (MH) pooled odds ratio without
the zero-cell correction was used to quantify the
risk of myelosuppression in TPMT heterozygotes
or intermediate activity [21,22] .
Two sensitivity analyses were carried out to
compare estimates of the odds ratio by including
and then excluding zero-cells and study design,
classified as case–control prospective cohort and
retrospective cohort.
Between-study heterogeneity was investigated
by estimation of the I2 statistic, the betweenstudy variance t2, and a c2 test of heterogeneity.
The hetero­geneity statistic I2 is interpreted as the
percentage of variability due to heterogeneity
between studies rather than sampling error. High
I2 values indicate increasing heterogeneity. Rucker
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179
Research Article
Higgs, Payne, Roberts & Newman
et al. show that I2 increases with the number of
patients included in the meta-ana­lysis and suggest using estimates of heterogeneity variance t2 ;
therefore, t2 was calculated in addition to I2 [23] .
A number needed to test (NNT) was estimated from the pooled odds ratio and the proportion of subjects in the control group suffering an event [102] . This was assumed to be
7% using data from the current study ( Online
Supplementary Table 2; www.futuremedicine.com/
doi/suppl/10.2217/pgs.09.155/suppl_file/suppl_
material.xls) and Gisbert and Gomollon [7] .
Results
„„
Study selection
The search strategy identified 725 studies. 67 studies were selected for inclusion in the systematic
review. Figure 1 summarizes reasons for exclusion
from the systematic review. The list of excluded
studies is available from the corresponding author
on request. A further two studies were identified
by searching the references of review articles [24,25],
which were e-publications ahead of print and,
therefore, not in the OVID/EMBASE databases
at the time of searching (29 September 2008).
O nline Supplementary Tables 3 & 4 & R eference L ist
(www.futuremedicine.com/doi/suppl/10.2217/
pgs.09.155/suppl_file/suppl_material.xls)�����
summarize the main characteristics of the studies included. Sample sizes ranged from 19
to 667 patients. The systematic review and
meta-ana­lysis included two case–control trials, 11 cohort prospective studies and 34 cohort
retrospective studies. Included in the systematic review, but not the meta-ana­lysis were
two RCTs, two case–control trials, five cohort
prospective studies and 11 cohort retrospective
studies. The follow-up time of the studies varied from 4 weeks to 14 years (mean: 2.5 years;
median: 6 months). Thiopurine drugs and doses
varied between and within studies. Azathioprine
was used in the majority of studies (n = 52), and
6-mercaptopurine was used in 15 studies.
There was a consistent relationship between
very low or absent TPMT activity or carriage of
two TPMT variant alleles and myelosuppression.
Of the 43 patients identified with two TPMT
variant alleles or low/absent TPMT activity
(Online Supplementary Table 4; www.futuremedicine.
com/doi/suppl/10.2217/pgs.09.155/suppl_file/
suppl_material.xls), 37 (86%) patients developed
severe myelosuppression.
Most studies had sample sizes of fewer than
100 patients. The two largest prospective studies,
with sample sizes of 394 [26] and 207 [24] , both
concluded that intermediate TPMT activity was
180
Pharmacogenomics (2010) 11(2)
a risk factor for myelosuppression. Gisbert et al.
reported that inflammatory bowel disease patients
with intermediate TPMT activity had four-times
more chance of developing myelosuppression
than individuals with normal TPMT activity [26] .
Ansari et al. followed 207 inflammatory bowel
disease patients, who were started on 2 mg/kg of
azathioprine, for 6 months [24] . They reported that
TPMT heterozygotes were more likely to suffer
from gastric intolerance and myelosuppression
than wild-type patients.
In the 67 studies, 655 cases of myelosuppression
were reported. It was possible to assign a phenotype or genotype to 492 of these; 37 (7.5%) were
TPMT variant homozygotes, compound heterozygotes or had low TPMT activity; 118 (24%)
were TPMT variant heterozygotes or had intermediate activity; and 337 (68.5%) were wild-type
or had high/normal TPMT activity. A number
of studies specifically selected myelosuppressed
patients, which explains the higher frequencies
of low and intermediate TPMT activity patients
than reported in population studies [1] . These
studies were excluded from the meta-ana­lysis but
not the systematic review. Importantly, different
ranges of white cell counts were used to define
leukopenia, varying from 1 × 109/l to less than
4 × 109/l (Table 1) , while 17 (25%) studies did not
define leukopenia.
Two RCTs have been carried out that measured
TPMT and based the dose of azathioprine on the
TPMT activity. However, Meggitt et al. compared
azathioprine-adjusted doses with no azathioprine; therefore, it is not possible to determine
if reducing the dose of azathioprine for patients
with intermediate TPMT activity reduces the
risk of myelosuppression [27] . The other RCT,
while it did compare patients on standard doses
and doses based on TPMT activity, had a sample
size of only 29, and only one case of myelosuppression occurred [28] . Neither study could be
used in the meta-ana­lysis: Meggitt et al. did not
provide the phenotypes for each patient [27] , and
no inter­mediate-activity patients were present in
the trial by Sayani et al. [28] .
„„
Meta-ana­lysis
A total of 47 studies were used in the metaanalysis [14,24,25,27,29–72] . Of the 20 studies
excluded from the meta-ana­lysis, this was generally because it was not possible to combine
the data in a meta-ana­lysis, as either all patients
in the study were myelosuppressed, or the raw
data required to calculate an odds ratio was not
provided [3,13,15,16,26–28,31,73–84] . No studies were
excluded because of poor methodological quality.
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TPMT activity & myelosuppression when taking thiopurine medications
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Number of potentially relevant
articles identified: 1122 from
electronic search
Duplicates: 397
Number of potentially relevant
articles after removing
duplicates: 725
Number of articles remaining:
103
Number of articles excluded: 622
Not relevant to TPMT testing: 69
No comparison of TPMT test result with hematological
adverse drug reactiosn to thiopurine treatment: 127
Not in English: 7
Nonhuman: 15
Review article, case report, letter, editorial or not
full text: 404
Full text retrieved
Identified through
searching
bibliographies: 2
Number of articles remaining:
65
Number of articles excluded: 38
No TPMT testing: 6
No comparison of TPMT test result with hematological
adverse drug reactions to thiopurine treatment: 24
Same patients as in a previous study: 4
Not in English: 2
Comment/editorial material, case study: 2
Total articles for systematic
review: 67
Number of articles excluded from meta-analysis: 20
All patients in the study myelosuppressed: 2
Data required to calculate an odds ratio not provided: 18
Total articles for
meta-analysis: 47
Figure 1. Study selection.
TPMT: Thiopurine S-methyltransferase.
Two (4%) of the 47 studies included in
the meta-ana­lysis were case–control designs
(150 patients), 11 (23%) studies were prospective
cohort studies (834 patients), and the remaining 34 studies (72%) were retrospective cohorts
(3322 patients). Ten studies had no cases of myelosuppression among TPMT variant hetero­zygotes/
intermediate-activity subjects. In all studies, some
wild-type subjects were event free (i.e., there were
no myelosuppression cases). Sensitivity ana­lysis
demonstrated that the odds ratio is similar for
each study design, whether zero-cells are included
or not (Table 2) . The summary odds ratio for leukopenia in intermediate TPMT activity or hetero­
zygote TPMT variant allele patients compared
with wild-type was 4.19 (95% CI: 3.20–5.48). A
c2 test of heterogeneity suggested there was heterogeneity between studies (p = 0.001) and the I2
was 44.2%, which also indicated that the studies
were heterogeneous. Pooling studies with an I2 of
less than 50% has been suggested as acceptable,
as values of greater than 50% indicate substantial
heterogeneity [101] . The magnitude of I2 statistic
may be inflated by the large number of patients
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included in this meta-ana­lysis (n = 4306) [23] . The
number of studies that cross one, indicating no
statistically significant difference, on the Forest
plot is 27 out of 47. However, most of the studies
show an effect in the same direction (Figure 2) .
Gisbert and Gomollon carried out a metaana­lysis that estimated the incidence of myelo­
suppression to be 7% [7] . The data from the
current study also estimate the frequency of
myelosuppression in patients with normal TPMT
activity to be 7% ( Online Supplementary Table 2;
www.futuremedicine.com/doi/suppl/10.2217/
pgs.09.155/suppl_file/suppl_material.xls). If 7%
is used as the baseline risk in order to calculate the
NNT to detect one patient at risk of leukopenia,
the NNT is six.
„„
Quality assessment
Quality-assessment tables are shown in full in
Online Supplementary Tables 1, 5–7 (���������������
www.futuremedicine.com/doi/suppl/10.2217/pgs.09.155/suppl_
file/suppl_material.xls). The majority of studies
described their aims, outcomes and interventions,
as well as patient characteristics.
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Higgs, Payne, Roberts & Newman
Table 2. Sensitivity analyses.
Study design
Case–control
Prospective cohort studies
Prospective cohort studies*
Retrospective cohort studies
Retrospective cohort studies*
Overall
Mantel Haenszel
odds ratio
95% CI
13.27
4.30
3.37
4.07
3.99
4.19
1.64–107.69
2.53–7.29
1.90–6.01
2.97–5.58
2.73–5.82
3.20–5.48
p-value Studies
(n)
Variance t
I2 (%)
p-value
0.016
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
1.48
0.94
0.91
0.64
0.95
56.4
48.6
43.1
43.2
44.2
0.011
0.049
0.005
0.024
0.001
2
11
9
34
19
47
Heterogeneity
2
*Excluding studies with a zero cell.
The recruited patients and study sites were
representative of the patient population and
normal treatment facilities. Randomization
and blinding of patients was carried out by the
two RCTs only, although more studies blinded
assessors (14 out of 67, 21%). Compliance was
considered adequate by all studies, although only
two studies formally measured compliance [74,53] .
The reporting of results was variable, and
30 out of 67 (45%) studies did not give their full
results and used averages or gave statistics without
presenting raw data. When used, p-values were
often rounded up (17 out of 57 cases [30%]),
rather than reported as absolute values.
The quality of the pharmacogenetic study
design was assessed using the Jorgensen and
Williamson criteria [20] . The reporting of methods and Hardy–Weinberg equilibrium (HWE)
could be improved. The lack of power calculations (45 out of 67 [67%] cases) reflects that the
majority of retrospective, cohort studies did not
perform a power calculation.
Most studies that performed a genetic test
(n = 48) carried out a literature review (81%)
that included reasons for choosing the SNPs
genotyped (30 out of 48 [63%]). In all cases,
they selected the common SNPs associated
with low TPMT enzyme activity based on the
current literature. The mode of inheritance of
TPMT was explicitly stated in 45 (67%) out
of 67 studies. None of the studies described a
sample size calculation or a priori defined the
power to detect effect sizes or checked for the
presence of population stratification. Two of
the four case–control studies clearly defined the
patient groups, but none genotyped the cases
and controls in mixed batches. The genotype
and/or phenotype tests were described by 57 of
the 67 (85%) studies. Quality-control measures
were described by only six (9%) studies, and of
these, four reported their findings. Independent
checking of results was not described by any
study. Blinding of assessors was described by 14
(21%) studies.
182
Pharmacogenomics (2010) 11(2)
Two of the studies reported on noncompliance,
and each withdrew those patients whose TPMT
metabolite levels indicated nonadherence with the
prescribed dose and medication [74,52] .
Of the 48 studies carrying out a genotype test,
only five (10%) checked for HWE. Two of the
studies reported using the c2 test to measure HWE
and one study used a software package [103] . The
remaining two studies did not report the method
used. None of the studies found deviation from
HWE, but none of the studies reported the statistical results or the p-values. One study reported
these details as online supplementary data.
Outcomes were justified and fully reported
by all the studies. The outcomes were defined in
51 (76%) of the studies. Missing data were not
mentioned by any of the studies reviewed. A relatively small number of SNPs were investigated;
therefore, missing data should be minimal.
Five additional quality assessment questions
were asked that were specific to TPMT testing (Online Supplementary Tables 6 & 7; www.futuremedicine.com/doi/suppl/10.2217/pgs.09.155/
suppl_file/suppl_material.xls). The follow-up
of the patients was 3 months (which is considered to be long enough to identify the majority of patients who will suffer a hematological
adverse drug reaction to thiopurines [7]) or longer
in 62 (93%) of the 67 studies. If using the phenotype test, patients should be tested before thiopurine use and at least 3 months after any blood
trans­fusion. This was achieved by ten (27%) out
of 37 of the studies, reflecting that the majority
were retrospective in design. High, intermediate
and low TPMT phenotypic activity was defined
by 27 (73%) out of 37 studies.
Several areas of heterogeneity have been
explored with sensitivity ana­lysis (e.g., ethnicity,
disease studied, azathioprine rather than other
thiopurine drug, leukopenia definition, if the
study stated the mode of inheritance and defined
outcomes, personnel blinding and number of
alleles tested). Where more than five studies were
combined, the odds ratio was between 3 and 9.1,
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Myelosuppression more likely with reduced
TPMT activity
Figure 2. Forest plot showing the odds ratio of myelosuppression for each study, separated by study design.
OR: Odds ratio; TPMT: Thiopurine S-methyltransferase.
Myelosuppression less likely with reduced
TPMT activity
4.19 (3.20–5.48)
Overall (I2 = 44.2%; p = 0.001)
12442
3.77 (0.15–95.82)
10.56 (0.61–183.37)
0.31 (0.01–7.05)
0.22 (0.01–4.15)
35.00 (1.63–750.31)
1.97 (0.08–49.80)
1.44 (0.42–4.95)
16.50 (0.86–315.19)
3.68 (1.08–12.50)
21.00 (0.74–598.29)
7.73 (1.24–48.39)
82.80 (8.27–828.68)
0.99 (0.28–3.47)
45.89 (2.02–1044.21)
8.60 (0.38–193.86)
1.92 (0.34–10.85)
19.00 (0.62–583.38)
3.46 (0.58–20.82)
0.85 (0.04–16.55)
3.51 (0.62–19.93)
7.47 (2.03–27.47)
9.46 (1.07–83.37)
3.00 (0.08–111.78)
43.00 (3.86–478.64)
88.20 (3.60–2160.37)
14.67 (1.00–215.31)
35.00 (6.36–192.63)
0.71 (0.04–14.15)
1.62 (0.07–35.63)
27.53 (1.28–592.93)
30.07 (0.97–932.78)
203.00 (7.60–5424.16)
0.88 (0.10–7.50)
4.31 (1.06–17.55)
4.07 (2.97–5.58)
Cohort, retrospective
Ando et al. (2001)
Ansari et al. (2002)
Bezier et al. (2008)
Breen et al. (2005)
Chocair et al. (1992)
Corominas et al. (2003)
Czaja et al. (2006)
De Ridder et al. (2006)
Fabre et al. (2004)
Formea et al. (2004)
Heckmann et al. (2005)
Hibi et al. (2003)
Hindorf et al. (2006b)
Ishioka et al. (1999)
Joji et al. (2003)
Jun et al. (2005)
Kader et al. (2000)
Langley et al. (2002)
McLeod et al. (1999)
Naughton et al. (1999)
Pandya et al. (2002)
Schedel et al. (2006)
Schutz et al. (1995)
Schwab et al. (2002)
Sebbag et al. (2000)
Snow et al. (1995)
Song et al. (2006)
Stocco et al. (2005)
Stocco et al. (2007)
Taja-Chayeb et al. (2008)
Tamori et al. (2007)
Tumer et al. (2007)
Winter et al. (2007)
Zelinkova et al. (2006)
Subtotal (I2 = 43.1%; p = 0.005)
1
33.21 (5.90–186.87)
451.00 (16.35–12441.78)
19.00 (1.15–314.97)
0.86 (0.10–7.60)
4.96 (0.46–53.24)
0.94 (0.16–5.45)
32.00 (1.96–522.76)
1.28 (0.23–7.19)
3.80 (1.16–12.46)
3.43 (0.19, 62.12)
3.89 (0.11–143.60)
4.30 (2.53–7.29)
Cohort, prospective
Ansari et al. (2008)
Black et al. (1998)
Derijks et al. (2004)
Dubinsky et al. (2000)
Gisbert et al. (2006a)
Hawwa et al. (2008)
Herrlinger et al. (2003)
Hindorf et al. (2006a)
Kurzawski et al. (2005)
Stolk et al. (1998)
Zhang et al. (2006)
Subtotal (I2 = 56.4%; p = 0.011)
8.0 × 10 -5
36.60 (1.37–978.57)
4.52 (0.15–132.83)
13.27 (1.63–107.69)
OR (95% CI)
Case–control
Gearry et al. (2003)
Reuther et al. (2004)
Subtotal (I2 = 0.0%; p = 0.385)
ID
Study
100.00
1.11
0.40
4.25
7.16
0.36
1.02
10.09
0.26
5.04
0.28
1.11
0.29
11.74
0.28
0.80
3.80
0.22
2.27
2.39
2.31
3.25
1.58
0.63
0.21
0.19
0.51
0.50
2.73
1.27
0.44
0.08
0.06
4.43
2.92
73.99
0.64
0.05
0.40
4.35
0.87
6.18
0.26
5.17
5.92
1.01
0.51
25.36
0.18
0.47
0.65
Weight (%)
TPMT activity & myelosuppression when taking thiopurine medications
Research Article
183
Research Article
Higgs, Payne, Roberts & Newman
with increased variability when fewer studies were
combined (Online Supplementary Table 8; www.futuremedicine.com/doi/suppl/10.2217/pgs.09.155/
suppl_file/suppl_material.xls). The I2 value is
not reduced by grouping the studies in this way,
remaining 40–50%.
Publication bias was explored using a funnel
plot. This shows evidence of publication bias, in
that small studies that do not show an increased
rate of myelosuppression in patients with inter­
mediate TPMT activity are under-represented
(Online Supplementary Figure 1; www.futuremedicine.
com/doi/suppl/10.2217/pgs.09.155/suppl_file/
suppl_material.xls).
Discussion
This study provides systematically collated evidence that low and/or absent TPMT activity
results in severe myelosuppression in individuals
treated with standard doses of thiopurines. It has
been this association that has driven forward the
adoption of TPMT testing into clinical practice
to identify individuals deficient in TPMT and
avoid this serious adverse drug reaction. More
controversial has been the relationship between
thiopurine-associated hematological adverse reactions in individuals with intermediate enzyme
activity. Our meta-ana­lysis demonstrates a fourfold raised risk of leukopenia in patients with one
TPMT variant or intermediate activity. Owing to
the hetero­geneity of the studies used in the metaana­lysis, the raised odds ratio should be interpreted with caution. The range of white cell counts
used to define leukopenia varied. Importantly,
the increased risk was of mild leukopenia rather
than severe leukopenia, neutropenia or infection.
Furthermore, lymphopenia is a normal response
to thiopurine immunosuppression [66] . Individuals
with higher levels of thiopurine active metabolites
are more likely to be lymphopenic [85] , and individuals with intermediate TPMT activity have
raised levels of active metabolites [86] . Therefore,
modest leukopenia may reflect effective treatment
with thiopurines, rather than a clinically relevant
adverse event. Some authors have argued that identification of individuals with intermediate TPMT
activity would allow reduced doses of thiopurine
to achieve effective immunosuppression, and careful early monitoring to reduce the likelihood of
myelosuppression [87] . Whether knowledge of
TPMT status and the potential for improved care
for patients with intermediate TPMT activity is
cost effective has not been established.
A key challenge for this study was how to
identify studies that included descriptions of
TPMT-associated adverse drug reactions because
184
Pharmacogenomics (2010) 11(2)
a variety of different terms were used to refer
to ADRs and myelosuppression. To address this
issue, the search terms were made as comprehensive as possible, and many words for adverse
drug reaction and myelosuppression were used in
the search strategy (Online Supplementary A ppendix 1;
www.futuremedicine.com/doi/suppl/10.2217/
pgs.09.155/suppl_file/suppl_material.xls).
There are a number of sources of methodo­
logical and clinical heterogeneity in the studies
selected for this review, which may account for
the statistical heterogeneity. Most of the studies
were small, and, in theory, combining their data
should give a more precise result. Although the
varying quality of methods and reporting of the
studies contributes to methodological hetero­
geneity, the studies included in the meta-ana­lysis
were of a similar methodological standard without obvious factors that would lead to an exaggerated or reduced measure of the effect of TPMT
activity on myelosuppression. Important sources
of clinical heterogeneity include the definitions of
leukopenia, length of follow-up, TPMT testing
methods, specific thiopurine drug and dose.
Gisbert and Gomollon showed that bone
marrow suppression can occur at any time after
commencing treatment [7] . Although it occurs
most commonly within the first few months,
studies with shorter follow-up time may miss
some cases of myelosuppression and underestimate the effect of TPMT status, reducing
the measured effect of TPMT activity. Side
effects, including nausea, may cause patients to
discontinue thio­purine treatment before myelosuppression occurs, again reducing the effect of
TPMT activity. However, there is no evidence
that patients who suffer from other side effects
have an increased incidence of myelosuppression.
The number of alleles tested varied between
studies. Some studies sequenced TPMT and were,
therefore, likely to detect all functional TPMT
variants, while others tested for the three most
common variant alleles only, which may lead
to underestimation of the effect of TPMT on
myelosuppression. Conversely, myelo­suppression
has a number of causes that are independent of
TPMT in individuals taking thiopurines; that
is, other immunosuppressants, viral (e.g., cytomegalovirus) and antithymocyte globulin in
transplant patients, the underlying disease and
idiosyncratic reactions [13] .
There are several sources of variability associated with the tests used for TPMT activity. The
studies used a number of different phenotypic
tests, and consequently, the units for TPMT
activity varied between studies; therefore,
future science group
TPMT activity & myelosuppression when taking thiopurine medications
direct comparison of values is not possible and
patients are allocated into high-, intermediateand low-activity groups in order to compare the
studies. This classification is set at an arbitrary
cutoff point defined by the individual studies,
because the concordance between the genetic
and phenotypic tests for TPMT is not 100% [88] .
Although some studies found perfect concordance when carrying out genetic and phenotype tests [15,30,89] , many did not [16,33,52,53,71,90] .
The concordance was as low as 77% in one
study [33] . In addition, TPMT activity ranges
differ between ethnic groups; notably, people
of Afro-Caribbean ethnicity have lower TPMT
activity than Caucasians and South Asians [91] .
Variation in test methods and definitions will
alter the sensitivity and specificity of TPMT
testing in clinical practice.
Although higher doses of azathioprine have
not been shown to increase the incidence of
myelosuppression [7] , this has not been analyzed
specifically in patients with intermediate TPMT
activity, and dose may be more important in
this group, as intermediate TPMT activity may
increase sensitivity to lower doses of thiopurines.
Four studies excluded thiopurines or used a
reduced thiopurine dose for low- or intermediateactivity/TPMT variant patients [29,27,41,53] . This
may lead to less cases of myelosuppression than
if standard doses had been used and therefore
under-estimates of the odds ratio produced by
the meta-ana­lysis.
A funnel plot indicated the presence of publication bias (Online Supplementary Figure 1; www.futuremedicine.com/doi/suppl/10.2217/pgs.09.155/
suppl_file/suppl_material.xls). However, ana­lysis
of the use of funnel plots suggests that there may
be other reasons for this effect in addition to publication bias; for example, inherent differences in
the design of small and large studies [92] .
Conclusion
Many studies have been conducted on the relationship between TPMT and hematological
adverse reactions. This systematic review and
Research Article
meta-ana­lysis supports the association between
low-activity TPMT and a high risk of myelosuppression in individuals treated with standard
doses of thiopurines. Individuals who are hetero­
zygous for TPMT variant alleles or patients
with intermediate TPMT activity had a fourfold increased risk of leukopenia compared with
wild-type. However, the clinical significance of
moderate leukopenia is more difficult to interpret
owing to the heterogeneity between studies.
We recommend that further retrospective
studies are unnecessary and unlikely to add to
the evidence base. Prospective studies that evaluate the cost–effectiveness and utility of the test
would be valuable. However, the use of TPMT
testing has been accepted, and the test is cheap
and routinely carried out. This study highlights
that the increased risk of myelo­suppression
for intermediate-activity patients, while present, is low and should not preclude the use of
thiopurine medications.
Acknowledgements
Thanks to Yenal Dundar, Specialty Registrar in Psychiatry,
and Brian H Willis for comments on the search strategies.
Financial & competing interests disclosure
William G Newman’s group is supported through funding
by the NIHR Manchester Biomedical Research Centre,
MAHSC and through the Department of Health
Pharmacogenetics Initiative (TARGET study) Ref.
PHGX09A. Katherine Payne is funded by an RCUK aca�
demic fellowship. ��������������������������������������
The authors have no other relevant affili�
����
ations or financial involvement with any organization or
entity with a financial interest in or financial conflict with
the subject matter or materials discussed in the manuscript
apart from those disclosed.
Ethical conduct of research
The authors state that they have obtained appropriate insti­
tutional review board approval or have followed the princi­
ples outlined in the Declaration of Helsinki for all human
or animal experimental investigations. In addition, for
investi­gations involving human subjects, informed consent
has been obtained from the participants involved.
Executive summary
ƒƒ Patients with absent thiopurine S-methyltransferase (TPMT) activity have an increased risk of becoming profoundly myelosuppressed if
given standard doses of thiopurine medications; for example, azathioprine.
ƒƒ Patients with intermediate TPMT activity (or one variant allele) also have an increased risk of myelosuppression following standard
thiopurine treatment; however, this is substantially less than homozygote/absent-activity individuals.
ƒƒ The increased risk of leukopenia for individuals with intermediate TPMT activity compared with wild-type treated with thiopurines
is fourfold.
ƒƒ Mild leukopenia may reflect response to treatment rather than an adverse event.
ƒƒ Prospective studies with well-defined and clinically significant end points (e.g., neutropenia and neutropenic sepsis) are required to
accurately assess the increased risk for intermediate-activity patients.
future science group
www.futuremedicine.com
185
Research Article
Higgs, Payne, Roberts & Newman
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Pharmacogenomics (2010) 11(2)
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