Thiopurine Methyltransferase Activity Research Articles

Review article: the management of autoimmune hepatitis beyond consensus guidelines

Consensus guidelines aid in the diagnosis and management of autoimmune hepatitis, but they are frequently based on low-quality clinical evidence, conflicting experiences and divergent opinions. Recommendations may be weak, discrepant or non-existent at critical decision points. To identify the decision points where guidelines are weak or non-existent and review the evidence essential in the decision process. Full-text articles published in English using the keyword 'autoimmune hepatitis' were identified by PubMed from 1972 to 2013. Personal experience and investigations in autoimmune hepatitis also identified important contributions. Seventy per cent of the guidelines developed by the American Association for the Study of Liver Diseases and 48% of those proposed by the British Society of Gastroenterology are based on low-quality evidence, conflicting experiences or divergent opinions. The key uncertainties in diagnosis relate to the timing of liver biopsy, recognising acute severe (fulminant) disease, interpreting coincidental nonclassical histological changes, accommodating atypical or deficient features in non-White patients, differentiating drug-induced from classical disease and identifying overlap syndromes. The key uncertainties in management relate to pre-treatment testing for thiopurine methyltransferase activity, treating asymptomatic mild disease, determining treatment end points, managing suboptimal responses, incorporating nonstandard medications as front-line and salvage agents, using azathioprine in pregnancy and instituting surveillance for hepatocellular carcinoma. Consensus guidelines are fraught with uncertainties in the diagnosis and management of autoimmune hepatitis. Each decision point must counterbalance the current available evidence and tailor the application of this evidence to the individual patient.

Preanalytical stringency: what factors may confound interpretation of thiopurine S-methyl transferase enzyme activity?

Measurement of red blood cell thiopurine S-methyl transferase (TPMT) enzyme activity before commencing thiopurine therapy is recommended to avoid severe bone marrow suppression in TPMT-deficient patients. Patient's samples go through preanalytical transportation and storage steps before measurement. We studied patient's TPMT activity sample data to assess the effect of preanalytical variables including transportation time. A total of 8524 TPMT enzyme activity analyses were conducted from 2002 to 2010 in a single laboratory, with samples sent from seven centres throughout New Zealand. TPMT activity was correlated with time of arrival at the reference laboratory, patient gender and age and centre of sample collection. The 6348 (74%) selected TPMT measurements that fulfilled selection criteria ranged from 0 to 25.8 IU/mL. Median delay to sample analysis was 42 h. Median TPMT activity was significantly lower for all centres compared with the reference centre (P < 0.001). Delay in sample arrival was significantly and independently correlated with TPMT enzyme activity (ANCOVA; P < 0.001), which showed a 0.011 (95% CI, 0.008-0.014) IU/mL decrease per extra hour of delay. After correcting for these data, one centre still had a significantly lower TPMT enzyme activity compared with the reference centre. There was a significant negative correlation between TPMT enzyme activity and delay from sample collection to analysis. Transportation time is therefore an important preanalytical variable influencing TPMT activity. Samples from one centre had a lower TPMT activity after correcting for transportation delay, suggesting that other factors related to sample processing may also be relevant.

Thiopurine methyltransferase genotype–phenotype discordance and thiopurine active metabolite formation in childhood acute lymphoblastic leukaemia

In children with acute lymphoblastic leukaemia (ALL) bone marrow activity can influence red blood cell (RBC) kinetics, the surrogate tissue for thiopurine methyltransferase (TPMT) measurements. The aim of this study was to investigate TPMT phenotype-genotype concordance in ALL, and the influence of TPMT on thiopurine metabolite formation. We measured TPMT (activity, as units ml(-1) packed RBCs and genotype) at diagnosis (n = 1150) and TPMT and thioguanine nucleotide (TGN) and methylmercaptopurine nucleotide (MeMPN) metabolites (pmol/8 × 10(8) RBCs) during chemotherapy (n = 1131) in children randomized to thioguanine or mercaptopurine on the United Kingdom trial ALL97. Median TPMT activity at diagnosis (8.5 units) was significantly lower than during chemotherapy (13.8 units, median difference 5.1 units, 95% confidence interval (CI) 4.8, 5.4, P < 0.0001). At diagnosis genotype-phenotype was discordant. During chemotherapy the overall concordance was 92%, but this fell to 55% in the intermediate activity cohort (45% had wild-type genotypes). For both thiopurines TGN concentrations differed by TPMT status. For mercaptopurine, median TGNs were higher in TPMT heterozygous genotype (754 pmol) than wild-type (360 pmol) patients (median difference 406 pmol, 95% CI 332, 478, P < 0.0001), whilst median MeMPNs, products of the TPMT reaction, were higher in wild-type (10 650 pmol) than heterozygous patients (3868 pmol) (P < 0.0001). In TPMT intermediate activity patients with a wild-type genotype, TGN (median 366 pmol) and MeMPN (median 8590 pmol) concentrations were similar to those in wild-type, high activity patients. In childhood ALL, TPMT activity should not be used to predict heterozygosity particularly in blood samples obtained at disease diagnosis. Genotype is a better predictor of TGN accumulation during chemotherapy.

Biological variation of thiopurine methyltransferase enzyme activity: when has a significant change taken place?

Thiopurine methyltransferase (TPMT) enzyme activity is measured before initiating thiopurine therapy to reduce the risk of severe drug-associated myelotoxicity in patients with low enzyme activity. TPMT activity may vary over time in relation to drug treatment and patient clinical condition. What constitutes a significant change in TPMT activity can be derived from biological variation and analytical imprecision. A large national laboratory database was used to identify patients with three or more TPMT activity measurements. Variance of TPMT activity was analysed by determining the total coefficient of variation (CVTOT) of repeated measurements and by correlation with parameters including gender and follow-up time. Between-run analytical imprecision (CVa) was determined by replicate analysis (n = 314). Of 7383 patients with TPMT measurements, 136 were identified as having three or more measurements over time (range 3-14). Median CVTOT for individual patient results was 14.5% (range 2.5-36.7%). Analytical imprecision (CVa) was 10.3%. A reference change value (or critical difference) with 95% probability was calculated as 42%. Therefore, a change in measured TPMT activity above 42% should lead to considering sources of variation other than biological variation and analytical imprecision. TPMT enzyme activity needs to change by at least 42% to determine that a true change has taken place beyond biological variation and analytical imprecision. A single measurement of TPMT activity is sufficient for most clinical purposes.

SAT0202 Identification of thiopurine methyltransferase (TPMT) activity can predict azathioprine-related hematotoxicity in the maintenance therapy of systemic lupus erythematosus patients

ObjectivesTo investigate the azathioprine-related adverse drug reactions (ARDs) in the maintenance therapy of systemic lupus erythematosus (SLE) patients and the relationship between thiopurine methyltransferase (TPMT) activity and these ARDs.MethodsTPMT activity...

OC-015 The Influence of Gender and Haemoglobin on TPMT Activity

IntroductionPre-treatment measurement of red blood cell (RBC) thiopurine-S-methyltransferase (TPMT) activity is recommended to guide initial dosing of azathioprine (AZA) and mercaptopurine (MP). TPMT exhibits a trimodal distribution, with low and...

Methotrexate binds to recombinant thiopurine S-methyltransferase and inhibits enzyme activity after high-dose infusions in childhood leukaemia

Important drugs in the treatment of childhood acute lymphoblastic leukaemia (ALL) are 6-mercaptopurine (6-MP) and methotrexate (MTX). Thiopurine methyltransferase (TPMT) is a polymorphic enzyme causing variability in 6-MP response and toxicity. The aim of this study was to investigate the fluctuation in TPMT enzyme activity over time and the effect of high-dose MTX infusions on TPMT enzyme activity and 6-MP metabolites in paediatric ALL patients. Fifty-three children with ALL treated according to the NOPHO-ALL 2000 protocol were included in the study. TPMT enzyme activity was measured at six different times starting from diagnosis until after the end of maintenance treatment. TPMT and 6-MP metabolites were measured before the initiation of high-dose MTX (HD-MTX) infusions and at 66 h post-infusion. The interaction between MTX and TPMT was investigated in vitro using recombinant TPMT protein and a leukaemic cell line. Forty percent of TPMT wild-type individuals had deceptively low TPMT enzyme activity according to genotype at the time of diagnosis. TPMT activity had decreased significantly 66 h after the start of HD-MTX infusions (-9.2 %; p = 0.013). MTX bound to recombinant TPMT protein severely inhibiting TPMT enzyme activity (remaining activity 16 %). Our results show that TPMT genotyping should be performed in children with ALL, since 40 % of the children in our study who carried the wild-type TPMT gene were at risk of initial underdosing of 6-MP in cases where only TPMT enzyme activity was determined. MTX inhibits the TPMT enzyme activity after HD-MTX infusions due to protein binding.

Relationship Between Azathioprine Dosage and Thiopurine Metabolites in Pediatric IBD Patients

Previous studies have reported no or only a very poor correlation between 6-methylmercaptopurine/6-thioguanine nucleotide (6-MeMPN/6-TGN) and azathioprine (AZA) dose in the treatment of inflammatory bowel disease (IBD). However, metabolite levels are often repeatedly measured yielding a hierarchical data structure that requires more appropriate data analysis. This study explored the relationship between the weight-based dosage of AZA and metabolites levels in 86 pediatric IBD patients using multilevel analysis. Other covariates related to patient characteristics and treatment were evaluated. This is the first study to demonstrate positive correlations between AZA dose and 6-TGN and 6-MeMPN levels and 6-MeMPN/6-TGN ratio (P < 0.001) in IBD children. Other novel predictors of metabolites were reported. Younger children exhibited lower 6-TGN and 6-MeMPN levels, probably suggesting age-related differences in metabolism and/or absorption of thiopurines. Coadministration of infliximab resulted in a significant increase in 6-TGN levels (P = 0.023). Moreover, alanine aminotransferase values positively correlated with 6-MeMPN levels (P = 0.032). The duration of AZA therapy, gender, and thiopurine methyltransferase activity were associated with metabolite levels. The wide interindividual variability in metabolite levels that accounted for 67.7%, 48.6%, and 49.4% of variance in the 6-TGN and 6-MeMPN levels and the ratio, respectively, were confirmed. The reliable AZA dose-metabolites relationship is useful for clinicians to guide the dosing regimen to maximize clinical response and minimize side effects or to consider alternative therapies when patients have preferential production of the toxic 6-MeMPN. These results may be of potential interest for optimizing thiopurine therapy to achieve safe and efficacious AZA use in pediatric IBD patients.

The effect of the adverse events with thiopurine S-methyltransferase gene mutation on outcome of childhood acute lymphoblastic leukemia

To investigate thiopurine S-methyltransferase (TPMT) activity and gene promoter polymorphism to probe its significance of individual chemotherapy in acute lymphoblastic leukemia (ALL) children. HPLC method was carried out to determine TPMT activity (n=100), which activity at newly diagnosed. At the same time determination of TPMT activity in healthy children (n=180), these children come from the health care clinic. Using online primer3 software design primers, PCR products were purified. To sequence TPMT gene of the patients with clinical events(n=30). According to the method to analysis of correlation between TPMT activity and toxicity. The average TPMT activities were (31.72±10.31) nmol·g⁻¹Hb·h⁻¹ and (30.70±9.67) nmol·g⁻¹Hb·h⁻¹ in ALL and healthy groups respectively, without gender differences of TPMT activities (P=0.45) in both groups. The TPMT activity with clinical events in newly diagnosed ALL patients (n=30) was (24.07±11.43) nmol·g⁻¹Hb·h⁻¹. There are significant differences of TPMT activities between severe bone marrow suppression [(20.96±7.24) nmol·g⁻¹Hb·h⁻¹] and ALL patients with clinical events groups (P<0.05). The TPMT activity of (40.46±8.18) nmol·g⁻¹Hb·h⁻¹ in recurrence children was also significantly different (P<0.05). TPMT activity in severe liver toxicity group was not significantly different (P=0. 930). Of TPMT gene sequencing in ALL patients with clinical events, only 3 children were heterozygosity mutations of TPMT*3C, while others homozygous genotype. There were significant differences of TPMT activities between heterozygosity genotype [(11.99±1.32) nmol·g⁻¹Hb·h⁻¹] and homozygous genotype groups [(24.95±11.32) nmol·g⁻¹Hb·h⁻¹] (P<0.05). There were five kinds of variations at the vicinity of the promoter region of -100 of tandem repeats (VNTR) polymorphism（*V3/*V3、*V3/*V4、*V4/*V4、*V5/*V5、*V4/*V6）without significant differences of TPMT activities among five kinds (P=0.186). TPMT activity was related to the gene polymorphism. TPMT activity determination had prognostic value and guided individualized treatment.

Role of Thiopurine Metabolite Testing and Thiopurine Methyltransferase Determination in Pediatric IBD

Thiopurines have been used in inflammatory bowel disease (IBD) for >30 years, and measurements of both thiopurine methyltransferase (TPMT) and thiopurine (TP) metabolites, 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine (6-MMP), have been readily available. The North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition (NASPGHAN) Committee on Inflammatory Bowel Disease thought it appropriate to review the present indications for use of TPMT and TP metabolite testing. Substantial evidence demonstrates that TP therapy is useful for both Crohn disease and ulcerative colitis. Review of the existing data yielded the following recommendations. TPMT testing is recommended before initiation of TPs to identify individuals who are homozygote recessive or have extremely low TPMT activity, with the latter having more reliability than the former. Individuals who are homozygous recessive or have extremely low TPMT activity should avoid the use of TPs because of concerns for significant leukopenia. TMPT testing does not predict all cases of leukopenia and has no value to predict hypersensitivity adverse effects such as pancreatitis. Any potential value to reduce the risk of malignancy has not been studied. All individuals taking TPs should have routine monitoring with complete blood cell count and white blood cell count differential to evaluate for leukopenia regardless of TPMT testing results. Metabolite testing can be used to determine adherence with TP therapy. Metabolite testing can be used to guide dose increases or modifications in patients with active disease. Consideration would include either increasing the dose, changing therapy or for those with elevated transaminases or an elevated 6-MMP, using adjunctive allopurinol to help raise 6-thioguanine metabolites and suppress formation of 6-MMP. Routine and repetitive metabolite testing has little or no role in patients who are doing well and taking an acceptable dose of a TP.

The effect of allopurinol and low-dose thiopurine combination therapy on the activity of three pivotal thiopurine metabolizing enzymes: Results from a prospective pharmacological study

IntroductionThiopurine therapy is often discontinued in inflammatory bowel disease (IBD) patients. The xanthine oxidase (XO) inhibitor allopurinol has previously shown to enhance thiopurine efficacy and to prevent adverse reactions, the mechanism of this beneficial interaction is not completely clarified. The aim of this study is to observe possible effects of allopurinol and low-dose thiopurine combination therapy on the activity of three pivotal thiopurine metabolizing enzymes. MethodsA prospective study of IBD patients failing thiopurine therapy due to a skewed thiopurine metabolism was performed. Patients were treated with allopurinol and azathioprine or mercaptopurine. Xanthine oxidase, hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and thiopurine S-methyl transferase (TPMT) activities, and thiopurine metabolites concentrations were measured during thiopurine monotherapy, and after 4 and 12weeks of combination therapy. ResultsOf fifteen IBD patients, XO activity decreased from 0.18 (IQR 0.08–0.3) during thiopurine monotherapy to 0.14 (IQR 0.06–0.2) and 0.11 (IQR 0.06–0.2; p=0.008) mU/hour/ml at 4 and 12weeks, respectively. HGPRT activity increased from 150 (IQR 114–176) to 180 (IQR 135–213) and 204nmol/(h×mg protein) (IQR 173–213; p=0.013). TPMT activity seemed not to be affected. 6-Thioguanine nucleotide concentrations increased from 138 (IQR 119–188) to 235 (223–304) and to 265pmol/8×10^8 (IQR 188–344), whereas 6-methyl mercaptopurine ribonucleotides concentrations decreased from 13230 (IQR 7130–17420) to 690 (IQR 378–1325) and 540 (IQR 240–790) pmol/8×10^8 at 4 and 12weeks of combination therapy (both p<0.001). ConclusionAllopurinol and thiopurine combination-therapy seems to increase HGPRT and decrease XO activity in IBD patients, which at least in part may explain the observed changes in thiopurine metabolite concentrations.

Azathioprine‐induced Sweet's syndrome and published work review

Hypersensitivity to azathioprine can manifest with a wide clinical spectrum. Azathioprine-induced Sweet's syndrome (SS) is rare and usually overlooked because it can mimic disease exacerbation and sepsis. This study aims to characterize the clinical findings of azathioprine-induced SS. A retrospective analysis of the records of three patients diagnosed with azathioprine-induced SS and a review of the relevant English-language published work was performed. Twelve (71%) of the 17 patients were male, ranging 9-89 years in age (mean, 47.2). The time of onset after starting azathioprine was 5-28 days (mean, 13.3). The most common associated disease was inflammatory bowel disease including ulcerative colitis and Crohn's disease (76%). The clinical features typically consisted of fever and classic rash of SS with pustules and vesicles. The lesions occurred most commonly on the face and trunk. Systemic involvement was rare and no hypotension or shock was reported as seen in azathioprine hypersensitivity syndrome. Thiopurine methyltransferase activity is not predictive of this type of adverse effect. Most patients dramatically responded to systemic corticosteroids. Azathioprine-induced SS may be underdiagnosed because it can be easily misinterpreted as inflammatory bowel disease-associated skin eruption. Patients with inflammatory bowel disease may be at higher risk of this condition. Early recognition and drug withdrawal can decrease morbidity of the patients.

Thiopurine S-Methyltransferase Phenotype and Genotype in Pediatric Patients with Inflammatory Bowel Disease; Implication for Azathioprine Treatment

Inflammatory Bowel Disease (IBD) is more prevalent in children than adults, and the incidence is increasing. IBD is treated with thiopurines, metabolized by thiopurine S-methyltransferase (TPMT) and inter-individual variability in activity of TPMT affecting therapy efficiency and drug toxicity arises from genetic polymorphisms, mainly TPMT*2, *3A, and *3C. The aim was to investigate the frequency distribution of TPMT activity, determine the penetration rate of TPMT*2, *3A, and *3C alleles in children, and compare TPMT activity in children and adults with IBD. The study included 85 children, 45% with Crohn’s disease (CD) and 55% with ulcerative colitis (UC), and 31 adults with IBD. TPMT activity was measured with radiochemistry. TPMT*2, *3A, and *3C alleles were investigated with PCR and restriction fragment length polymorphism analyses. Children showed median TPMT activities of 13.12 and 13.19 U/ml RBC in CD and UC, respectively, with 4.8-fold variability (range, 4.74 - 22.56 U/ml RBC). TPMT activity was similar in children and adults; ranges: 5.56-21.34 vs. 9.61-17.84 U/ml RBC, respectively, in CD; and 4.74-22.56 vs. 5.19-21.98 U/ml RBC, respectively, in UC. Patients with CD and UC treated with azathioprine displayed similar TPMT activities, similar adverse event frequencies, and similar numbers of non-responders. One out of 85 patients (1.18%) was heterozygous with TPMT*1/TPMT*2 (TPMT activity: 5.19 ± 0.05 U/ml RBC). Individuals with low-intermediate TPMT activity (<8 U/ ml RBC) did not carry mutant alleles *3A or *3C. TPMT phenotypes were similar in children and adults with inflammatory bowel disease.

Validation of New Allele-Specific Real-Time PCR System for Thiopurine Methyltransferase Genotyping in Korean Population

Introduction. Thiopurine drugs are metabolized via S-methylation and catalyzed by thiopurine S-methyltransferase (TPMT). Patients with very low TPMT activity are at high risk of fatal bone marrow toxicity when standard doses of thiopurine drugs are administered. TPMT genotyping can predict TPMT activity and is not affected by transfusion or red blood cell defects. Here, we report a new allele-specific real-time polymerase chain reaction (PCR) system for thiopurine methyltransferase genotyping that is validated in Korean population. Materials and Methods. Three major TPMT single-nucleotide polymorphisms (TPMT∗2, ∗3B, and ∗3C) were genotyped using real-time PCR with the allele-specific primers and probes. Internal positive controls were included in each well, and an automatic interpretative algorithm was applied. This system was validated using 244 clinical samples and 2 commercial DNA samples that had been previously genotyped using PCR-direct sequencing. Results. All of the obtained results are concordant with those of the reference method. All of the internal positive control reactions were successful. The allele frequency of TPMT∗3C was 2.05% (10 of 488 alleles). All of the patients with variant alleles were heterozygotes, and no homozygotes were detected. No TPMT∗2, ∗3A, or ∗3B alleles were observed in this Korean population. Conclusion. This rapid, accurate, and user-friendly genotyping system can be readily used to improve the efficacy and safety of thiopurine treatments in clinical practice.

Thiopurines related malignancies in inflammatory bowel disease: Local experience in Granada, Spain

To investigate the incidence of neoplasms in inflammatory bowel disease (IBD) patients and the potential causative role of thiopurines. We performed an observational descriptive study comparing the incidence of malignancies in IBD patients treated with thiopurines and patients not treated with these drugs. We included 812 patients which were divided in two groups depending on whether they have received thiopurines or not. We have studied basal characteristics of both groups (age when the disease was diagnosed, sex, type of IBD, etc.) and treatments received (Azathioprine, mercaptopurine, infliximab, adalimumab or other immunomodulators), as well as neoplasms incidence. Univariate analysis was performed with the student t test, χ(2) test or Wilcoxon exact test as appropriate. A logistic regression analysis was performed as multivariate analysis. Statistical significance was establish at P values of less than 0.05, and 95%CI were used for the odds ratios. Among 812 patients included, 429 (52.83%) have received thiopurines: 79.5% azathioprine, 14% mercaptopurine and 6.5% both drugs. 44.76% of patients treated with thiopurines and 46, 48% of patients who did not receive this treatment were women (P > 0.05). The proportion of ulcerative colitis patients treated with thiopurines was 30.3% compare to 66. 67% of patients not treated (P < 0.001). Mean azathioprine dose was 123.79 ± 36.5 mg/d (range: 50-250 mg/d), mean usage time was 72.16 ± 55.7 mo (range: 1-300 mo) and the accumulated dose along this time was 274.32 ± 233.5 g (1.5-1350 g). With respect to mercaptopurine, mean dose was 74.7 ± 23.9 mg/d (range: 25-150 mg/d), mean usage time of 23.37 ± 27.6 mo (range: 1-118 mo), and the accumulated dose along this time was 52.2 ± 63.5 g (range: 1.5-243 g). Thiopurine S-methyltransferase activity was tested in 66% of patients treated with thiopurines, among which 98.2% had an intermediate or high activity. Among the patients treated with thiopurines, 27.27% (112 patients) and 11.66% (50 patients) received treatment with Infliximab and Adalimumab respectively, but only 1.83% (7 patients) and 0.78% (3 patients) received these drugs in the group of patients who did not received thiopurines (P < 0.001 and P < 0.001 respectively). Finally, 6.8% (29 patients) among those treated with thiopurines have received other immunosuppressants (Methotrexate, Tacrolimus, Cyclosporin), compare to 1% (4 patients) of patients not treated with thiopurines (P < 0.001). Among patients treated with thiopurines, 3.97% developed a malignancy, and among those not treated neoplasms presented in 8.1% (P = 0.013). The most frequent neoplasms were colorectal ones (12 cases in patients not treated with thiopurines but none in treated, P < 0.001) followed by non-melanoma skin cancer (8 patients in treated with thiopurines and 6 in not treated, P > 0.05). In our experience, thiopurine therapy did not increase malignancies development in IBD patients, and was an effective and safe treatment for these diseases.

Successful Azathioprine Treatment with Metabolite Monitoring in a Pediatric Inflammatory Bowel Disease Patient Homozygous forTPMT*3C

Thiopurine S-methyltransferase (TPMT) methylates purine analogues, showing TPMT activity in inverse relation to concentrations of active metabolites such as 6-thioguanine nucleotide (6-TGN). With conventional dosing of thiopurines, patients with homozygous variant TPMT alleles consistently suffer from severe myelosuppression. Here, we report a patient with TPMT*3C/*3C who managed successfully with monitoring of thiopurine metabolites. The patient was an 18-year-old male diagnosed with Crohn's disease. The standard dose of azathioprine (AZA) (1.8 mg/kg/day) with mesalazine (55.6 mg/kg/day) was prescribed. Two weeks after starting AZA treatment, the patient developed leukopenia. The DNA sequence analysis of TPMT identified a homozygous missense variation (NM_000367.2: c.719A>G; p.Tyr240Cys), TPMT*3C/*3C. He was treated with adjusted doses of azathioprine (0.1-0.2 mg/kg/day) and his metabolites were closely monitored. Leukopenia did not reoccur during the follow-up period of 24 months. To our knowledge, this is the first case of a patient homozygous for TPMT*3C successfully treated with azathioprine in Korea. While a TPMT genotyping test may be helpful to determine a safe starting dose, it may not completely prevent myelosuppression. Monitoring metabolites as well as routine laboratory tests can contribute to assessing drug metabolism and optimizing drug dosing with minimized drug-induced toxicity.

Should Thiopurine Methyltransferase Genotypes and Phenotypes be Measured Before Thiopurine Therapy in Patients With Inflammatory Bowel Disease?

Not all of the adverse effects to thiopurine therapy can be explained by thiopurine methyltransferase (TPMT) polymorphisms. This study was intended to evaluate the value of TPMT genotype and phenotype measurement during the first year of thiopurine therapy. Consecutive patients with inflammatory bowel disease (IBD) who were receiving azathioprine or 6-mercaptopurine were followed up for 12 months. TPMT genotypes and phenotypes were examined in patients with IBD before thiopurine therapy and in unrelated healthy volunteers by polymerase chain reaction and high-performance liquid chromatography. A total of 199 patients and 300 healthy volunteers were included at 2 centers. Forty-seven of the 199 patients (23.62%) exhibited adverse effects during the entire course of thiopurine therapy. Two (1%) patients carrying TPMT*3C developed leucopenia at week 4 of azathioprine treatment. The TPMT*3C had a specificity of 100% (163/163) but a sensitivity of 5.56% (2/36) for predicting leucopenia. The calculated optimal cutoff activity for high TPMT activity and decreased TPMT activity was 4.75 U/mL red blood cells. The risk of leucopenia increased in the decreased TPMT group (odds ratio: 20.25; 95% confidence interval: 2.19-187.17; P = 0.004) and increased more during the initial 3 months of thiopurine therapy (odds ratio: 34.80; 95% confidence interval: 3.71-326.77; P = 0.001). Leucopenia occurred more frequently in the patients cotreated with 5-aminosalicylates than in those not cotreated (32.81% versus 11.11%, respectively, P < 0.001). The results of this study suggest that the value of TPMT genotyping before thiopurine therapy is limited in Chinese patients with IBD, considering the low sensitivity of predicting leucopenia, and that phenotyping is a more cost-effective tool that can be successfully used in patients. The coadministration of 5-aminosalicylates results in a high frequency of leucopenia in patients receiving azathioprine or 6-mercaptopurine.

Host Thiopurine Methyltransferase Status Affects Mercaptopurine Antileukemic Effectiveness

AbstractAbstract 3560Thiopurines are routinely used to treat many hematopoietic malignancies, including acute lymphoblastic leukemia (ALL), as well as inflammatory and autoimmune diseases. Common polymorphic null alleles among humans in thiopurine methyltransferase (TPMT) render 1 in 400 patients equivalent to homozygous knock-outs (−/−), 10% of patients equivalent to hemizygous (+/−), and 90% of patients comparable to wild-type (+/+) status for TPMT enzyme activity. TPMT activity is inversely related to the accumulation of active thioguanine nucleotide metabolites among patients, resulting in a higher risk for myelosuppressive adverse effects if routine doses are given to −/− compared to +/− compared to +/+ patients. However, the impact of the host TPMT polymorphism on antileukemic efficacy in the clinic is less clear.We generated a murine model of ALL by transducing bone marrow cells of Arf-null mice on the 129X1SvJ background (Tpmt +/+) with a retroviral vector encoding p185 BCR-ABL and luciferase, and culturing them for 7–10 days. We injected 2000 BCR-ABL+ pro/pre-B cells into Tpmt-deficient (−/−), heterozygous (+/−), or wild type (+/+) unconditioned syngenic recipients and compared the antileukemic efficacy when treating with a relatively low dose of mercaptopurine (equivalent to 7–9 mg/m2—such as would be tolerated by patients homozygous for null TPMT alleles). Treatment was started one day after injection of leukemia cells and continued for 70 days or until mice were sacrificed due to moribund state (usually due to illness from leukemia). To mimic clinically relevant treatment regimens, methotrexate was injected intraperitoneally (IP) at 1 mg/kg, once a week. Mercaptopurine was administered in the drinking water at 10 mg/L, for an estimated delivered dose of 2 – 2.5 mg/kg/day of mercaptopurine.Erythrocyte TPMT activity segregated by Tpmt genotype as expected and was comparable to the differences observed in patients: 0.6 U/ml in Tpmt −/−, 6.7 U/ml in Tpmt +/−, and 11.6 U/ml in Tpmt +/+ mice (p =0.008, n=2 for each genotype). As expected, the median survival (± standard error) was similar among Tpmt genotypes in untreated mice: 16 (± 0.4) days for Tpmt +/+, 17 (± 0.7) days for Tpmt +/−, and 18 (± 0.4) days for Tpmt −/−. The difference in leukemia-free survival by Tpmt genotype was profound: at 30 days after the start of treatment, 5% (± 9%) of Tpmt +/+ mice, 47% (± 26%) of Tpmt +/− mice, and 85% (± 14%) of Tpmt −/− mice were surviving (p = 5×10−8, Figure), indicating a substantial impact of host Tpmt status on thiopurine antileukemic effectiveness. In vivo imaging of luciferase-marked ALL cells confirmed an intermediate response phenotype for the Tpmt heterozygous mice (p = 0.00001). With higher doses (closer to the doses tolerated by patients homozygous for wild-type alleles, equivalent to 50–70 mg/m2/day), leukemia-free survival improved in the Tpmt +/+ and Tpmt +/− mice, comparable to the survival of Tpmt −/− mice treated with the low dose of mercaptopurine. These findings support the notion that germline polymorphisms in Tpmt, even in the heterozygous state, affect not only host tissue toxicity, but antitumor effectiveness as well. [Display omitted] Disclosures:Evans:St. Jude Children’s Research Hospital: Dr. William Evans receives a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms., Dr. William Evans receives a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms. Patents & Royalties. Relling:St. Jude Children’s Research Hospital: Dr. Mary Relling receives a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms and GGH polymorphisms., Dr. Mary Relling receives a portion of the income St. Jude receives from licensing patent rights related to TPMT polymorphisms and GGH polymorphisms. Patents & Royalties; Sigma-Tau Pharmaceuticals: Research Funding.

Establishment of Thiopurine S-Methyltransferase Gene Knockdown in Jurkat T-lymphocytes

Thiopurine S-methyltransferase (TPMT) is an excellent example of an enzyme whose pharmacogenetic polymorphisms affect efficacy and toxicity of a drug. The association between TPMT activity and thiopurine-related myelosuppression is well recognized. To study the significance of TPMT deficiency in thiopurine metabolism and immunosuppressive activity in vitro, we established RNA interference-based TPMT knockdown (kd) in a Jurkat cell line. In Jurkat TPMT kd cells, TPMT expression was reduced to 73% at the RNA level and 83% at the protein level. TPMT kd cells were more sensitive to 6-mercaptopurine (6-MP) (10 μmol/L) and 6-thioguanine (6-TG) (8 μmol/L) than wild-type (wt) cells, (32% versus 20%) and (18% versus 9%), respectively. Both Jurkat wt and kd cells were more sensitive to 6-TG-induced apoptosis than to 6-MP. 6-TG activity was also more affected by TPMT levels than was 6-MP as reflected by IC60, concentrations that is, 6-MP [4.6 μmol/L (wt) and 4.7 μmol/L (kd)], 6-TG [2.7 μmol/L (wt) and 0.8 μmol/L (kd)]. IC60 concentrations induced significant apoptosis in both Jurkat wt and kd cells (257%, versus 314%) with 6-MP and (323% versus 306%) with 6-TG, respectively. At IC60 (6-MP) 6-thioguanine nucleotides (6-TGN) accumulation in cells was 518 versus 447 pmol/million cells in wt and kd cells, respectively. On the other hand 6-TGN accumulation at IC60 (6-TG) was 477 versus 570 pmol/million cells in wt and kd cells, respectively. 6-Methylated mercaptopurine (6-MeMP) concentrations were more affected than 6-TGN by TPMT kd (194 versus 10 pmol/million cells) in wt and kd cells, respectively. We conclude that TPMT kd cells are an appropriate in vitro model to investigate the significance of TPMT deficiency with thiopurine therapy and could be helpful in understanding possible clinical consequences of TPMT polymorphism.

Azathioprine Induced Allergic Hepatitis in a Patient with Crohnʼs Disease

Purpose: Azathioprine (AZA) is a widely used immunomodulator in the treatment of inflammatory bowel disease (IBD). AZA has been associated with hepatotoxicity. Such hepatotoxicity can occur as an idiosyncratic reaction in the form of cholestatic jaundice or a hypersensitivity reaction with hepatocellular injury. Alternatively, it can be dose-dependent in the form of transaminitis, nodular regenerative hyperplasia, veno-oclusive disease, peliosis hepatitis, etc. Here, we report a rare case of AZA induced immunoallergic hepatitis: A 50-year-old female with a 5-year history of ileo-colonic Crohn's disease (CD) was started on AZA due to diarrhea and lower abdominal pain that were not controlled on 5-aminosalicylates. The patient's thiopurine methyltransferase activity was normal. Weekly monitoring of hepatic function profile for 5 weeks showed normal results. On week 6, the ALT and AST rose for the first time to 190 and 117 U/L. At week 7, ALT and AST further increased to 290 and 159 U/L, respectively. AZA was stopped with rapid decline of the enzymes back to normal within 10 days. During this period, the patient stated she may have had an upper respiratory infection, but denied any Tylenol, herbal medicine, alcohol or recreational drug intake. Viral serology for hepatitis A, B and C were all negative. Serum ANA, ASMA, ceruloplasmin, iron, transferrin saturation, ferritin were normal. Quantitative PCR for CMV in blood was negative. Peripheral smear did not suggest EBV infection. US and MRI of the abdomen revealed no evidence of biliary obstruction or other liver diseases. Concomitant treatments were lanzoprazole, hydrochlorothiazide, metoprolol, and mesalamine; and none of these had been recently introduced or withdrawn from the patient. After discussing potential treatment options and the risks of rechallenge, AZA was restarted at 25 mg daily. Upon rechallenge with a single dose, the patient began having flu-like symptoms, fatigue, nausea, vomiting and diarrhea within 24 hours. Further treatment with AZA was stopped. Her ALT and AST rose to 374 and 280 U/L in 24 hours; and continued to peak at 823 and 652 U/L, respectively, at 48 hours. Peak AP was 229 U/L and peak total bilirubin was 1.4 mg/dl. Of the six IBD cases reported to have idiosyncratic reactions to AZA to date, most are male, and the two CD cases have had cholestatic injury. This is the first case of CD with clear cut hepatocellular hypersensitivity reaction after a single ½ tablet rechallenge. The rapid rise in enzymes with minimal challenge within hours to levels far above the previous levels of elevation in our case suggests further caution, and requirement for daily LFTs in any patient undergoing a re-challenge protocol.