Normal Thiopurine Methyltransferase Research Articles

Additive effects of TPMT and NUDT15 on thiopurine toxicity in children with acute lymphoblastic leukemia across multiethnic populations.

Thiopurines such as mercaptopurine (MP) are widely used to treat acute lymphoblastic leukemia (ALL). Thiopurine-S-methyltransferase (TPMT) and Nudix hydrolase 15 (NUDT15) inactivate thiopurines, and no-function variants are associated with drug-induced myelosuppression. Dose adjustment of MP is strongly recommended in patients with intermediate or complete loss of activity of TPMT and NUDT15. However, the extent of dosage reduction recommended for patients with intermediate activity in both enzymes is currently not clear. MP dosages during maintenance were collected from 1768 patients with ALL in Singapore, Guatemala, India, and North America. Patients were genotyped for TPMT and NUDT15, and actionable variants defined by the Clinical Pharmacogenetics Implementation Consortium were used to classify patients as TPMT and NUDT15 normal metabolizers (TPMT/NUDT15 NM), TPMT or NUDT15 intermediate metabolizers (TPMT IM or NUDT15 IM), or TPMT and NUDT15 compound intermediate metabolizers (TPMT/NUDT15 IM/IM). In parallel, we evaluated MP toxicity, metabolism, and dose adjustment using a Tpmt/Nudt15 combined heterozygous mouse model (Tpmt+/-/Nudt15+/-). Twenty-two patients (1.2%) were TPMT/NUDT15 IM/IM in the cohort, with the majority self-reported as Hispanics (68.2%, 15/22). TPMT/NUDT15 IM/IM patients tolerated a median daily MP dose of 25.7 mg/m2 (interquartile range = 19.0-31.1 mg/m2), significantly lower than TPMT IM and NUDT15 IM dosage (P < .001). Similarly, Tpmt+/-/Nudt15+/- mice displayed excessive hematopoietic toxicity and accumulated more metabolite (DNA-TG) than wild-type or single heterozygous mice, which was effectively mitigated by a genotype-guided dose titration of MP. We recommend more substantial dose reductions to individualize MP therapy and mitigate toxicity in TPMT/NUDT15 IM/IM patients.

Impact of Duodenal Pathology on Oral Drug Bioavailability and Disease Outcomes in Pediatric Crohn’s Disease

Crohn's disease with upper gastrointestinal tract involvement occurs more often in children than adults and has the potential to interfere with oral drug absorption. We aimed to compare disease outcomes in children receiving oral azathioprine for the treatment of Crohn's disease with (DP) and without (NDP) duodenal pathology at diagnosis. Duodenal villous length, body mass index (BMI), and laboratory studies were compared in DP vs. NDP during the first year post-diagnosis, using parametric/nonparametric tests and regression analysis (SAS v9.4); the data are reported as the median (interquartile range) or the mean ± standard deviation. Thiopurine metabolite concentration (pmol/8 × 108 erythrocytes) 230-400 was considered therapeutic for 6-thioguanine nucleotides (6-TGN), and >5700 was considered hepatotoxic for 6-methylmercaptopurine (6-MMPN). Twenty-six of the fifty-eight children enrolled (29 DP, 29 NDP) started azathioprine for standard medical care, including nine DP and ten NDP who had normal thiopurine methyltransferase activity. Duodenal villous length was significantly shorter in DP vs. NDP (342 ± 153 vs. 460 ± 85 μm; p < 0.001) at diagnosis; age, sex, hemoglobin, and BMI were comparable between groups. A trend toward lower 6-TGN was observed in the DP vs. NDP subset receiving azathioprine (164 (117, 271) vs. 272 (187, 331); p = 0.15). Compared to NDP, DP received significantly higher azathioprine doses (2.5 (2.3, 2.6) vs. 2.2 (2.0, 2.2) mg/kg/day; p = 0.01) and had an increased relative risk of sub-therapeutic 6-TGN. At 9 months post-diagnosis, children with DP had significantly lower hemoglobin (12.5 (11.7, 12.6) vs. 13.1 (12.7, 13.3) g/dL; p = 0.01) and BMI z-scores (-0.29 (-0.93, -0.11) vs. 0.88 (0.53, 0.99); p = 0.02) than children with NDP. For children with Crohn's disease, duodenal pathology, marked by villous blunting, increased the risk of sub-therapeutic 6-TGN levels, despite higher azathioprine dosing during the first year post-diagnosis. Lower hemoglobin and BMI z-scores at 9 months post-diagnosis suggest the impaired absorption/bioavailability of nutrients, as well as oral drugs, in children with duodenal disease.

Anagen Effluvium due to Azathioprine in a Patient With Normal Thiopurine S-Methyltransferase Levels: A Case Report Using Videodermoscopic Analysis

Introduction: Anagen effluvium (AE) is caused by impaired mitotic activity of matrix cells in the hair follicles. It presents with sudden loss of hair and myelosuppression. The diagnosis is based on the clinical history, trichoscopy, and trichogram. We herein report a case in which videodermoscopy revealed new trichoscopic features of AE caused by azathioprine in a patient with normal thiopurine S-methyltransferase (TPMT) levels. Case presentation: A 20-year-old woman presented with sudden loss of hair following treatment with oral azathioprine. Evaluation of the TPMT were within normal limits both in genotype and phenotype levels. Hair pull test was positive. Trichoscopy revealed yellow dots and multiple black dots, empty yellow dots, dark lines, upright regrowing hairs, and circle hairs. Azathioprine was stopped, and minoxidil 5% and oral multivitamin were started. Posttreatment trichoscopic analysis revealed terminal hairs and upright regrowing hairs. Discussion: AE is characterized by abrupt loss of anagen hairs due to impaired metabolic activity. Trichoscopy in AE shows coudability hairs, black dots, exclamation mark hairs, multiple broken shafts, and regrowing hairs. Empty yellow dots, yellow dots with black dots, pigtail hairs, and dark lines were additional features in this study. Conclusion: Trichoscopy assists in the accurate diagnosis of AE. Adverse effects of azathioprine can be observed even with normal TPMT levels. Weekly monitoring is therefore of prime importance.

Azathioprine-induced severe pancytopenia: A serious complication in a patient with normal TPMT activity

Azathioprine is an immunomodulator commonly used in renal transplant recipients and to treat lupus erythematous, and inflammatory bowel disease, and as second-line therapy in cases of rheumatoid arthritis. Here, we present a case of non-segmental vitiligo treated with low-dose azathioprine developing life-threatening pancytopenia with febrile neutropenia and trephine biopsy-proven drug-induced myelosuppression. The patient needed broad-spectrum antibiotics and granulocyte colony-stimulating factor (G-CSF) support with intermittent transfusion of blood products to completely recover from myelosuppression. It took almost a month for the patient to completely recover from the cytotoxicity caused by azathioprine. Interestingly, the patient had normal thiopurine methyl transferase (TPMT) activity and wild-type TPMT allele. We tried to outline how these types of patients can be managed and to emphasize the need for continuous monitoring of blood counts in patients receiving azathioprine to prevent life-threatening cytopenia even with normal TPMT activity and wild-type TPMT allele.

Azathioprine‐induced pancytopenia: A case report

AbstractAzathioprine (AZA) is commonly used as immunosuppressive therapy for autoimmune diseases, including systemic lupus erythematosus (SLE). Myelosuppression is a common side effect of AZA. Here we report a case of severe myelosuppression following AZA therapy in a 15‐year‐old girl despite a normal thiopurine methyltransferase (TPMT) level. She had been receiving AZA for SLE and presented with neutropenic fever and pancytopenia. AZA was stopped. After stopping AZA, her blood counts steadily improved. When TPMT genotyping results were normal, AZA was reintroduced. Pancytopenia reappeared after starting AZA, despite normal TPMT genotype. AZA was replaced with mycophenolate mofetil which consequently resulted in improvement of blood counts. It is essential to understand the temporal relationship between AZA use and pancytopenia onset in patients with normal TPMT activity. This case illustrates that regular monitoring of blood cell counts should be routine practice after starting AZA regardless of TPMT activity.

LC-MS/MS Method for Measurement of Thiopurine Nucleotides (TN) in Erythrocytes and Association of TN Concentrations With TPMT Enzyme Activity.

Monitoring concentrations of thiopurine metabolites is used clinically to prevent adverse effects in patients on thiopurine drug therapy. We developed a LC–MS/MS method for the quantification of 6-thioguanine (6-TG) and 6-methylmercaptopurine (6-MMP) in red blood cells (RBCs). This method utilizes an automated cell washer for RBC separation from whole blood samples and washing of the separated RBCs. The lower limit of quantification of the method was 0.2 μmol/L for 6-TG (∼50 pmol/8 × 108 RBC) and 4 μmol/L for 6-MMP (∼1,000 pmol/8 × 108 RBC). The total imprecision of the assay was <3.0%. The upper limit of linearity for 6-TG and 6-MMP was 7.5 μmol/L and 150 μmol/L, respectively. The stability of the thiopurine metabolites under pre- and post-analytically relevant conditions was also evaluated. A good agreement was observed between this method and validated LC–MS/MS methods from three laboratories, except for ∼40% low bias for 6-MMP observed in one of the methods. The assessment of the association between 6-TG and 6-MMP concentrations with thiopurine S-methyltransferase (TPMT) phenotype and genotype demonstrated a statistically significant difference in the thiopurine metabolite concentrations between the TPMT groups with normal and intermediate activity of 6-MMP (p < 0.0001), while the difference in 6-TG concentrations was statistically not significant (p = 0.096). Among the samples with normal TPMT activity, higher concentrations of 6-MMP (p = 0.015) were observed in pediatric samples than in the samples of adults. No statistically significant differences were observed in the distributions of 6-TG and 6-MMP concentrations among the evaluated genotypes.

Low-dose azathioprine and allopurinol versus azathioprine monotherapy in patients with ulcerative colitis (AAUC): An investigator-initiated, open, multicenter, parallel-arm, randomised controlled trial.

SummaryBackgroundRetrospective studies suggest that for patients with ulcerative colitis (UC) combination therapy with low-dose azathioprine and allopurinol (L-AZA/ALLO) may result in higher remission rates than monotherapy with azathioprine (AZA). We prospectively investigated the effects of these drugs for remission in patients with moderate-to-severe UC.MethodsOpen-label, unblinded, randomised, controlled, investigator-initiated, multicentre study conducted at eight hospital sites in Denmark. Adult patients with established UC, who were steroid dependent/refractory, thiopurine naïve, had a normal thiopurine methyltransferase, and achieved remission with steroids or infliximab were eligible for inclusion. Patients were randomly assigned by the investigators (1:1) to 52 weeks of treatment with once daily oral AZA (median dose 50 mg) combined with ALLO 100 mg versus AZA monotherapy (median dose 200 mg), using a computer-generated randomisation list with blocks of six. The trial was open without masking. All randomised patients who received at least one dose of study drug were included in primary and safety analyses (intention to treat population). The primary outcome was steroid and infliximab free remission after 52 weeks, defined as a Mayo Score of ≤1 and no rectal bleeding. The trial is completed and is registered in ClinicalTrials.gov (ClinicalTrials.gov NCT03101800).FindingsBetween January 9, 2017 and February 10, 2021, 47 patients were randomised to l-AZA/ALLO and 42 to AZA and received at least one dose of the study drug. After 52 weeks, 20 of 47 (43%) patients in the l-AZA/ALLO group and nine of 42 (21%) patients in the AZA group achieved remission (odds ratio 2·54 [95% CI 1·00 to 6.78, p < 0·048]). Fourteen patients (30%) in the l-AZA/ALLO group and 16 (38%) in the AZA group were withdrawn from the study due to adverse events.InterpretationThis study suggests that after one year l-AZA/ALLO therapy may be associated with a beneficial effect on steroid- and infliximab-free clinical remission in patients with moderate-to-severe UC and should be considered as first line therapy.FundingFunding for AAUC was provided by The Capital Region of Denmark (Regionernes Medicinpulje (6062/16)).

797 High Thiopurine Methyltransferase Activity Does Not Affect Concentration of Azathioprine Metabolite 6-Thioguanine Nucleotide in Inflammatory Bowel Disease

INTRODUCTION: Azathioprine (AZA) is commonly used to treat inflammatory bowel disease (IBD). Thiopurine methyltransferase (TPMT) converts AZA to key active metabolite 6-thioguanine nucleotide (6-TGN). Low TPMT activity is associated with high 6-TGN and side effects. Conversely, low 6-TGN is associated with lack of clinical response and has been associated with high TPMT activity but has not been examined in an IBD population. The aim of this study was to identify the independent effect of normal to high TPMT activity on 6-TGN concentrations in an IBD population. METHODS: We performed a retrospective chart review of patients aged &gt;18 with IBD treated with AZA and with documented TPMT activity and 6-TGN levels from 2010 to 2017. To account for different assays, TPMT activity was normalized against the established low-normal TPMT activity, with low activity defined as less than 1 and high activity defined as above the 75th quantile of TPMT activity in the cohort. Those with low TPMT were excluded. Linear regression was used to determine the effect of TPMT activity on 6-TGN accounting for potential confounders. Further assessment of the interaction between dose and TPMT activity on 6-TGN was measured through the Average Causal Mediation Effects (ACME). Statistics performed in R v3.6.0 using mediation package. This study was IRB approved. RESULTS: Dosing of AZA in mg/kg, TPMT activity, and 6-TGN concentration were documented in 63 patients, of which 10 had low TPMT activity and were subsequently excluded, leaving 53 patients for analysis. Overall 37% of the cohort had a therapeutic 6-TGN level of &gt;240 pmol/8 × 108 RBC. No patient under 1mg/kg achieved a therapeutic 6-TGN level, whereas 50% of patients taking 2.5 mg/kg did. The median 6-TGN in the normal TPMT range was 191.5 (IQR: 78.8, 296.3) versus 201 (IQR: 45, 312) in the high group (P = 0.57). There was a weak positive correlation between dosing (mg/kg) and 6-TGN (R2 = 0.08), while no correlation was seen between TPMT and 6-TGN (R2 = 0.0). Linear regression to assess the effect of TPMT activity and dose on 6-TGN concentrations including potential confounders of sex, age, smoking status only identified dose (mg/kg) to have a significant effect on 6-TGN concentration (Table 1). CONCLUSION: There was no effect on 6-TGN as TPMT activity increased, although 6-TGN was positively affected by dose. Despite normal TPMT activity, therapeutic 6-TGN concentrations were only found in 50% of those dosed at 2.5 mg/kg.

Retrospective Data Analysis of the Influence of Age and Sex on TPMT Activity and Its Phenotype-Genotype Correlation.

Therapeutic efficacy and toxicity of thiopurine drugs (used as anticancer and immunosuppressant agents) are affected by thiopurine S-methyltransferase (TPMT) enzyme activity. TPMT genotype and/or phenotype is used to predict the risk for adverse effects before drug administration. Inosine triphosphate pyrophosphatase (ITPA) is another enzyme involved in thiopurine metabolism. In this study, we aimed to evaluate (a) frequency of various TPMT phenotypes and genotypes, (b) correlations between them, (c) influence of age and sex on TPMT activity, and (d) distribution of ITPA variants among various TPMT subgroups. TPMT enzyme activity was determined by LC-MS/MS. TPMT (*2,*3A-C) and ITPA (rs1127354, rs7270101) genotypes were determined using a customized TaqMan® OpenArray®. TPMT enzyme activity varied largely (6.3-90 U/mL). The frequency of low, intermediate, normal, and high activity was 0.5% (n = 230), 13.1% (n = 5998), 86.1% (n = 39448), and 0.28% (n = 126), respectively. No significant difference in TPMT activity in relation to age and sex was found. Genotype analysis revealed the frequency of variant TPMT alleles was 6.73% (*3A, n = 344), 0.05% (*3B, n = 2), 2.22% (*3C, n = 95), and 0.42% (*2, n = 19). Analysis of paired phenotype and genotype showed that TPMT activity in samples with variant allele(s) was significantly lower than those without variant alleles. Lastly, an equal distribution of ITPA variants was found among normal and abnormal TPMT activity. This retrospective data analysis demonstrated a clustering of variant TPMT genotypes with phenotypes, no significant influence of age and sex on TPMT activity, and an equal distribution of ITPA variants among various TPMT subgroups.

Frequency of serious adverse events of thiopurine treatment in normal thiopurine S-methyltransferase genotype children with inflammatory bowel disease

Genetic polymorphism of thiopurine S-methyltransferase, the key enzyme in metabolism of thiopurines (azathioprine and 6-mercaptopurine) used in the treatment of inflammatory bowel disease, results in different enzyme activities. Decreased enzyme activity causes myelosuppression whereas abnormally high activity results in hepatotoxicity at standard thiopurine doses. Four allele variants (TMPT*2, TMPT*3A, TPMT*3B and TPMT*3C) account for decreased activity in more than 95% of cases. To evaluate the frequency of severe side effects, such as myelosuppression and hepatotoxicity, at standard or decreased azathioprine doses in children with inflammatory bowel disease who do not exhibit any of the four most common variant alleles. Retrospective analysis of children with inflammatory bowel disease treated with azathioprine at a single tertiary referral center. 51 patients were identified (44: Crohn's disease, 7: ulcerative colitis; male ratio: 28/51; mean age at diagnosis: 12.4 years). Two patients discontinued azathioprine arbitrarily whereas in one patient it was stopped due to serious pancreatitis and in another one because of severe flu-like symptoms. None of the remaining 47 patients exhibited hepatotoxicity suggesting abnormally high thiopurine S-methyltransferase activity. Four patients (8.5%) had profound myelosuppression on less than 1 mg/kg/day azathioprine requiring discontinuation of the drug, and all of them showed complete bone marrow recovery subsequently. No myelosuppression occurred in the remaining 43 patients on 2.17 ± 0.31 mg/kg/day (mean ± SD) azathioprine treatment. Regular blood tests are necessary on thiopurine therapy despite normal thiopurine S-methyltransferase genotype because of the risk of myelosuppression. The four most common variant alleles were identified in routine genotyping only, therefore most likely rare variant allele(s) or polymorphism of other enzymes involved in thiopurine metabolism account for the aforementioned four cases with profound bone marrow suppression. Thiopurine metabolite monitoring is the key for individualized treatment when optimal dosing can be achieved with the least side effects. Orv Hetil. 2019; 160(5): 179-185.

P677 Azathioprine dosing and metabolite measurement in paediatric inflammatory bowel disease—does one size fit all?

Azathioprine (AZA) is an effective immunomodulatory maintenance therapy in children with inflammatory bowel disease (IBD). Current recommended dosing is 2–2.5 mg/kg/dose in the presence of normal thiopurine methyltransferase (TPMT) level. Elevated levels of the AZA metabolites 6-TGN and 6-MMP have been associated with increased risk of myelotoxicity and hepatotoxicity, however evidence suggests that monitoring metabolites can help optimise effectiveness and safety of thiopurines. We aim to assess association between AZA weight-based dosing, thiopurine metabolites and disease activity index in a cohort of paediatric IBD patients. Retrospective data review (2015–2017) from the paediatric gastroenterology department at Evelina London Children’s Hospital. Children with IBD who were taking AZA and had regular thiopurine metabolites measurement were included. PCDAI and PUCAI were used to assess disease activity. A total of 41 patients, 38% female, 62% male with mean (±SD) age of diagnosis 12.2 (±3.4) years, were included. Twelve patients had ulcerative colitis (UC), 27 Crohn’s disease (CD) and 2 very early onset IBD (VEOIBD). Mean AZA dose, in mg/kg (±SD), 1.3 (±0.08). Mean 6TGN value (±SEM) 260 (±25.2), and mean MMT value (±SEM) 1023 (±168). Thiopurine metabolites were measured at (mean ± SEM) after (75 ± 13.5) days after starting AZA, 5 patients had elevated 6TGN levels >450. Mean lymphocyte count at time of metabolite measurement (±SD) 1.7 (±0.8). Activity index at the start of thiopurine were (±SD) 22.5 (±16.3) and when metabolites were measured 8.1 (±12). We have identified that the majority of our patients maintain clinical remission (defined by an activity index—PUCAI/PCDAI—of less than 10) on lower AZA doses than currently recommended. We have also identified a number of patients with elevated 6TGN levels without signs of myelosuppression. Higher doses of AZA are associated with higher metabolite levels, and we propose that regular routine measurement of AZA metabolites will help optimise therapy, minimise side effects and identify children at risk of hepato- and myelotoxicity.

Randomized clinical trial: a pilot study comparing efficacy of low-dose azathioprine and allopurinol to azathioprine on clinical outcomes in inflammatory bowel disease

Background: Treating inflammatory bowel diseases (IBD) using thiopurines is effective; however, a high rate of adverse effects and lack of efficacy limit its use. Retrospective studies have suggested that treatment with low-dose thiopurines in combination with allopurinol is associated with higher remission rates and lower incidence of adverse events.Aim: To compare the rates of clinical remission and the rates of adverse events in IBD patients treated with either standard treatment with azathioprine or low-dose azathioprine in combination with allopurinol.Methods: A prospective, open-label study, randomizing thiopurine-naïve IBD patients with normal thiopurine methyltransferase to 24 weeks of treatment with either standard azathioprine dose or low-dose azathioprine and allopurinol.Results: A total of 46 patients with ulcerative colitis or Crohn’s disease were randomized. We conducted an intention to treat analysis and found a significant (69.6%) proportion of the patients treated with low-dose azathioprine in combination with allopurinol was in clinical remission without the need for steroid or biologic treatment at week 24 compared to 34.7% of the patients treated with azathioprine monotherapy (RR, 2.10 [95% CI: 1.07–4.11]). In the azathioprine group, 47.8% of the patients compared to 30.4% of the patients in the azathioprine–allopurinol group had to withdraw from the study due to adverse events (RR, 1.47 [95% CI: 0.76–2.85])Conclusions: This study indicated that by changing the treatment strategy from standard weight-based dosing of azathioprine to weight-based low-dose azathioprine in combination with allopurinol, we can increase remission rates in patients with IBD.

Association between Thiopurine S-Methyltransferase Polymorphisms and Azathioprine-Induced Adverse Drug Reactions in Patients with Autoimmune Diseases: A Meta-Analysis.

PurposeAzathioprine (AZA) is widely used as an immunosuppressive drug in autoimmune diseases, but its use is limited by significant adverse drug reactions (ADRs). Thiopurine S-methyltransferase (TPMT) is an important enzyme involved in AZA metabolism. Several clinical guidelines recommend determining TPMT genotype or phenotype before initiating AZA therapy. Although several studies have investigated the association between TPMT polymorphisms and AZA-induced ADRs, the results are inconsistent. The purpose of this study is to evaluate whether there is an association between TPMT polymorphisms and AZA-induced ADRs using meta-analysis.MethodsWe explored PubMed, Web of Science and Embase for articles on TPMT polymorphisms and AZA-induced ADRs. Studies that compared TPMT polymorphisms with-ADRs and without-ADRs in patients with autoimmune diseases were included. Relevant outcome data from all the included articles were extracted and the pooled odds ratios (ORs) with corresponding 95% confidence intervals (CIs) were calculated using Revman 5.3 software.ResultsEleven published studies, with a total of 651 patients with autoimmune diseases, investigated associations between TPMT polymorphisms and AZA-induced ADRs, were included in this meta-analysis. Our meta-analysis demonstrated that TPMT polymorphisms were significantly associated with AZA-induced overall ADRs, bone marrow toxicity and gastric intolerance; pooled ORs were 3.12 (1.48–6.56), 3.76 (1.97–7.17) and 6.43 (2.04–20.25), respectively. TPMT polymorphisms were not associated with the development of hepatotoxicity; the corresponding pooled OR was 2.86 (95%CI: 0.32–25.86). However, the association in GI subset could be driven by one single study. After this study was excluded, the OR was 2.11 (95%CI: 0.36–12.42); namely, the association became negative.ConclusionsOur meta-analysis demonstrated an association of TPMT polymorphisms with overall AZA-induced ADRs, bone marrow toxicity and gastric intolerance, but not with hepatotoxicity. The presence of the normal TPMT genotypes cannot preclude the development of ADRs during AZA treatment, TPMT genotyping prior to commencing AZA therapy cannot replace, may augment, the current practice of regular monitoring of the white blood cell. Because of small sample sizes, large and extensive exploration was required to validate our findings.

Imputation of TPMT defective alleles for the identification of patients with high-risk phenotypes.

Background: The activity of thiopurine methyltransferase (TPMT) is subject to genetic variation. Loss-of-function alleles are associated with various degrees of myelosuppression after treatment with thiopurine drugs, thus genotype-based dosing recommendations currently exist. The aim of this study was to evaluate the potential utility of leveraging genomic data from large biorepositories in the identification of individuals with TPMT defective alleles.Material and methods: TPMT variants were imputed using the 1000 Genomes Project reference panel in 87,979 samples from the biobank at The Children's Hospital of Philadelphia. Population ancestry was determined by principal component analysis using HapMap3 samples as reference. Frequencies of the TPMT imputed alleles, genotypes and the associated phenotype were determined across the different populations. A sample of 630 subjects with genotype data from Sanger sequencing (N = 59) and direct genotyping (N = 583) (12 samples overlapping in the two groups) was used to check the concordance between the imputed and observed genotypes, as well as the sensitivity, specificity and positive and negative predictive values of the imputation.Results: Two SNPs (rs1800460 and rs1142345) that represent three TPMT alleles (*3A, *3B, and *3C) were imputed with adequate quality. Frequency for the associated enzyme activity varied across populations and 89.36–94.58% were predicted to have normal TPMT activity, 5.3–10.31% intermediate and 0.12–0.34% poor activities. Overall, 98.88% of individuals (623/630) were correctly imputed into carrying no risk alleles (553/553), heterozygous (45/46) and homozygous (25/31). Sensitivity, specificity and predictive values of imputation were over 90% in all cases except for the sensitivity of imputing homozygous subjects that was 80.64%.Conclusion: Imputation of TPMT alleles from existing genomic data can be used as a first step in the screening of individuals at risk of developing serious adverse events secondary to thiopurine drugs.

Tu1938 Identification of New Genetic Variants Related to Thiopurine-Induced Myelotoxicity in Inflammatory Bowel Disease (IBD) Patients With Normal Thiopurines-Methyltransferase (TPMT): A Genome-Wide Association Study

Tu1938 Identification of New Genetic Variants Related to Thiopurine-Induced Myelotoxicity in Inflammatory Bowel Disease (IBD) Patients With Normal Thiopurines-Methyltransferase (TPMT): A Genome-Wide Association Study

Su1416 Thiopurines in the Management of Crohn's Disease: Safety and Efficacy Profile in Patients With Intermediate and Normal Thiopurine Methyltransferase Activity, a Retrospective Study

Su1416 Thiopurines in the Management of Crohn's Disease: Safety and Efficacy Profile in Patients With Intermediate and Normal Thiopurine Methyltransferase Activity, a Retrospective Study

Augmenting Clinical Interpretability of Thiopurine Methyltransferase Laboratory Evaluation

Objective: Individuals with decreased thiopurine methyltransferase (TPMT) activity are at risk of adverse effects of thiopurine administration whereas its increased activity may inactivate drugs faster. We evaluated genotype-phenotype correlations in patients with suspected hematological malignancies and inflammatory bowel disease from our region based on findings of nonlinear TPMT enzyme kinetics previously unreported. Patients and Methods: The study group comprised 267 individuals. They were screened for the most common variants of low TPMT activity. TPMT activity was measured in erythrocytes using the HPLC rate-blanked method. Results: Thirty-three patients (12.4%) were heterozygous (26 were TPMT*1/*3A, 5 TPMT*1/*2, 2 TPMT *1/*3C) and 1 was a compound heterozygote (*2/*3A). Normal and low normal TPMT activities substantially overlapped in wild-type and heterozygous individuals, whereas high activities were found in 29 wild-type genotyped patients. Extreme and life-threatening toxicity was observed in the compound heterozygote patient. Conclusion: Activity measurement performed at diagnosis provides clinicians with information on immediate pharmacokinetic-related adverse events and/or hypermetabolism, and genotyping may indicate the rate of pharmacodynamic thioguanine nucleotide accumulation due to slower overall thiopurine metabolism.

P651 Identification of new genetic variants related to thiopurine-induced myelotoxicity in inflammatory bowel disease (IBD) patients with normal thiopurines-methyltransferase (TPMT): a genome-wide association study

analyzed by gene array, immunofluorescence, flow cytometry and thymidine incorporation. Results Anti-TNF induced regulatory macrophages displayed increased numbers of autophagosomes as well as an increased expression of autophagy related transcripts including atg5, atg7, atg9 and atg16l2, suggesting induction of autophagy by IFX treatment. Of all donors, 7 were homozygously carrying the CD associated risk allele, 14 were heterozygous and 7 were homozygous for the WT allele. The number of CD14+ regulatory macrophages correlated significantly with the number of WT alleles present in each individual culture, with the largest number of macrophages found in cultures containing 3 or 4 WT alleles (2 WT donors or 1 WT and 1 heterozygous). Similarly, expression of CD206, which is associated with the immunosuppressive function of macrophages, positively correlated with the number of WT alleles present. To confirm the functional consequences of these findings, IFX mediated suppression of T cell proliferation was determined. Again, the level of suppression correlated significantly with the number of WT alleles present in the respective donor combinations. Conclusion Induction of regulatory macrophages by IFX is associated with induction of autophagy. In vitro, the number and function of IFX induced macrophages correlated with the number of WT alleles for the autophagy related gene ATG16L1. This suggests that an intact autophagy pathway is important for effectiveness of anti-TNF therapy.

Commentary: individual vs. weight‐based dosing of azathioprine in Crohn's disease

This study illustrates the difficulties of running a clinical study. There were several attempts to change the protocol, but low enrolment numbers resulted in an underpowered negative study. The primary end point of this study was clinical remission at 16 weeks. There was no difference in remission rates at 16 weeks, although the per-protocol analysis showed a trend towards better results in the individualised group (clinical remission in 3/12 in weight-based vs. 9/15 in the individualised group). These remission rates were below predicted levels, partly because a high proportion of patients had steroid dependency or steroid-refractory disease. The secondary outcomes were remission at 28 and 52 weeks, but only 4 patients were still in the study at week 52. This study shows that dose escalation does not always achieve the target concentration. For the individualised group, the mean 6-tioguanine (thioguanine) (6TGN) concentrations among normal thiopurine methyltransferase (TPMT) metabolisers was 198 pmol/8 9 10 red blood cells (significantly below the therapeutic target of 250, and not different from the mean 6TGN for the weight-based arm). These disappointingly low 6TGN levels may also explain the low remission rates. This study adds to the data showing the clinical benefit of achieving higher 6TGN levels and is consistent with a meta-analysis of 6TGN and IBD activity. There was a significant difference in 6TGN between patients achieving remission and those not achieving remission (216 vs. 149), although a wide range and significant overlap in values were observed. The study showed a modest difference in dose between individualised and weight-based groups (3.4 compared with 2.3 mg/kg for normal TPMT and 1.5 compared with 2.4 mg/kg for intermediate levels), but the trial was not long enough to observe a more significant divergence. A study by Gardiner et al. showed a two-fold divergence in dosing after 9 months of monitoring and dose change, although they predicted that a three-fold difference was likely to be more appropriate. The most useful test may be the initial TMPT activity with reduction of starting dose to 1.0 – 1.5 mg/kg for patients with intermediate TPMT (in this study, the numbers with intermediate TMPT were low and it is difficult to reach any conclusions). The only similar study is a randomised, open-label study where dose adjustment was based on 6TGN alone. Recruitment was also difficult, and the numbers in each group were small. Similar to this study, mean concentrations of 6TGN were below target in the dose-adjusted group and not different from the weightbased group. It is difficult to see this study being repeated. The concept of dose optimisation of azathioprine dose is now well accepted, and there will be reluctance among clinicians to enrol patients into a weight-based arm. There is a strong body of data, including studies with azathioprine/allopurinol combination, supporting this approach. Dose optimisation may be a slow process and not easy to replicate in a clinical trial. However, the disappointing remission rates in this study should not discourage use of azathioprine for an extended period with dose optimisation.

P-176 Thiopurine S-methyltransferase Analysis Does Not Correctly Predict the Risk of Toxicity in Hispanic Children with Inflammatory Bowel Disease

Thiopurine S-methyltransferase (TPMT) catalyzes the methlyation of thiopurine compounds, and is measured prior to using medications such as 6-mercaptopurine or Azathioprine. TPMT can be measured as blood enzyme levels or allelic variations (phenotype or genotype assay), both of which are important in distinguishing the possibility of desired clinical effects versus toxicity. The aim of our study was to evaluate the prevalence of variations in TPMT activity in pediatric Hispanic Inflammatory Bowel Disease (IBD) patients in our South Florida based Pediatric Gastroenterology practice, and to evaluate the reliability of TPMT levels in predicting toxicity risk in our studied population. Sixty-four pediatric Hispanic patients treated with either 6-mercaptopurine or Azathioprine for IBD were retrospectively studied. Either a radiochemical method to measure TPMT phenotype or TPMT genotype was analyzed to detect allelic variations and predisposition to myelotoxicity, hepatotoxicity, and pancreatotoxicity. Of the 64 Hispanic patients included (mean age: 16 years): 54.6% with Crohn's disease, 37.5% with Ulcerative Colitis, 4.6% with nonspecific colitis, and 3.1% with other unspecified abdominal complaints. TPMT phenotypes were documented in 50 patients and Genotype analysis was documented in 14 patients. TPMT phenotype was distributed as: Normal level 80% (40/50), Intermediate 18% (9/50), Low 2% (1/50). TPMT genotypic variations were distributed as: Normal 92.8% (13/14), Intermediate 7.1% (1/14), Low 0% (0/14). Toxicity was characterized by leukopenia with or without neutropenia, anemia, pancytopenia, elevated transaminases, or pancreatitis. The percentage of patients with toxicity regardless of normal or abnormal TPMT levels or alleles were 37.5%. This was further evaluated to find that 12.5% had liver toxicity, 15% had bone marrow toxicity, and 10% had both. The percentage of patients with toxicity despite normal TPMT phenotype was 22.5%. The percentage of patients with toxicity despite normal genotype was 23.07%. Our study demonstrates that 17% (11/64) of pediatric Hispanic patients in our practice have either an abnormal phenotype or genotype TPMT enzyme analysis, which is greater than previously documented in other ethnic groups. Additionally, the risk of toxicity despite normal TPMT enzyme levels and alleles was higher in our studied group compared to other previously studied populations, indicating that pediatric Hispanic patients must be monitored carefully at regular intervals to monitor for adverse reactions to thiopurine based medications.