6-thioguanine Nucleotide Levels Research Articles

Long-term efficacy and tolerability of dose-adjusted thiopurine treatment in maintaining remission in inflammatory bowel disease patients with NUDT15 heterozygosity.

Background/AimsThiopurines are key drugs for inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn’s disease (CD). Recently, NUDT15 polymorphism (R139C, c.415C > T) has been shown to be associated with thiopurine-induced adverse events in Asian populations. In patients with the C/T genotype, low-dose thiopurine treatment is recommended, but its long-term efficacy and tolerability remain unclear. This study aimed to uncover the long-term efficacy and appropriate dosage of thiopurine for IBD patients with the C/T genotype.MethodsA total of 210 patients with IBD (103 UC and 107 CD) determined to have NUDT15 R139C variants were enrolled. Clinical data were retrospectively reviewed from medical records.ResultsOf 46 patients (21.9%) with the C/T genotype, 30 patients (65.2%) were treated with thiopurines. Three of whom (10.0%) discontinued thiopurine treatment due to adverse events and 27 of whom continued. The median maintenance dosage of 6-mercaptopurine was 0.25 mg/kg/day (range, 0.19–0.36 mg/kg/day), and 6-thioguanine nucleotides level was 230 (104–298) pmol/8 × 108 red blood cells. Cumulative thiopurine continuation rates for 120 months for patients with the C/C and C/T genotypes were not significantly different (P = 0.895). Cumulative non-relapse rates in the patients with UC treated with thiopurine monotherapy and surgery-free rates in CD patients treated with combination therapy (thiopurines and anti-tumor necrosis factor-α agents) for maintenance remission were not significantly different at 60 months (C/C vs. C/T, P = 0.339 and P = 0.422, respectively).ConclusionsLow-dose thiopurine treatment is an effective and acceptable treatment for patients with C/T genotype.

Systematic Review and Meta-Analysis of Thiopurine Metabolite Levels and Biochemical Remission in Autoimmune Hepatitis.

The utility of measuring thiopurine metabolites (TM) to individualize therapy in autoimmune hepatitis (AIH) has not been defined, and the evidence regarding its use in clinical practice is heterogeneous. This systematic review and meta-analysis aimed to compare the mean concentration of TM between patients in biochemical remission and those not in remission. A systematic literature search was conducted using PubMed, Scopus, the Cochrane Library, and Google Scholar for keywords related to TM and AIH. Two reviewers independently searched and selected studies comparing the levels of 6-methyl mercaptopurine (6-MMP) and 6-thioguanine nucleotide (6-TGN) and their ratio in cases of AIH in remission and otherwise. Meta-analysis was performed by calculating the weighted mean difference using the inverse variance heterogeneity model. A total of 1066 records were identified through systematic search; of which, 7 (n = 3 pediatric, n = 4 adults) were considered for inclusion, and 442 TM measurements (n = 128 in children) were analyzed. Mean 6-TGN levels were significantly higher among patients in remission than in those who were not, with a pooled weighted mean difference (WMD) of 15.67 [95% confidence interval (CI), 6.68-24.66] pmol/8 × 108 red blood cells (RBC). The difference was higher in the pediatric age group (WMD, 56.11; 95% CI, 13.60-98.62) than in adults (WMD, 13.77; 95% CI, 4.58-22.97). There was no significant difference in the 6-MMP levels (WMD, -431.7; 95% CI, -1237.4 to 373.9 pmol/8 × 108 RBC; I2 = 82%; n = 3 studies) or 6-MMP/6-TGN ratio among the patients who were in biochemical remission and those who were not (WMD, -0.97; 95% CI, -5.77 to 3.84; I2 = 82%; n = 3 studies). This meta-analysis suggests a link between 6-TGN levels and biochemical remission in AIH. Further high-quality studies are required to determine the therapeutic cutoff of 6-TGN.

Thalidomide Combined With Azathioprine as Induction and Maintenance Therapy for Azathioprine-Refractory Crohn's Disease Patients.

The combination therapy of thalidomide and azathioprine (AZA) offers an alternative in clinical practice for Crohn's disease (CD) patients experiencing a loss of response to AZA monotherapy. However, little is known about the efficacy and safety of this combination therapy for patients with CD. This was a retrospective study of 122 consecutive CD patients who lost response to AZA therapy and had switched to a combination therapy of thalidomide and AZA. The primary outcomes were clinical response and clinical remission rates at week 24. Patients who had an initial response to combination therapy were continued on the treatment for remission maintenance. The secondary outcomes were the proportion of clinical relapse throughout maintenance. The Kaplan–Meier method was used to calculate cumulative rates, and Cox regression analysis was used for multivariate analysis. During induction, 80.3% (98/122) patients achieved clinical response within a median duration of 6.5 weeks, (interquartile range, 4.3–8.1 weeks). The rate of clinical remission at 24 weeks was 70.5%. During follow-up, 22.4% (22/98) of the patients that were maintained on combination therapy experienced clinical relapse. The proportions of patients in remission status at 12, 24, and 36 months were 85.1, 78.3, and 70.1%, respectively. Multivariate analysis revealed C-reactive protein >10 mg/L at disease relapse on AZA monotherapy [adjusted hazard ratio (HR), 4.72; 95% CI, 1.19–18.75, P = 0.027] and 6-thioguanine nucleotides level ≥235 pmol/8 × 108 erythrocytes at AZA monotherapy (adjusted HR, 5.32; 95% CI, 1.40–20.14, P = 0.014) were associated with disease relapse on combination therapy. The endoscopic remission rate was 63.6%. Mucosal healing was achieved in 23.6% of the patients. Both Crohn's Disease Endoscopic Index of Severity (13.4 ± 4.92 vs. 6.12 ± 5.24, P < 0.001) and Rutgeerts scores (3.23 ± 0.73 vs. 1.77 ± 1.59, P = 0.003) were significantly decreased with the use of combination therapy. Adverse events occurred in 62 (50.8%) patients, but only 13 (10.7%) necessitated therapy discontinuation. Thalidomide combined with AZA was effective in inducing clinical remission and sustaining long-term steroid-free remission in CD patients who lost response to AZA monotherapy.

Thiopurines and their optimization during infliximab induction and maintenance: A retrospective study in Crohn's disease.

Combining therapy with a thiopurine is favored when commencing infliximab in Crohn's disease; however, the optimal 6-thioguanine nucleotide (TGN) level and how long to continue thiopurines after induction are uncertain. We aimed to compare outcomes after induction and during maintenance in combination therapy versus infliximab monotherapy in Crohn's and to examine whether TGN levels were associated with outcomes. Crohn's patients induced with infliximab with or without concomitant thiopurines were retrospectively identified. Response to induction and clinical outcomes in subsequent 6-month maintenance semesters were analyzed. A TGN level ≥235pmol/8×108 red blood cells was considered therapeutic. In 89 patients, response to induction was higher in combination therapy than monotherapy (74 vs 47%, P=0.04). This benefit was only seen in patients with a therapeutic TGN (odds ratio 3.72, confidence interval 1.07-13.0, P=0.04). Combination therapy during induction yielded a three times longer time to subsequent need for treatment escalation or treatment failure compared with monotherapy (29 vs 9months, P=0.01), with both therapeutic and subtherapeutic TGNs independent predictors on multivariate analysis. Among 370 semesters, there was no difference in outcomes between combination therapy and monotherapy (P=0.42), nor when combination semesters were stratified by therapeutic versus subtherapeutic TGN (P=0.56). In semester 1 only, a significantly higher remission rate was observed with therapeutic compared with subtherapeutic TGN (76% vs 33%, P=0.02). Combination therapy dosed with an optimized thiopurine was superior to infliximab monotherapy for induction of response, durability of response, and clinical outcomes in the first 6months following induction. Thereafter, combination therapy yielded no clinical advantage, supporting consideration of thiopurine withdrawal on a case-by-case basis.

Thiopurines' Metabolites and Drug Toxicity: A Meta-Analysis.

Many questions remain unanswered regarding therapeutic drug monitoring (TDM) utility with thiopurines. This study aims to establish a relationship between thiopurines’ metabolites and drug toxicity. We performed a systematic review with inclusion of studies evaluating the relationship between thiopurines’ metabolites and drug toxicity. Meta-analysis of mean difference (MD), correlations and odds ratio (OR) was performed. We identified 21,240 records, 72 of which were eligible for meta-analysis. Levels of 6-thioguanine nucleotides (6-TGN) were higher in patients with leukopenia (MD 127.06 pmol/8 × 108 RBC) and gastrointestinal intolerance (MD 201.46 pmol/8 × 108 RBC), and lower in patients with hepatotoxicity (MD −40.6 pmol × 108 RBC). We established a significant correlation between 6-TGN and leukocytes (r = −0.21), neutrophils (r = −0.24) and alanine aminotransferase levels (r = −0.24). OR for leukopenia in patients with elevated 6-TGN was 4.63 (95% CI 2.24; 9.57). An optimal cut-off of 135 pmol/8 × 108 RBC for leukopenia was calculated (sensitivity 75.4%; specificity 46.4%). 6-methylmercaptopurine ribonucleotides (6-MMPR) were significantly associated with hepatotoxicity (MD 3241.2 pmol/8 × 108 RBC; OR 4.28; 95% CI 3.20; 5.71). Levels of 6-MMPR measured in the first 8 weeks of treatment were associated with leukopenia. We conclude that TDM could be used to prevent thiopurines’ toxicity. As optimal metabolites level may vary according to indication, physicians may adapt posology to decrease toxicity without compromising efficacy.

Adjustment of azathioprine dose should be based on a lower 6-TGN target level to avoid leucopenia in NUDT15 intermediate metabolisers.

The association between NUDT15 polymorphisms and thiopurine-induced leucopenia is well known. To investigate the association between NUDT15 polymorphisms and time-to-leucopenia in paediatric patients with inflammatory bowel disease (IBD) receiving azathioprine and to determine the relationship between NUDT15 polymorphisms and 6-thioguanine nucleotide (6-TGN) levels. This retrospective observational study included Korean paediatric patients with IBD who were treated with azathioprine and underwent NUDT15 and TPMT genotyping. Azathioprine doses were adjusted by regular thiopurine metabolite monitoring. Factors associated with time-to-leucopenia and the relationship between NUDT15 polymorphisms and 6-TGN levels were analysed. Among the 167 patients included, leucopenia was observed in 16% (19/119), 44% (20/45) and 100% (3/3) of the NUDT15 normal, intermediate and poor metabolisers respectively (P<0.001). NUDT15 polymorphism was significantly associated with time-to-leucopenia (HR=5.26, 95% CI=2.74-10.09, P<0.001). There was a positive association between 6-TGN levels and leucopenia among the NUDT15 intermediate/TPMT normal metabolisers (median 361.3 vs 263.8pmol/8×108 RBC, P=0.013). The most accurate 6-TGN cut-off level associated with leucopenia was 308.2pmol/8×108 RBC (AUC=0.742, 95% CI=0.569-0.915, sensitivity 80.0%, specificity 72.7%, P<0.001) in this subgroup. When the specificity was set to <15%, the 6-TGN cut-off level was 167.1pmol/8×108 RBC (sensitivity 93.3%, specificity 13.6%). NUDT15 polymorphisms were associated with time-to-leucopenia during azathioprine treatment in Korean paediatric patients with IBD. In order to reduce the development of thiopurine-induced leucopenia (<15%) in NUDT15 intermediate metabolisers, adjustment of azathioprine doses should be based on a lower 6-TGN target level (<167.1pmol/8×108 RBC).

Jsou změny krevního obrazu použitelným ukazatelem velikosti dávky azathioprinu?

The 6-thioguanine nucleotide (6-TGN) level, may be used to estimate dose-adequacy of azathioprine (AZA) therapy. 6-TGN test is not commercially available. The aim of the study was to determine whether a blood cell changes correlate also with the dose of AZA and may serve as a predictor of the dose adequacy (for MCV &gt; 6 fl).Retrospective, multicentre study in subjects with IBD treated with azathioprine. Demographic data, leukocyte, platelet counts, erythrocyte (MCV) and thrombocyte (MPV) volume, azathioprine dose, inflammatory activity in the 3rd, 6th and 12th months of treatment and presence of sideropenia were recorded.103 subjects analysed. To increase the MCV by 6 fl, the AZA dose above 2 mg/kg is needed (p = 0.04). The MCV increases within 165 days (95% CI, 154-181 days, p = 0.002). Sideropenia has no impact on the MCV change. Number of leukocytes and thrombocytes decreases during treatment (p &lt; 0.001). Change in their number as well as MPV, does not correlate with MCV change and is not affected by activity of the inflammation.The MCV dynamics (&gt; 6 fl within 6 months) is the only relevant indicator during AZA treatment. Changes in the number of leukocytes, platelets and their volume can not be used to assess the sufficiency of the AZA dose. Sideropenia has no impact on the dynamics of MCV.

Review article: opportunities to improve and expand thiopurine therapy for autoimmune hepatitis.

Thiopurines in combination with glucocorticoids are used as first-line, second-line and maintenance therapies in autoimmune hepatitis and opportunities exist to improve and expand their use. To describe the metabolic pathways and key factors implicated in the efficacy and toxicity of the thiopurine drugs and to indicate the opportunities to improve outcomes by monitoring and manipulating metabolic pathways, individualising dosage and strengthening the response. English abstracts were identified in PubMed by multiple search terms. Full-length articles were selected for review, and secondary and tertiary bibliographies were developed. Thiopurine methyltransferase activity and 6-tioguanine (6-thioguanine) nucleotide levels influence drug efficacy and safety, and they can be manipulated to improve treatment response and prevent myelosuppression. Methylated thiopurine metabolites are associated with hepatotoxicity, drug intolerance and nonresponse and their production can be reduced or bypassed. Universal pre-treatment assessment of thiopurine methyltransferase activity and individualisation of dosage to manipulate metabolite thresholds could improve outcomes. Early detection of thiopurine resistance by metabolite testing, accurate estimations of drug onset and strength by surrogate markers and adjunctive use of allopurinol could improve the management of refractory disease. Dose-restricted tioguanine (thioguanine) could expand treatment options by reducing methylated metabolites, increasing the bioavailability of 6-tioguanine nucleotides and ameliorating thiopurine intolerance or resistance. The efficacy and safety of thiopurines in autoimmune hepatitis can be improved by investigational efforts that establish monitoring strategies that allow individualisation of dosage and prediction of outcome, increase bioavailability of the active metabolites and demonstrate superiority to alternative agents.

P478 Thiopurines’ metabolites levels and drug toxicity: a systematic review and meta-analysis

Abstract Background Besides Inflammatory Bowel Disease, thiopurines are used in a broad range of clinical conditions. Even though therapeutic drug monitoring (TDM) is widely used with biologics, there are still many unanswered questions regarding TDM for thiopurines. This study aimed to establish a relationship between the levels of thiopurines’ metabolites and treatment toxicity. Methods Literature search was carried out in 3 databases (Pubmed, Web of Science and Scopus) following PRISMA guidelines. Studies evaluating the relationship between the levels of thiopurines’ metabolites and their toxicity (haematological and hepatoxicity, pancreatitis, intolerance and infection), regardless of the disease to which they were prescribed, were included. Results From the 16194 identified records 74 were eligible for further analysis. A meta-analysis of mean comparisons, correlations and odds ratio (OR) were performed. The levels of 6-thioguanine nucleotides (6-TGN) were significantly higher in patients with general toxicity and leukopenia when the Lennard method was used for quantification (Figure 1). Significant correlations were obtained between the 6-TGN levels and leucocytes (r = -0.21;p &amp;lt; 0.001), neutrophils (r = -0.34;p &amp;lt; 0.001) and alanine aminotransferase (ALT) levels (r = -0.24;p &amp;lt; 0.001). The pooled OR (95%IC) for general toxicity, haematological toxicity and leukopenia in patients with 6-TGN concentrations above the predefined thresholds was 2.78(1.54–5.00), 8.54(3.55–20.56) and 5.26(3.34–8.15), respectively. Regarding 6-methylmercaptopurine metabolites (6-MMP), there was a significant correlation of their levels with general toxicity and leukopenia when the Lennard method was used for quantification (Figure 2), and with hepatotoxicity. 6-MMP levels were significantly correlated with ALT (r = 0.35;p &amp;lt; 0.001). The pooled OR (95%IC) for haematological toxicity, leukopenia and hepatotoxicity in patients with 6-MMP above the predefined thresholds was 4.12(1.80–9.47), 4.09(1.98–8.45) and 4.31(3.21–5.78), respectively. The studies evaluating the relationship between the ratio 6-MMP/TGN and thiopurines’ toxicity did not find differences between the mean ratio in patients with haematological toxicity and leukopenia. The pooled OR for hepatotoxicity in patients with a ratio above the predefined threshold was 4.44(IC 95%2.92–6.75). Conclusion These results reinforce that TDM may be used to prevent the occurrence of toxicity during thiopurines’ treatment. However, the cut-off values for this association may depend on the method used to measure metabolites’ levels.

P453 Comparison of therapeutic effects between groups of thiopurine alone and combination of thiopurine with 5-ASA after remission introduced by oral tacrolimus for patients with severe ulcerative colitis

Abstract Background Although thiopurine is recommended to be used for maintenance after remission, the reliable data of maintenance introduced by tacrolimus is limited for patients with ulcerative colitis (UC). 5-aminosalicylic acid (5-ASA) is reported to induce 6-thioguanine nucleotides (6-TGN) levels higher in patients with inflammatory bowel diseases. However, the data of 5-ASA are few reported among East Asians. Methods A retrospective cohort study was conducted evaluating the 70 patients with severe UC who were primary responders to oral tacrolimus from April 2015 to March 2018. Twenty-seven patients were administered maintenance treatment with thiopurine. We evaluated the efficacy of thiopurines with and without 5-ASA in these patients, using ΔMCV, lowest WBC, highest 6TGN between groups of thiopurine alone and thiopurine+ 5-ASA. Kaplan–Meier analysis was used to assess time to relapse between groups of thiopurine and thiopurine+5-ASA. Results The median follow-up period was 430 days (interquartile range 207–952 days). The statistical significances were not found in patients background between groups of thiopurine and thiopurine+5-ASA. ΔMCV were significantly greater (p &amp;lt; 0.01), lowest WBC were significantly lower (p = 0.02) in the thiopurines+5-ASA group than in thiopurines alone group. The highest 6-TGN levels tended to be higher in thiopurine+5-ASA group than in thiopurine alone group (p = 0.09). The rate of relapse was significantly higher in the thiopurine alone group than in thiopurines+5-ASA group (p = 0.03). Kaplan–Meier curves confirmed that thiopurine+5-ASA group appeared to protect against relapse (log-rank test, p &amp;lt; 0.01). Conclusion Thiopurine+5-ASA induced significantly lower relapse than thiopurine alone after remission introduced by tacrolimus in the patients with severe UC, along with significantly greater the ΔMCV and lower the lowest WBC.

Nucleoside diphosphate-linked moiety X-type motif 15 R139C genotypes impact 6-thioguanine nucleotide cut-off levels to predict thiopurine-induced leukopenia in Crohn's disease patients.

BACKGROUNDThiopurine-induced leukopenia (TIL) is a life-threatening toxicity and occurs with a high frequency in the Asian population. Although nucleoside diphosphate-linked moiety X-type motif 15 (NUDT15) variants significantly improve the predictive sensitivity of TIL, more than 50% of cases of this toxicity cannot be predicted by this mutation. The potential use of the 6-thioguanine nucleotide (6TGN) level to predict TIL has been explored, but no decisive conclusion has been reached. Can we increase the predictive sensitivity based on 6TGN by subgrouping patients according to their NUDT15 R139C genotypes?AIMTo determine the 6TGN cut-off levels after dividing patients into subgroups according to their NUDT15 R139C genotypes.METHODSPatients’ clinical and epidemiological characteristics were collected from medical records from July 2014 to February 2017. NUDT15 R139C, thiopurine S-methyltransferase, and 6TGN concentrations were measured.RESULTSA total of 411 Crohn’s disease patients were included. TIL was observed in 72 individuals with a median 6TGN level of 323.4 pmol/8 × 108 red blood cells (RBC), which was not different from that of patients without TIL (P = 0.071). Then, we compared the 6TGN levels based on NUDT15 R139C. For CC (n = 342) and CT (n = 65) genotypes, the median 6TGN level in patients with TIL was significantly higher than that in patients without (474.8 vs 306.0 pmol/8 × 108 RBC, P = 9.4 × 10-5; 291.7 vs 217.6 pmol/8 × 108 RBC, P = 0.039, respectively). The four TT carriers developed TIL, with a median 6TGN concentration of 135.8 pmol/8 × 108 RBC. The 6TGN cut-off levels were 411.5 and 319.2 pmol/8 × 108 RBC for the CC and CT groups, respectively.CONCLUSIONThe predictive sensitivity of TIL based on 6TGN is dramatically increased after subgrouping according to NUDT15 R139C genotypes. Applying 6TGN cut-off levels to adjust thiopurine therapies based on NUDT15 is strongly recommended.

Level of UV Exposure, Skin Type, and Age Are More Important than Thiopurine Use for Keratinocyte Carcinoma Development in IBD Patients.

Retrospective studies observe an increased risk of keratinocyte carcinomas (KCs) in patients with inflammatory bowel disease (IBD) on thiopurine (TP) medication. The role of traditional risk factors such as skin type and sun protection behavior has not been studied in this population. This study aimed to examine traditional KC risk factors and thiopurine use on skin cancer development in an IBD cohort. Consecutive IBD patients were recruited from four specialist centers in Australia and New Zealand, each with varying UV exposure indices. Data pertaining to race, skin color, freckling and sun protection behavior, dose of TP therapy, and skin cancer development were elicited through a self-reported questionnaire. A total of 691 IBD patients were included with 62 reporting KC development. Thiopurine usage was similar among patients who developed skin cancer compared with those who did not (92% vs. 89%, p = 0.3). There was no statistically significant association between KC development and TP dose or 6-thioguanine nucleotide levels. In multivariate modeling, four factors were independently and significantly associated with KC: age over 61years old versus less than 30years old (OR 6.76; 95% CI 2.38-19.18), residing in Brisbane versus Christchurch (OR 3.3; 95% CI 1.6-6.8), never staying in the shade versus staying in the shade ≥ 50% of the time (OR 3.8; 95% CI 1.4-10.5), and having a skin type that never tanned versus other skin types (OR 6.9; 95% CI 2.9-16.0). Skin type, age, and sun protection behavior are more important risk factors for KC development than thiopurine medication use in this IBD population.

Low 6-thioguanine nucleotide level: Effective in maintaining remission in Chinese patients with Crohn's disease.

Measuring 6-thioguanine nucleotide (6-TGN) level is useful in optimizing dose of azathioprine (AZA) and monitoring for toxicity. Lower dose of AZA was suggested for maintenance of clinical remission in Asian patients than Caucasian patients with Crohn's disease (CD). However, the optimal 6-TGN threshold required in Asian patients is undetermined. Therefore, the aim of the current study is to explore the optimal 6-TGN threshold required in Asian patients with CD for maintenance of clinical remission. A retrospective cohort study in a tertiary referral center recruited 252 CD patients. The primary endpoint was disease relapse. The levels of 6-TGN and AZA dose were compared in remission group and relapse group. Remission rate was compared across the increased 6-TGN level and dose range. Patients with 6-TGN range of 0-180.94pmol/8×108 red blood cells (RBC) had lower remission rate compared with those with 180.94-255.50pmol/8×108 RBC (P=0.020). Quartile analysis showed that increasing 6-TGN level beyond 180pmol/8×108 RBC produced negligible gain in rate of remission. Frequency of adverse events significantly increased in patients with 6-TGN level>355pmol/8×108 RBC (8.0% with 6-TGN>355pmol/8×108 RBC vs 2.7% with 6-TGN<355pmol/8×108 RBC, P=0.035). Our study suggested that optimal 6-TGN threshold required to maintain clinical remission in Chinese patients was 180-355pmol/8×108 RBC.

NT5C2 germline variants alter thiopurine metabolism and are associated with acquired NT5C2 relapse mutations in childhood acute lymphoblastic leukaemia.

The antileukaemic drug 6-mercaptopurine is converted into thioguanine nucleotides (TGN) and incorporated into DNA (DNA-TG), the active end metabolite. In a series of genome-wide association studies, we analysed time-weighted means (wm) of erythrocyte concentrations of TGN (Ery-TGN) and DNA-TG in 1009 patients undergoing maintenance therapy for acute lymphoblastic leukaemia (ALL). In discovery analyses (454 patients), the propensity for DNA-TG incorporation (wmDNA-TG/wmEry-TGN ratio) was significantly associated with three intronic SNPs in NT5C2 (top hit: rs72846714; P = 2.09 × 10-10, minor allele frequency 15%). In validation analyses (555 patients), this association remained significant during both early and late maintenance therapy (P = 8.4 × 10-6 and 1.3 × 10-3, respectively). The association was mostly driven by differences in wmEry-TGN, but in regression analyses adjusted for wmEry-TGN (P < 0.0001), rs72846714-A genotype was also associated with a higher wmDNA-TG (P = 0.029). Targeted sequencing of NT5C2 did not identify any missense variants associated with rs72846714 or wmEry-TGN/wmDNA-TG. rs72846714 was not associated with relapse risk, but in a separate cohort of 180 children with relapsed ALL, rs72846714-A genotype was associated with increased occurrence of relapse-specific NT5C2 gain-of-function mutations that reduce cytosol TGN levels (P = 0.03). These observations highlight the impact of both germline and acquired mutations in drug metabolism and disease trajectory.

The Impact of Thiopurine Metabolite Monitoring on the Durability of Thiopurine Monotherapy in Pediatric IBD.

Thiopurine metabolite monitoring to proactively dose optimize to achieve therapeutic levels has not been used consistently, and it is unclear if this would lead to better outcomes. We aimed to compare 6-month outcomes between standard and optimized dosing strategies and define long-term predictors of thiopurine durability. Two hundred sixteen pediatric IBD patients with at least 2 6-thioguanine nucleotide (6-TGN) levels were grouped for analysis by start dose: >2.5 mg/kg/day AZA (group 1) or <2.5 mg/kg/day (group 2) and further subgrouped depending on whether dosing was optimized to achieve 6-TGN >235 pmol/8 × 108 RBC. The metabolites, 6TGN and 6MMP, were univariate and multivariate analyses tested associations among metabolite levels, laboratory data, and the primary outcome of 6-month steroid-free clinical remission (SFR) (HBI ≤4 for CD; partial Mayo Score [pMS] ≤2 for UC). Thiopurine durability was measured using Kaplan Maier survival analysis. 6-MP, azathioprine, pediatrics, therapeutic drug monitoring, pediatrics were measured a median 59 (43-76) days after initiation of thiopurine. Both dosing strategies led to similar initial 6-TGN levels (group 1 = median 209 [IQR: 155-272] with 25% of patients >235; group 2 = 196 [139-274] with 29% >235). Steroid-free clinical remission was achieved in 74% of the 180 still on thiopurines at 6 months. Start dose was not associated with 6-month SFR-73% in group 1 and 77% in group 2 within those on thiopurines at 6 months (P = 0.61). Fixed- and optimized-dosing subgroups had similar 6-month 6-TGN levels, SFR rate, and percentage 6-TGN > 235. Only 6-TGN level >235 at 6 months predicted thiopurine durability (3 years [1.7-7.7] vs 2.5 years [0.83-5]; log-rank P < 0.001), and this did not retain significant in a multivariate model. Initial dosing strategy, first 6-TGN level, 6-month SFR, 6MMP:6TGN ratio, and delta-MCV did not predict durability. The rate of adverse events was 22%. Steroid-free clinical remission and 6-TGN levels at 6 months were no different between a standardized, fixed dosing strategy and a metabolite-driven, optimized dosing strategy.

6-Thioguanine Nucleotide Levels Are Associated With Mucosal Healing in Patients With Crohn's Disease.

Level of 6-thioguanine nucleotides (6-TGN) has been reported to be associated with clinical remission in patients with Crohn's disease (CD) receiving maintenance treatment with thiopurines. Whether 6-TGN levels are associated with mucosal healing (MH) has seldom been investigated. We aimed to assess the correlation between 6-TGN levels and MH in patients with CD. This was a retrospective, cross-sectional, observational, multicenter study of 119 patients with CD treated with thiopurines in 3 inflammatory bowel disease referral centers (France, Australia, and China) between June 2012 and April 2016. Established CD patients who underwent ileocolonoscopy during thiopurine treatment were included. MH was defined as simple endoscopic score-CD <3. Univariate and multivariable regression analyses were used to evaluate variables associated with MH. The mean concentration of 6-TGN in the MH group was higher compared with that in the non-MH group (359.0 ± 226.7 pmol/8 × 108 red blood cell count [RBC] vs 277.1 ± 170.5 pmol/8 × 108 RBC; P = 0.017). The cutoff 6-TGN concentration of 397.3 pmol/8 × 108 RBC was 86.7% specific to MH, with a sensitivity of 35.3% and area under curve (AUC) of 0.631 (P = 0.010). On multivariable analysis, 6-TGN levels were associated with MH (odds ratio [OR], 3.287; 95% confidence interval [CI], 1.348-8.017; P = 0.009) whereas late initiation of AZA (longer duration from disease onset) was inversely associated with MH (OR, 0.972; 95% CI, 0.954-0.991; P = 0.004). Higher 6-TGN levels are independently associated with a reduced rate of endoscopically active disease and a higher rate of mucosal healing in CD patients. Prospective studies of adequate sample size are required to confirm these findings.

MSH6 haploinsufficiency at relapse contributes to the development of thiopurine resistance in pediatric B-lymphoblastic leukemia.

Survival of children with relapsed acute lymphoblastic leukemia is poor, and understanding mechanisms underlying resistance is essential to developing new therapy. Relapse-specific heterozygous deletions in MSH6, a crucial part of DNA mismatch repair, are frequently detected. Our aim was to determine whether MSH6 deletion results in a hypermutator phenotype associated with generation of secondary mutations involved in drug resistance, or if it leads to a failure to initiate apoptosis directly in response to chemotherapeutic agents. We knocked down MSH6 in mismatch repair proficient cell lines (697 and UOCB1) and showed significant increases in IC50s to 6-thioguanine and 6-mercaptopurine (697: 26- and 9-fold; UOCB1: 5- and 8-fold) in vitro, as well as increased resistance to 6-mercaptopurine treatment in vivo. No shift in IC50 was observed in deficient cells (Reh and RS4;11). 697 MSH6 knockdown resulted in increased DNA thioguanine nucleotide levels compared to non-targeted cells (3070 vs. 1722 fmol/μg DNA) with no difference observed in mismatch repair deficient cells. Loss of MSH6 did not give rise to microsatellite instability in cell lines or clinical samples, nor did it significantly increase mutation rate, but rather resulted in a defect in cell cycle arrest upon thiopurine exposure. MSH6 knockdown cells showed minimal activation of checkpoint regulator CHK1, γH2AX (DNA damage marker) and p53 levels upon treatment with thiopurines, consistent with intrinsic chemoresistance due to failure to recognize thioguanine nucleotide mismatching and initiate mismatch repair. Aberrant MSH6 adds to the list of alterations/mutations associated with acquired resistance to purine analogs emphasizing the importance of thiopurine therapy.

Role of TPMT and ITPA variants in mercaptopurine disposition.

To explore the levels of thioguanine incorporated into DNA (DNA-TG), and erythrocyte levels of 6-thioguanine nucleotides (Ery-TGN) and methylated metabolites (Ery-MeMP) during 6-mercaptopurine (6MP)/Methotrexate (MTX) therapy of childhood acute lymphoblastic leukemia (ALL) and the relation to inosine triphosphatase (ITPA) and thiopurine methyltransferase (TPMT) gene variants. Blood samples were drawn during 6MP/MTX maintenance therapy from 132 children treated for ALL at Rigshospitalet, Copenhagen. The samples were analysed for thiopurine metabolites and compared to TPMT (rs1800460 and rs1142345) and ITPA (rs1127354) genotypes. Median DNA-TG (mDNA-TG) levels were higher in TPMT and ITPA low-activity patients as compared to wildtype patients (TPMTLA 549 vs. 364 fmol/µg DNA, p = 0.007, ITPALA 465 vs. 387 fmol/µg DNA, p = 0.04). mDNA-TG levels were positively correlated to median Ery-TGN (mEry-TGN)(rs = 0.37, p = 0.001), but plateaued at higher mEry-TGN levels. DNA-TG indices (mDNA-TG/mEry-TGN) were 42% higher in TPMTWT patients as compared to TPMTLA patients but no difference in DNA-TG indices was observed between ITPAWT and ITPALA patients (median 1.7 vs. 1.6 fmol/µg DNA/ nmol/mmol Hb, p = 0.81). DNA-TG indices increased with median Ery-MeMP (mEry-MeMP) levels (rs = 0.25, p = 0.001). TPMT and ITPA genotypes significantly influence the metabolism of 6MP. DNA-TG may prove to be a more relevant pharmacokinetic parameter for monitoring 6MP treatment intensity than cytosolic metabolites. Prospective trials are needed to evaluate the usefulness of DNA-TGN for individual dose adjustments in childhood ALL maintenance therapy.

P551 6-Thioguanine nucleotide levels are associated with mucosal healing in patients with Crohn’s disease: A multi-centre, international study

The level of 6-thioguanine nucleotides (6-TGN) has been reported to be associated with clinical remission in patients with Crohn’s disease (CD) receiving maintenance treatment with thiopurines. Whether 6-TGN levels are associated with mucosal healing (MH) has seldom been investigated. We aimed to assess the correlation between 6-TGN levels and MH in patients with CD. This was a retrospective cross-sectional observational multi-centre study of 119 patients with CD treated with thiopurines in three IBD referral centres (France, Australia and China) between June 2012 and April 2016. Established CD patients who underwent ileocolonoscopy during thiopurine treatment were included. MH was defined as simple endoscopic score-CD <3. Univariate and multivariable regression analyses were used to evaluate variables associated with MH. The mean concentration of 6-TGN in MH group was higher compared with that in non-MH group (359.0 ± 226.7 pmol/8 × 108 RBC vs. 277.1 ± 170.5 pmol/8 × 108 RBC) (p = 0.017). The cut-off 6-TGN concentration of 397.3 pmol/8x108RBC was 86.7% specific for MH, with a sensitivity of 35.3% and area under curve (AUC) of 0.631 (p = 0.010). On multivariate analysis, 6-TGN levels were associated with MH (odds ratio [OR] 3.287, 95% confidence interval [CI] 1.348–8.017; p = 0.009) while late initiation of AZA (longer duration from disease onset) was inversely associated with MH (OR 0.972, 95% CI 0.954–0.991; p = 0.004). Higher 6-TGN levels are independently associated with a reduced rate of endoscopically active disease and a higher rate of mucosal healing in CD patients. Prospective studies of adequate sample size are required to confirm these findings.

Addition of Allopurinol for Altering Thiopurine Metabolism to Optimize Therapy in Patients with Inflammatory Bowel Disease.

Thiopurine drugs, including azathioprine and 6-mercaptopurine, are used commonly in patients with inflammatory bowel disease for maintenance of remission. Although generally well tolerated, adverse effects lead to discontinuation in a significant minority of patients. Pharmacogenomic studies have suggested that metabolic breakdown of azathioprine in an individual is genetically determined. Coupled with the fact that certain thiopurine metabolites, notably 6-thioguanine nucleotide and 6-methylmercaptopurine, are associated with antiinflammatory effects and adverse effects, respectively, some investigators have examined intentionally shunting the metabolism of azathioprine toward increasing 6-thioguanine nucleotide levels by using low doses of the xanthine oxidoreductase inhibitor allopurinol to improve efficacy and decrease toxicity of azathioprine in patients with inflammatory bowel disease. We performed a search of the MEDLINE and Embase databases for basic and clinical research reports of this modality. Pertinent articles were retrieved, reviewed, and assessed by the authors. Case series, cohort studies, and one randomized trial have investigated adding allopurinol to azathioprine therapy in patients with inflammatory bowel disease. Most reports primarily examined metabolite levels in these patients. In general, the literature suggests that this modality was successful at significantly increasing 6-thioguanine nucleotide levels while decreasing 6-methylmercaptopurine levels. Several small reports have suggested that patients with increased 6-thioguanine nucleotide levels had improved symptoms or symptom remission. Adverse effects and discontinuation rates remained similar or were improved in patients who were taking a thiopurine and started allopurinol. In conclusion, the addition of allopurinol may be an option for optimizing thiopurine metabolite production in select patients with low 6-thioguanine nucleotide levels. Appropriate care and monitoring of these patients are mandatory to prevent neutropenia or other adverse effects.