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Risk factors for thiopurine‐induced myelosuppression and infections in inflammatory bowel disease patients with a normal TPMT genotype

Summary Background Leucopenia is a common side effect in patients treated with thiopurines. Variants in the thiopurine S‐methyltransferase (TPMT) gene are the best‐known risk factor, but only explain up to 25% of leucopenia cases. Aim To identify the clinical risk factors for thiopurine‐induced leucopenia in patients without a common TPMT variant, and explore if these patients are at increased risk for infections. Methods Post hoc analysis of the Thiopurine response Optimisation by Pharmacogenetic testing in Inflammatory bowel disease Clinics (TOPIC) trial. For this analysis, patients without a variant in TPMT (*2, *3A or*3C) were included. Uni‐ and multivariate Cox‐proportional hazard models were used to identify risk factors for leucopenia and infections. Leucopenia was defined as a white blood cell (WBC) count <3.0 × 109/L and infections were classified according to the Common Terminology Criteria for Adverse Events. Results Sixty hundred and ninety‐five patients (90.6%) included in the TOPIC‐trial had no variant in TPMT, of which 45 (6.5%) developed leucopenia. Median time to leucopenia was 56 (29‐112) days. Multivariate analysis showed that use of mercaptopurine compared to azathioprine was associated with leucopenia (hazard ratio [HR] 2.61 [95% CIs, 1.39‐4.88; P < .01]) and a higher baseline WBC count was protective (HR 0.80 [95% CIs, 0.71‐0.89; P < .01]). Risk factors for infections were older age (per 10 year; HR 2.07 [95% CIs, 1.18‐3.63; P = .01]) and concomitant use of biologic drugs (HR 2.15 [95% CIs, 1.14‐4.07; P = .02]). Conclusions Low baseline WBC count and mercaptopurine, due to a relatively higher dose, were risk factors for thiopurine‐induced leucopenia in patients without a TPMT variant.


| INTRODUCTION
Thiopurines are immunosuppressive drugs that have an important role in the treatment of inflammatory bowel diseases (IBD), both as monotherapy and as combination treatment with biologics. 1-3 Yet many patients have to discontinue treatment in an early phase due to ineffectiveness or side effects. Leucopenia as a result of thiopurine use is a frequent reason for dose reductions and/or (temporary) treatment discontinuation in patients with IBD, leading to suboptimal treatment conditions. 4,5 Dose reductions or treatment discontinuation in case of leucopenia is mainly driven by fear for infectious complications. 4 The best-known risk factor for the development of thiopurineinduced leucopenia is the presence of genetic variants in the thiopurine S-methyltransferase (TPMT) gene, which leads to a reduced or negligible TPMT activity and subsequently to excessive cytotoxic 6-thioguanine nucleotide (6-TGN) metabolite formation. 6,7 In the Caucasian population, 95% of the decreased TPMT enzyme activity follows from three common variants (TPMT*2, TPMT*3A or TPMT*3C). 7 Especially patients with a homozygous genetic variant in TPMT (~0.3% of the population), and to a lesser degree patients with a heterozygous variant in TPMT (~10% of the population) are at increased risk for severe, life-threatening, myelosuppression when treated with the standard recommended dose of azathioprine (AZA) or mercaptopurine (MP). [8][9][10] Leucopenia can be prevented by genotyping for these variants prior to initiation of thiopurine treatment and subsequently taper the thiopurine dose to 50% of the original dose in those heterozygous for a variant in TPMT and 0%-10% of the original dose in patients with a homozygous variant. [11][12][13] However, only up to 25% of the leucopenia cases can be explained by variants in TPMT, which makes that leucopenia is frequently observed in patients without a TPMT variant. 1,5,[14][15][16] This suggests that also other genes and environmental or clinical factors may contribute to the risk of leucopenia during thiopurine treatment. [17][18][19][20][21] It is unknown to what degree leucopenia attributes to the risk for infections. Recent studies in the treatment of hepatitis C did not reveal a relation between interferon-induced leucopenia and infectious complications. 22,23 If this also applies for thiopurine treatment in IBD, strict dose reductions in case of leucopenia might be redundant.
In the present post hoc analysis of the "Thiopurine response Optimisation by Pharmacogenetic testing in Inflammatory bowel disease Clinics" (TOPIC) trial, we aimed to (1) identify clinical risk factors for the development of TPMT-independent leucopenia in patients with IBD treated with thiopurines and (2) evaluate whether TPMT-independent leucopenia is a risk factor for the occurrence of infections in these patients.

| Patients
We designed a case-control study with patient data from the TOPIC trial, which evaluated the efficacy of pre-treatment TPMT genotyping on the occurrence of myelosuppression during thiopurine treatment. 14 In brief, in the TOPIC trial thiopurine-naive patients with IBD with an indication for thiopurine treatment (indicated by the treating physician) were randomised 1:1 for the intervention group with prior to treatment genotyping of the three most common genetic variants (*2, *3A and *3C) in TPMT vs the standard of care group with thiopurine dosing according to current standard of care; AZA (2.0-2.5 mg/kg) and MP (1.0-1.5 mg/kg). In patients allocated to the intervention arm, a 50% dose reduction was recommended in case of a heterozygous TPMT variant and in case of a homozygous TPMT variant 10% of the standard dose or an alternative treatment was advised. Patients were followed for 20 AE 6 weeks with laboratory measurements including white blood cell (WBC) count at week 0, 1, 2, 4, 6, 8, 20 and when indicated by the treating physician.
Neutrophil count was not routinely measured in the TOPIC trial.
Physicians were free in their choice for AZA or MP and were advised to start full dose. After the follow-up period, TPMT-genotype was assessed in patients randomised to the standard of care arm and in addition TPMT activity was assessed in all patients. Main exclusion criteria of the TOPIC trial were previous use of thiopurines, co-treatment with allopurinol, baseline WBC count <3.0 9 10 9 /L, baseline liver test abnormalities (alanine transaminase, aspartate transaminase, alkaline phosphatase and/or gamma glutamate transpeptidase ≥ two times the normal upper limit), known TPMT enzyme activity or TPMT-genotype. 6-Methylmercaptopurine ribonucleotides (6-MMPR) and 6-TGN levels were assessed at week eight in the first 301 patients included in the trial. For a detailed description of the study design, patient selection and patient data, we refer to Coenen et al 14 The TOPIC trial was approved by the institutional ethics committee and all patients provided written informed consent (clinicaltrials.gov, NCT00521950).
The primary aim of the current study was to indentify risk factors for leucopenia in patients without one of the tested genetic variants in TPMT. All randomised patients who started with a thiopurine and tested negative for a variant in TPMT were included in an intention-to-treat analysis ( Figure 1). Patients allocated to the genotyping arm with a TPMT variant (treated with 50% of the regular dose), as well as patients assigned to the standard of care arm with a TPMT variant (treated with 100% of the regular dose) were excluded. The Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 was used to classify the severity of leucopenia. 24 According to these criteria, mild leucopenia (grade 1) is defined as a WBC count between the lower limit of normal and ≥3.0 9 10 9 /L, moderate leucopenia (grade 2) was defined as a WBC count between ≥2.0 9 10 9 /L and <3.0 9 10 9 /L and severe leucopenia (grade 3) if the WBC count was <2.0 9 10 9 /L. For the current analysis, only cases of moderate and severe leucopenia were included. Leucopenia which developed 2 weeks or later after discontinuation of the thiopurine was not considered to be associated with thiopurine use.
The second goal of this study was to explore if patients with TPMT-independent leucopenia during thiopurine treatment were at increased risk for infections. For this analysis, infections were classified according to the CTCAE. 24 The source of infection was reported 2.2 | TPMT enzyme activity and TPMT genotyping TPMT enzyme activity was measured in red blood cells (RBCs) using a high-performance liquid chromatography method described previously. 14 Genotyping of variants *2, *3A and *3C in TPMT was described previously. 14   The variable leucopenia was retained in this model, since we wanted to explore whether these patients were at increased risk for infections. A P < .05 was considered as statistically significant.

| Patients
Of the 796 patients with IBD randomised in the TOPIC trial, 29 patients were excluded because they did not start with a thiopurine or were lost to follow-up. Of the remaining 767 patients who actually started with thiopurine treatment, 72 (9.4%) carried a genetic variant in TPMT and were excluded, which leaves 695 patients with IBD for this analysis (Figure 1 Table 1 shows the characteristics of patients with and without TPMT-independent leucopenia during thiopurine treatment. Table 2 provides the results of the uni-and multivariate Cox-proportional hazard model and shows that the choice for MP compared to AZA, Hazard ratio (HR) 2.61 (95% CIs 1.39-4.88; P < .01) was an independent risk factor for TPMT-independent thiopurine-induced leucopenia. Furthermore, a higher baseline WBC count protected the patient from TPMT-independent thiopurine-induced leucopenia, HR 0.80   Table 3. Characteristics of patients with and without an infection are presented in Table 4.

| DISCUSSION
In this study, we showed that in patients with IBD without a variant in TPMT, baseline WBC count and the choice of MP compared to AZA, were independent risk factors for thiopurine-induced leucopenia during the first five months of treatment. However, this did not result in a higher risk for infections. Infections during thiopurine treatment were more frequently seen in elderly and in patients with concomitant use of biologic drugs.
The overall incidence of thiopurine-induced leucopenia is estimated to be 7%. 4 Importantly, in 75% of the patients this is not correlated with a genetic variant in TPMT. As shown in this study, leucopenia still occurs in 6.5% of the patients without a genetic variant in TPMT, often leading to dose reductions or treatment discontinuation. 5 Thiopurine-induced leucopenia is at least partially dosedependent. 26 In a previous analysis of all patients included in the TOPIC trial, we already showed that patients treated with MP were relatively higher dosed than patients treated with AZA and that this resulted in amongst others a higher rate of leucopenia. 25 This difference annulled in a secondary analysis with adjustment for dose and metabolite levels, therefore an interdrug difference between AZA and MP seems unlikely. Also, other studies showed a relatively higher dose and leucopenia rate in patients on MP compared to AZA. 20,21,27 The difference might be attributed to the absence of MP tablets with a content of 25 mg, which allow a more personalised dosing. Another explanation might be derived from the conversion rate of 2.08, which is used to calculate the MP dose from the AZA dose. This conversion rate includes assumptions such as a 100% bioavailability. 28 The higher rate of dose-dependent side effects might be prevented with a slight reduction of the MP starting dose. 25 In our study, 6-TGN and 6-MMPR metabolite levels were not increased in patients with leucopenia. It must be stressed that most patients with leucopenia were subjected to dose reductions or treatment discontinuation before week eight. Therefore, steady-state week eight 6-TGN levels were available only in a limited number of cases, which most likely explains why we found no difference.
Besides 6-TGN levels, also excessively elevated 6-MMPR levels have been associated with leucopenia. 21 The increased risk for leucopenia with a lower baseline WBC count has also been reported in the T A B L E 1 Characteristics of the patients with and without TPMTindependent thiopurine-induced leucopenia setting of ganciclovir-and chemotherapy-induced leucopenia. 29,30 In contrast to a previous study, we did not find a correlation between concomitant 5-ASA use and myelosuppression. 31 It has been suggested that 5-ASA might increase 6-TGN levels by a weak inhibition of the TPMT enzyme activity and therefore may increase the risk of myelosuppression. 32 Previous studies using data of the TOPIC trial already showed that 6-TGN levels were not significantly higher in patients on 5-ASA, which explains why we found no increased leucopenia rate in this group. 33,34 It is known that smoking leads to an increase of WBC count of 0.5-1.0 9 10 9 /L as a result of a lowgrade systemic inflammation. 35 Indeed, in our study univariate analysis showed that smokers had less often leucopenia, however, this did not reach significance in the multivariate analysis.
Pharmacogenetic testing of TPMT is a cost-effective strategy to prevent severe, life-threatening, myelosuppression, in those who are TPMT-deficient. 36,37 This strategy is strongly recommended by several guidelines. 9,11,38,39 TPMT-related and TPMT-independent leucopenia may be considered as two separate entities. Most studies reporting on thiopurine-induced leucopenia provide no information regarding the TPMT status, which hampers extrapolation to daily clinical practice. [40][41][42] For this reason, we specifically focused on the patients without a variant in TPMT. Thiopurine-induced leucopenia in patients without genetic variants in TPMT occurred at any moment during the first 5 months of therapy, with a slight peak in the first 2 months. Therefore, the current routine laboratory tests should remain mandatory to identify patients with myelosuppression in an early stage. 1 In more than 90% of the cases leucocyte levels did not drop below 2.0 9 10 9 /L. Yet thiopurine dosage was reduced or treatment was discontinued in two-thirds of these patients, probably due to fear for infectious complications. Overall, most cases of TPMT-independent leucopenia are mild and therefore did not require immediate treatment discontinuation or dose reduction. However in the setting of an unknown TPMT-genotype, all cases of leucopenia in the first months of thiopurine treatment should be considered as potentially TPMT-induced, because otherwise life-threatening myelosuppression might occur in the patients whom actually have a genetic variant in TPMT. 1 Previous studies estimated a cumulative infection rate of 6.5% among patients with thiopurine-induced leucopenia. 4 In the present study, we showed that infection rates are comparable for patients with and without TPMT-independent leucopenia. Both viral and bacterial infections were seen, however in most of the cases no specific pathogen was isolated. The risk for bacterial infections increases with neutrophil levels below 0.5 9 10 9 /L. 43 These levels are often observed in patients with a homozygous variant in TPMT when treated with standard thiopurine doses due to the very high 6-TGN levels. 1,44 In our study, neutrophil levels at time of leucopenia were available in~50% of the patients and a neutrophil count below 0.5 9 10 9 /L was observed in only one patient, which suggests that severe neutropenia is not frequently observed in patients without a TPMT variant. This, in combination with the fact that in most cases leucopenia was only moderate (WBC count ≥2.0 9 10 9 /L), might explain why we did not find a higher infection rate in patients with a thiopurine-induced leucopenia. It must be mentioned that in 69% of patients with leucopenia the thiopurine dose was reduced or treatment was discontinued. It cannot be excluded that this prevented the aggravation of the leucopenia and/ or the development of infectious complications, which is an important limitation of our study. Yet, our findings corroborate studies exploring the risk of infections in hepatitis C treatment. In these studies, interferon-induced leucopenia was not a risk factor for infectious complications. 22,23 The lack of association between leucopenia and infections might have great implications for clinical practice. Some studies showed a relation between mild leucopenia (defined as WBC count ≥3.0 and <5.0 9 10 9 /L) and treatment efficacy. 45,46 This increased treatment efficacy in case of leucopenia T A B L E 2 Uni-and multivariate Cox-proportional hazards regression analysis to explore factors associated with development of thiopurineinduced leucopenia in the first five months of thiopurine treatment in patients without a Age and concomitant use of biologics were the only factors associated with the occurrence of infections. Increasing age is a known risk factor for infections, which is thought to result from an impaired immune response in the elderly. 47,48 This has also been reported in patients with IBD. 49,50 In contrast to many clinical trials, there was no upper age limit with inclusion in the TOPIC trial. As a consequence, 105 (15.1%) of the patients included in current study were 60 years or older, of whom 24 patients were 70 years or older. This makes it a representative sample for the real life setting in the era of an ageing IBD population. 51 The higher risk for infections with concomitant use of biologic drugs is not unexpected, since use of biologics is an independent risk factor for (opportunistic) infections. 49,52 This observation is not limited to IBD, but is also observed in other immune-mediated diseases. 53,54 In contrast to previous studies, we found no relation with concomitant steroid use. 49 A likely explanation is that steroid use was not protocolised and as a consequence the group of patients on oral steroids was very heterozygous and  Table 3, 45 patients experienced leucopenia, however in 3 patients with leucopenia during thiopurine treatment who also had an infection, the infection was not considered to be related to leucopenia, either because leucopenia was already resolved for more than 2 weeks (2 patients), or the infection was already known before the leucopenia occurred (1 patient). For the analysis, these cases were counted as an infection without leucopenia.
included patients on low maintenance dose, short-term use during thiopurine initiation, but also patients on a high dose for a longer period. Furthermore, patients currently taking no steroids still have an increased infection risk, when treated with steroids in the past 2 years. 55 We found no association between diabetes mellitus or smoking and infection risk, probably because of the relatively small study size and short follow-up period. 56,57 This study comes with some limitations. First, current analyses were not included in the original study protocol. Second, patients in the TOPIC trial were only tested for the three common variants in TPMT (*2, *3A and *3C), which cover more than 90% of the prevalence of genetic variants in TPMT in Caucasians. Therefore, it is possible that we missed some patients with other TPMT variants. 58 This was circumvented by sequencing the coding region of TPMT in all patients with a low TPMT activity or those who developed leucopenia; which did not reveal other variants (unpublished data).
Furthermore, we assumed that when the treating physician started anti-infectious therapy there was a true suspicion of an infection.
This comes with some restrictions, since exacerbations of IBD or flulike side effects (which might include elevation of C-reactive protein) can mimic (gastro-intestinal) infections leading to the prescription of antibiotics. Finally, (viral) infections may induce leucopenia. 19 However, if this is the case it would have resulted in an overestimation of 'leucopenia-associated infections' and further bolster our findings that (moderate) leucopenia itself is not a risk factor for infectious complications.
In conclusion, in the present study we showed that the choice for MP compared to AZA (as a result of a relatively higher dose) and baseline WBC count are risk factors for the development of thiopurine-induced leucopenia in patients without a genetic variant in TPMT; however, this was not associated with an increased risk for infections. Multivariate analysis showed that older patients and patients with concomitant use of biologics had a higher risk for infections during thiopurine treatment and should therefore be treated with more caution. Our data suggest that moderate TPMTindependent leucopenia during thiopurine treatment might be accepted in the light of close monitoring of WBC count and metabolite levels.

ACKNOWLEDG EMENT
We thank the patients for participation in the study. We thank Rene  Infections were classified according to the Common Terminology Criteria for Adverse Events 4.0 (version 4.0). Only infections grade 2 or higher were included. WBC, white blood cell.