6-mercaptopurine Research Articles

Asparaginase May Affect Mercaptopurine Tolerability in the Context of Multi-Agent Therapy for Acute Lymphoblastic Leukemia

▪Background: Mercaptopurine (MP) and asparaginase (ASP) are critical components in the treatment of acute lymphoblastic leukemia (ALL). Dose-limiting toxicities of the two drugs are common, resulting in therapy interruption, which has been associated with inferior treatment outcome in some studies. However, the interaction between these drugs has not been clearly identified. Merryman et al (Pediatr Blood Cancer 2012) reported that in DFCI ALL 05-01, patients had lower blood counts and more dosage reductions of MP during consolidation therapy (with concomitant ASP treatment) than during continuation therapy (identical treatment without concomitant ASP). Among groups of homogeneously treated patients with ALL, variability in ASP exposure due to inactivating antibodies can affect ASP pharmacodynamics: we have reported that ASP antibodies were associated with lower plasma ASP activity and higher dexamethasone (DEX) clearance, leading to a lower risk of osteonecrosis and a higher risk of CNS relapse (Liu, Leukemia 2012; Kawedia, Blood 2012). Here we studied the possible effect of ASP antibodies on MP tolerability in St. Jude Children's Research Hospital Total XV, a clinical trial featured intensive ASP treatment.Methods: A total of 390 children with ALL treated on St. Jude Total XV protocol were evaluable. TPMT genotype was used to guide starting doses of MP. During maintenance treatment, planned MP doses were higher on the low-risk arm (LR; n = 202) than on the standard/high-risk arm (SHR; n = 188). MP dose intensity was estimated as (prescribed dose)/(protocol dose) for weeks 1-146 (boys) or 1-120 (girls) for patients on the LR and SHR arms of maintenance therapy.Native E.coli-ASP (Elspar) was administered intramuscularly at 10000 U/m2 thrice weekly for 6 or 9 doses during remission induction. During maintenance therapy, patients on the LR arm received ASP only during reinductions I (weeks 7-9) and II (weeks 17-19), whereas those on the SHR arm received 19 weekly doses at 25000 U/m2 during weeks 1-19. Patients were tested for serum anti-Elspar antibodies at days 5, 19, 34 of remission induction, day 1 of reinduction I and day 1 of reinduction II, and were grouped based on whether they were ever positive for antibodies at any time during therapy or not. The area under the antibody concentration-time curve (AUC) for the entire period up to week 19 was also estimated in 360 patients.Result: Overall MP dose intensity was higher in those with vs without ASP antibodies in patients on the LR (median 83 vs 75%, P = 0.003) and SHR arms (median 86 vs 76%, P = 3.3 × 10-5; Figure 1A), and MP dose intensity was correlated with ASP antibody AUC in patients on both treatment arms (LR, P = 7.7 × 10-3 and SHR, P = 2.4 × 10-4; Figure 1B). In a multivariate model including age, sex, risk arm, ancestry, TPMT status, NUDT15 genotype and ASP antibody status, TPMT genotype was the strongest determinant of MP dose intensity (-17% in heterozygotes, P = 1.9 × 10-8), followed by ASP antibody positivity (+8.9% dose intensity in those with antibodies, P = 5.8 × 10-6). The model also confirmed previously identified associations of higher MP dose intensity with higher African ancestry (Bhatia et al. Blood 2014) (P = 1.8 × 10-4) and lower Asian ancestry (P = 0.05) (Yang et al. J Clin Oncol 2015).Conclusion: Interindividual differences in ASP systemic exposure, as reflected by ASP antibodies, had a strong impact on MP tolerance, especially in patients on the SHR arm who received intensive ASP therapy. We have previously shown that patients who are positive for ASP antibodies not only have lower exposure to ASP but also to dexamethasone (Kawedia, Blood 2012; Liu, Leukemia 2012). These data further emphasize the capacity for variation in ASP exposure to impact yet another critical component of ALL therapy. [Display omitted] DisclosuresEvans:Prometheus Labs: Patents & Royalties: Royalties from licensing TPMT genotyping. Relling:Prometheus Labs: Patents & Royalties: Royalties from licensing TPMT genotyping.

Rac1 as a Potential Pharmacodynamic Biomarker for Thiopurine Therapy in Inflammatory Bowel Disease

Azathioprine and mercaptopurine (MP) are effective in treating patients with inflammatory bowel disease (IBD). Immunosuppressive effects of thiopurines involve T-cell apoptosis after inhibition of GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1). This study aimed to assess whether expression and activity of Rac1 or phosphorylated ezrin-radixin-moesin (pERM) in patients with IBD could provide a useful biomarker for the pharmacodynamic thiopurine effect and might be related to clinical effectiveness. This was a 2-stage study: stage 1 concerned a cross-sectional cohort of patients with IBD clinically in remission and treated with (n = 10) or without stable weight-based thiopurine therapy (n = 11) and healthy controls (n = 6); stage 2 concerned a prospective study regarding IBD patients with clinically active disease who initiated MP therapy (n = 11) compared with healthy controls (n = 11). Expression and activity of Rac1 and ERM and pERM were determined. The median Rac1 expression was statistically significantly reduced by thiopurine maintenance therapy {0.54 [interquartile range (IQR) 0.47-0.88] versus 0.80 arbitrary units [IQR 0.64-1.46]} compared with patients without immunosuppressive therapy (P = 0.042), but not Rac1 activity and pERM. In responders to MP therapy (n = 6), both median active Rac1 [93 (IQR 81-151) to 76 ng Rac1/mg protein (IQR 62-98)] and Rac1 expression [16.2 (8.8-29.4) to 1.5 arbitrary units (0.9-5.3)] decreased (P = 0.028). In nonresponders (n = 3), Rac1 expression and activity increased. IBD patients treated with thiopurines had a lower expression of Rac1 compared with those not treated with thiopurine. Effective MP therapy led to decreasing concentrations of Rac1-GTP and Rac1 expression. Therefore, Rac1-GTP and expression of Rac1, but not phosphorylation of ERM, form potentially pharmacodynamic markers of therapeutic thiopurine effectiveness in patients with IBD.

Clinical Value of Mercaptopurine After Failing Azathioprine Therapy in Patients With Inflammatory Bowel Disease.

Thiopurines have been widely accepted as immunosuppressive therapy in inflammatory bowel disease. However, many patients have to discontinue thiopurines due to intolerance or ineffectiveness. A therapeutically beneficial effect of switching from azathioprine (AZA) to mercaptopurine (MP) after developing adverse events (AEs) has been reported. The authors assessed the clinical value of MP therapy after AZA discontinuation due to intolerance and, secondary, due to ineffectiveness. In this retrospective single-center study, the authors analyzed data from patients in whom AZA therapy had failed and who were subsequently treated with MP. Thirty-eight patients initiated MP therapy after intolerance to AZA. Intolerance reoccurred in 22 (58%) patients and the remaining 16 (42%) tolerated MP. In 18 out of 48 patients (38%), the AEs that led to discontinuation of MP were similar to those of AZA. A longer duration of prior AZA use was more common in patients who were subsequently tolerant for MP (5.3 versus 1.2 months; P = 0.04). Twenty-two patients discontinued AZA due to ineffectiveness. Eight (36%) patients had clinical benefit from a switch to MP. Six out of these 8 (75%) patients used allopurinol alongside MP, due to ineffectiveness based on a skewed thiopurine metabolism. Patients were more likely to have clinical benefit if the interval between both thiopurines was longer (4.4 versus 0.01 months; P < 0.05). The authors showed that a noteworthy number of patients benefitted therapeutically from a switch from AZA to MP when failing due to intolerance or ineffectiveness; however, the percentage was lower than previously reported in literature.

Pharmacogenomics for leukemia treatment.

The pharmacokinetics and pharmacodynamics of therapeutic drugs can greatly vary among individuals. For example, it is sometimes necessary to alter the treatment of childhood acute lymphoblastic leukemia from the standard protocol. Genetic variation is one important factor, which can exert a wide range of effects on sensitivities and responses to therapeutic agents. Thiopurine S-methyl transferase (TPMT) is a useful test for predicting 6-mercaptopurine (6-MP) sensitivity in Caucasians. However, it is not effective for predicting the 6-MP therapeutic responses of Japanese patients because the frequency of TPMT deficiency is lower in the Japanese population (approximately 1% versus approximately 10% in Caucasians). Recently, NUDT15 polymorphisms have been reported to be predictive factors contributing to responsiveness to thiopurine therapy in Asians. The associations between genetic variants and therapeutic responses have been reported in Western countries. However, questions remain about whether results studying other races are applicable to Japanese due to differences in genetic variant frequencies among races. To provide personalized therapy based on genetic factors, we need to ascertain the relationships between genetic variants and therapeutic responses in Japanese childhood acute lymphoblastic leukemia cases.

PTH-085 Deoxythioguanosine (DTG) in DNA as a Pharmacological Endpoint of Thiopurine Treatment in Inflammatory Bowel Disease Patients

Introduction We have used a sensitive assay to measure incorporated dTG in the DNA isolated from peripheral blood cells of patients treated with thiopurines with the aim of identifying a more appropriate pharmacological marker of response for patients on therapy with the thiopurines azathioprine (AZA) or mercaptopurine (MP). Methods DNA was isolated from blood samples collected from 38 IBD patients; 23 on AZA and 15 on MP. DNA was denatured, digested using P1 nuclease followed by treatment with alkaline phosphatase and finally diluted in MilliQ water prior to analysis. A total of 0.2 µg of DNA in 50 µL was injected for chromatographical separation followed by analysis on an API4000 triple quadrupole LC-MS/MS. Standard curves and controls were validated and samples analysed to determine number of moles of dTGN/ 1055 moles of dA. Results A cohort of patients with Crohn’s Disease (CD) on AZA (n = 11) or MP (n = 5) or Ulcerative Colitis (UC); AZA (n = 12), MP (n = 10) established on thiopurine therapy for more than 25 weeks and in clinical remission were analysed. For those on AZA only there was a statistically significant correlation between dTG levels and AZA dose (Kendall Rank correlation, P 0.05; median =1.05, mean = 1.0, SD = 1.0; range 0.0–3.3). When disease was taken into account there was no statistical difference in dTG levels between those taking AZA (Mann-Whitney, P > 0.9; for CD patients, median =0.89, mean = 2.13, SD = 3.34; for UC patients, median =1.11, mean = 1.19, SD = 2.61). However, for those taking MP there was a statistically-significant difference (Mann-Whitney, P Conclusion This pilot study shows that the levels of dTG in treated patients are similar whether the patients are on AZA (average dose 118 mg) or MP (average dose 39 mg equivalent to approximately 81 mg AZA). However, there were clear differences between the two drug-treatment groups with respect to the correlation of dTG and drug dose and in the relationship between dTG incorporation and disease type. These results justify a larger cohort study to investigate the role of drug-DNA interactions in clinical responses to thiopurines Disclosure of Interest None Declared

OC-072 The Toppic Trial: A Randomised, Double-Blind Parallel Group Trial of Mercaptopurine VS Placebo to Prevent Recurrence of Crohn’s Disease Following Surgical Resection in 240 Patients

<h3>Introduction</h3> Up to 65% of patients with Crohn’s disease (CD) require an operation to control the disease within the first ten years of the condition. This study assessed whether the use of mercaptopurine (MP) can prevent or delay post-operative recurrence in CD. <h3>Methods</h3> In this randomised, placebo-controlled, double-blind, parallel group trial patients with a confirmed diagnosis of CD undergoing intestinal resection were recruited from 29 UK hospitals. Following informed consent, each patient was randomised to receive a daily oral dose of MP or placebo. Dose was adjusted by weight and thiopurine methyltransferase (TPMT) status and safety monitoring was blinded. Treatment was for a maximum of 36 months. The primary endpoint was defined by clinical recurrence of CD (CDAI &gt; 150 plus 100 point rise) and the need for anti-inflammatory rescue therapy or primary surgical intervention. Secondary endpoints included endoscopic recurrence. <h3>Results</h3> 240 patients (median age 38 years, 60.8% female, 22.9% smokers were enrolled and received at least one dose of study drug. 128 (53%) were randomised to receive MP and 112 (47%) to placebo. No randomised patients were excluded from the analysis. More patients reached primary endpoint within the placebo (n = 26,23.2%) vs MP groups (n = 16,12.5%) with an adjusted p-value of 0.073 (Hazard Ratio (HR) 0.535, 95% CI 0.27–1.06) and an unadjusted p value of 0.046 (HR 0.527, 95% CI 0.28–0.99) Smokers were more likely to reach primary endpoint than non-smokers (p = 0.018 (HR 0.127,95% CI 0.04–0.46 NNT = 3 amongst smokers, HR 0.898, 95% CI 0.42–1.94 NNT = 31 amongst non-smokers). Smoking habit (HR 2.06, 1.09–3.90) but not age at diagnosis, duration of disease, sex, previous surgery or previous thiopurine or anti-TNF exposure predicted primary outcome. A higher proportion of patients on MP compared with placebo maintained complete endoscopic remission (Rutgeerts i0) at weeks 49 (29.7 v 14.4%, p = 0.006), and 157 (22.5% v 12.5%, p = 0.041) on post-hoc analysis. <h3>Conclusion</h3> TOPPIC is the largest, double-blind trial of thiopurines to prevent post-operative recurrence in CD. MP modestly reduces the frequency of clinical post-operative recurrence of CD. This was significant amongst smokers, but not in non-smokers. Adverse events did not differ between groups. <h3>Disclosure of Interest</h3> None Declared

PWE-028 A Novel Assay for The Direct Detection of Deoxythioguanosine (DTG) in DNA from Patients On Azathioprine Using Liquid Chromatography and Tandem Mass Spectrometry (LC-MS/MS)

Introduction The correlation between thiopurine drug metabolite levels in red blood cells (RBC) and clinical response is currently under debate, with some studies reporting a good correlation whereas others report a poor correlation. However, without a suitable alternative assay this is the only method presently available to guide dosing of patients treated with thiopurine drugs: azathioprine (AZA), mercaptopurine (MP) or thioguanine. Biologically, this method is not perfect given that RBC lack the critical drug-metabolising enzyme inosine-monophosphate dehydrogenase, which is central to the bioconversion of MP to thioguanine nucleotides that are then incorporated as fraudulent bases into nucleic acids. To try to get a better indicator of drug response and hence clinical outcome, we have developed a sensitive liquid chromatography- tandem mass spectrometry (LC-MS/MS) assay to measure incorporated dTG in the DNA isolated from peripheral blood mononuclear cells of patients treated with thiopurines Methods DNA was isolated from blood samples collected from IBD patients on thiopurines. DNA was denatured then digested using P1 nuclease followed by treatment with alkaline phosphatase and finally diluted in MilliQ water prior to analysis. A total of 0.2 μg of DNA in 50 µL was injected for chromatographic separation followed by analysis on an API4000 triple quadrupole mass spectrometer. Standard curves and controls were validated and samples analysed to determine number of moles of dTG/105 5 moles of dA. Results From a small cohort of 20 AZA treated IBD patients (10 Crohn’s Disease and 10 Ulcerative Colitis) with dTG levels of 0.18 to 11.3 moles dTG/105 5 moles dA (approximately 5 to 340 femtomoles/μg DNA) were detected with no detectable dTGs in un-treated patients. The intra- and inter-assay variability was below 7.8% and 17.0% respectively with a detection limit of 15.5 pg (54.7 femtomoles) and was quantified in DNA samples relative to endogenous dA. Conclusion This method enables the direct detection of a cytotoxic thiopurine metabolite in an easily obtainable, stable sample and will facilitate a better understanding of the mechanisms of action of these inexpensive yet effective drugs. Disclosure of Interest None Declared

Does Immunosuppressive Therapy Increase the Risk for Cancer Recurrence

The immunomodulatory agents azathioprine and 6-mercaptopurine increase risk for lymphoma and nonmelanoma skin cancers. Anti–tumor necrosis factor (TNF)

853 The TOPPIC Trial: A Randomized, Double-Blind Parallel Group Trial of Mercaptopurine vs Placebo to Prevent Recurrence of Crohn's Disease Following Surgical Resection in 240 Patients

Introduction Up to 65% of patients with Crohn’s disease (CD) require an operation to control the disease within the first ten years of the condition. This study assessed whether the use of mercaptopurine (MP) can prevent or delay post-operative recurrence in CD. Methods In this randomised, placebo-controlled, double-blind, parallel group trial patients with a confirmed diagnosis of CD undergoing intestinal resection were recruited from 29 UK hospitals. Following informed consent, each patient was randomised to receive a daily oral dose of MP or placebo. Dose was adjusted by weight and thiopurine methyltransferase (TPMT) status and safety monitoring was blinded. Treatment was for a maximum of 36 months. The primary endpoint was defined by clinical recurrence of CD (CDAI > 150 plus 100 point rise) and the need for anti-inflammatory rescue therapy or primary surgical intervention. Secondary endpoints included endoscopic recurrence. Results 240 patients (median age 38 years, 60.8% female, 22.9% smokers were enrolled and received at least one dose of study drug. 128 (53%) were randomised to receive MP and 112 (47%) to placebo. No randomised patients were excluded from the analysis. More patients reached primary endpoint within the placebo (n = 26,23.2%) vs MP groups (n = 16,12.5%) with an adjusted p-value of 0.073 (Hazard Ratio (HR) 0.535, 95% CI 0.27–1.06) and an unadjusted p value of 0.046 (HR 0.527, 95% CI 0.28–0.99) Smokers were more likely to reach primary endpoint than non-smokers (p = 0.018 (HR 0.127,95% CI 0.04–0.46 NNT = 3 amongst smokers, HR 0.898, 95% CI 0.42–1.94 NNT = 31 amongst non-smokers). Smoking habit (HR 2.06, 1.09–3.90) but not age at diagnosis, duration of disease, sex, previous surgery or previous thiopurine or anti-TNF exposure predicted primary outcome. A higher proportion of patients on MP compared with placebo maintained complete endoscopic remission (Rutgeerts i0) at weeks 49 (29.7 v 14.4%, p = 0.006), and 157 (22.5% v 12.5%, p = 0.041) on post-hoc analysis. Conclusion TOPPIC is the largest, double-blind trial of thiopurines to prevent post-operative recurrence in CD. MP modestly reduces the frequency of clinical post-operative recurrence of CD. This was significant amongst smokers, but not in non-smokers. Adverse events did not differ between groups. Disclosure of Interest None Declared

Use of 6-Mercaptopurine in Autoimmune Hepatitis Patients Intolerant to Azathioprine

Approximately 10% of patients with autoimmune hepatitis (AIH) do not respond to treatment with azathioprine (AZA) or are intolerant of it. Although effective second-line agents exist (e.g., mycophenolate mofetil), some require drug-level monitoring, are expensive, or have significant adverse effects. Although AZA is a prodrug of 6-mercaptopurine (6-MP), AZA intolerance does not necessarily lead to 6-MP intolerance. Researchers retrospectively …

NT5C2 As a Major Contributor to Thiopurine Resistance at ALL Relapse Via Multiple Mechanisms

Background Relapse remains as a formidable challenge for acute lymphoblastic leukemia (ALL), particularly because it is associated with dramatic drug resistance and thus dismal prognosis. It is crucial to understand the genetic basis of relapsed ALL to develop new therapeutic strategies. Recently, somatic mutations in NT5C2 were identified as one of the most common genomic lesions specific to relapsed ALL and were directly linked to thiopurine resistance (Nat Genet 2013, Nat Med 2013, Nat Commun 2015). A nucleotidase, NT5C2 dephosphorylates purine monophosphate nucleotides and is important for cellular nucleic acid homeostasis. However, the exact mechanisms by which NT5C2 mutations influence thiopurine resistance in ALL are not completely understood.Methods Wildtype and mutant human NT5C2 (p.R39Q, p.R238W, and p.R367Q) were expressed in E. Coli and purified by affinity chromatography followed by gel filtration. Nucleotidase activity was measured by quantifying the release of free phosphate with the Malachite Green assay. NT5C2 enzyme kinetics (Vmax and Km) were determined for endogenous purine nucleotide substrates (inosine monophosphate [IMP] and guanosine monophosphate [GMP]) and thiopurine drug metabolite substrates (thioinosine monophosphate [TIMP] and thioguanosine monophosphate [TGMP]). Human ALL cell lines (Nalm6 and REH) stably overexpressing wildtype or mutant NT5C2 were established by lentiviral transduction. Thiopurine transport was characterized in vitro by quantifying the influx and efflux of 14 C labeled mercaptopurine (MP) and its metabolites.Results To characterize NT5C2 substrate specificity, we first determined the nucleotidase activity of wildtype and mutant proteins with endogenous purine nucleotides (IMP and GMP) and thiopurine metabolites (TIMP and TGMP). Wildtype NT5C2 exhibited nucleotidase activity against all 4 nucleotide metabolites, but IMP and TIMP were clearly the preferred substrates with up to 4-fold higher reaction velocities compared to GMP and TGMP, respectively. All leukemia-derived NT5C2 mutations resulted in substantial increase in nucleotidase activity across substrates, with the p.R367Q mutant consistently exhibiting the greatest gain of activity. By comparison, mutant NT5C2 proteins showed similar substrate preference of IMP over GMP (e.g., reaction rate of 26.6 pmol/min and 10.4 pmol/min for IMP and GMP, respectively, with the p.R367Q NT5C2). However, this inosine over guanosine bias in substrate specificity was reversed with thiopurine metabolites: leukemia-derived mutant NT5C2s were substantially more efficient to dephosphorylate TGMP than TIMP (e.g., reaction rate of 27.8 pmol/min and 41.5 pmol/min for TIMP and TGMP, respectively, with the p.R367Q NT5C2). The "mutant to wildtype" ratio of NT5C2 nucleotidase activity (e.g., the relative increase of Vmax) was significantly greater for thiopurine metabolites than what was observed when endogenous purine nucleotides were used as substrates (e.g., 15.2- and 1.6-fold increase in Vmax for TIMP and IMP with the p.R367Q NT5C2). These results suggest that ALL relapse-associated NT5C2 mutations likely confer not only increase in endogenous nucleotidase activity but also novel enzymatic functions that specially affect thiopurine metabolism.In addition to the increase in thiopurine resistance, human ALL cell lines expressing mutant NT5C2 also showed significant reduction in intracellular levels of thiopurine metabolites after drug treatment, pointing to influence of NT5C2 mutations on thiopurine drug transport. Using 14 C labeled MP, we traced both the influx and efflux of drug metabolites in isogenic cell lines expressing wildtype or mutant NT5C2 s. In both Nalm6 and REH cells, intracellular uptake of MP was reduced by 51% in mutant cells compared to cells with wildtype protein. In contrast, the level of MP metabolites in the extracellular medium was 3 times higher with mutant cells, reflecting increased drug efflux. Therefore, expression of mutant NT5C2 resulted in a net reduction of intracellular exposure to thiopurine, suggesting disturbance of the drug transport pathway as a distinct mechanism for NT5C2-mediated thiopurine resistance.ConclusionsNT5C2 mutations specifically influence thiopurine metabolism and transport, and therefore are critical determinants for drug resistance in relapsed ALL. DisclosuresNo relevant conflicts of interest to declare.

Early Combined Immunosuppression in Crohn Disease: A Community-Based Trial

In treatment-naive patients with Crohn disease, early treatment with a combination of a tumor necrosis factor (TNF) blocker and an antimetabolite (azathioprine, 6-mercaptopurine, or methotrexate), or early combined immunosuppression (ECI), is recognized to result in the highest remission rates. In an industry-funded, cluster-randomized study of community practices, investigators tested the efficacy of ECI versus conventional step-up management. The primary outcome was the rate of steroid-free remission at month 12. Patients were followed for 24 months. In ECI-randomized practices, after initial mandated steroid trials (4 weeks in …

Tu1328 Final Thiopurine Metabolite Levels and Shunter Status Can Be Predicted After Six Weeks of Thiopurine Therapy - Biochemical Outcomes From the EATME Study

Background: Dosing of thiopurines [azathioprine (AZA) and mercaptopurine (MP)] in the management of IBD has been based upon patient weight. However, this appears unreliable in predicting final levels of thiopurine metabolites, leading to suboptimal clinical outcomes because of underand over-dosing, and ‘shunting' in 15% where toxic 6-methylmercaptopurine (6MMP) is preferentially produced over the efficacious 6-thioguianine nucleotides (6TGN), with 6MMP:6TGN ratio ≥20. Aims: 1. To determine prospectively the clinical value of timing of measurement of thiopurine metabolites during dose-escalating regimen of thiopurine initiation in a consecutive cohort of IBD patients being initiated on a thiopurine. 2. To determine if the 6MMP:6TGN ratio changes during thiopurine dose escalation. Methods: In this single centre, prospective, open label study patients were commenced on either a daily dose of 50mg AZA or 25mg MP (physician discretion). Doses increased fortnightly by 50mg for AZA or 25mg for MP until target was achieved, aiming for 2-2.5mg/kg for AZA and 1-1.5mg/kg for MP. Hematology, CRP and liver function tests were performed fortnightly to monitor toxicity. Thiopurine metabolites were measured fortnightly until steady-state was achieved, but results were not used for dosing decisions. Clinical and biochemical outcomes were recorded. Landmark analyses of shunter status used logistic regression models. Metabolite levels were analysed using linear regression models. Results: 64 patients (52 Crohn's, 11 ulcerative colitis and 1 IBD-U) were enrolled. Final metabolite outcomes were: 11 (17%) patients were shunters, 27 (42%) were underdosed (6TGN level of 450) and 21 (33%) were therapeutic. 6MMP:6TGN ratios escalated over time in shunters, but not in others. After 2 weeks of therapy, a 6MMP:6TGN ratio above 9 (p=0.058) was suggestive of shunting, while after 6 weeks, a ratio ≥12 (95% CI 7-28, p=0.01) was predictive of shunting. 6TGN levels at week 2 (median 106, range 0-328) predicted final 6TGN outcomes (median282, range 50-746, r=0.681, p .10). Conclusions: 6MMP:6TGN ratios increase during dose escalation in shunters only. Patients with a 6MMP:6TGN ratio 12 or above after 6 weeks should be considered for optimisation with allopurinol rather than waiting until 12 weeks of therapy. Weight-based dosing of thiopurines should no longer be used in standard practice.

A Rapid and Robust HPLC- DAD Method for the Monitoring of Thiopurine Metabolites in Whole Blood: Application to Paediatric Patients with Inflammatory Bowel Disease

Mercaptopurine (MP) and azathioprine (AZA) are thiopurine drugs that are used in paediatrics for the treatment of inflammatory bowel disease (IBD), Therapeutic drug monitoring (TDM) of thiopurine active metabolites, 6-methylmercaptopurine (6MMP) and 6-thioguanine nucleotides (6-TGN) is a common practice for the assessment of toxicity surveillance and treatment optimization. In this paper we described the development and validation of a new High Performance Liquid Chromatography coupled to Diode Array Detection (HPLC-DAD) method for the simultaneous quantification of 6-TGN and 6-MMP in whole blood with a total runtime of 7.5 minutes over a wide range of concentrations (50 – 2000 pmol/8x10^8 RBC for 6-TG and 20 – 15000 pmol/8x10^8 RBC for 6-MMP respectively). The new method has been validated following international guidelines on bioanalytical method validation. Within- and between-run precision and accuracy results for quality control samples were always ≤ 15% of the nominal concentrations. The correlation with our reference method was excellent. The new HPLCDAD method that we describe is robust, rapid, cost effective and suitable for application to the routine TDM analyses.

Outstanding Outcome for Children with Standard Risk-Low (SR-Low) Acute Lymphoblastic Leukemia (ALL) and No Benefit to Intensified Peg-Asparaginase (PEG-ASNase) Therapy: Results of Children’s Oncology Group (COG) Study AALL0331

▪Introduction: Overall, children with new-onset SR (age 1-9 years (yr), WBC <50,000/microliter) B-ALL enrolled on COG AALL0331 achieved excellent 5-yr event free (EFS) (89%) and overall survival (OS) (96%) (Maloney, ASH 2013). AALL0331 utilized a common 3-drug induction (IND), with post-IND assignment into refined risk groups per clinical, cytogenetic, and early response criteria. The SR-Low group was defined by favorable cytogenetics (triple trisomies of chromosomes 4+10+17 or ETV6-RUNX1), no central nervous system or testicular leukemia, and rapid marrow response (<5% blasts by day (d) 15 and end-IND minimal residual disease (MRD) <0.1%). Given an expected EFS >90%, the study sought to improve outcome in this group without adding significant toxicity. Published data had shown that hyperdiploid B-ALL blasts and those with the ETV6-RUNX1 fusion have enhanced in vitro ASNase sensitivity, and ASNase intensification had been effective and well tolerated in several pediatric trials. Thus, AALL0331 randomized SR-Low patients (pts) to standard post-IND therapy with or without 4 additional doses of PEG-ASNase given at 3-week intervals during consolidation (CON) and interim maintenance (IM). We now report the efficacy results for this group.Methods: AALL0331 enrolled 5377 SR-ALL pts from 4/2005-5/2010. All pts received standard IND (vincristine (VCR), dexamethasone (DEX), PEG-ASNase, intrathecal methotrexate (IT MTX)). At end-IND, 1857 pts meeting SR-Low criteria were randomized to one of two regimens, Low Risk Standard (LRS) or Low Risk ASNase (LRA), with identical CON (mercaptopurine (MP) 75 mg/M2 d1-28, VCR 1.5 mg/M2d1, IT MTX d1,8,15) and IM (DEX d1-5,29-33, MP d1-50, oral MTX weekly x8, IT MTX d29) phases except for additional PEG-ASNase (2500 IU/M2/dose) in LRA (CON d1,22 and IM d15,36). Subsequent delayed intensification (DI) and maintenance (MTC) phases were identical. After 6/2008, based on CCG-1991 SR-ALL efficacy analyses, the IM backbone was changed to escalating dose intravenous (IV) MTX (VCR d1,11,21,31,41, MTX d1,11,21,31,41, IT MTX d31) in both regimens (LRS-IV and LRA-IV).Results: The 5-yr continuous complete remission (CCR) and OS rates (SE) for SR-Low pts (n=1857) were 95.2% (0.6) and 98.8% (0.3). Consistent with the results of CCG-1991, the 3-yr EFS was numerically higher with IV MTX (99.0% (0.4) vs 97.0% (0.5), p=0.16) but the difference did not reach statistical significance. PEG-ASNase intensification did not significantly improve outcome, with 5-yr CCR rates for LRA/LRA-IV vs LRS/LRS-IV of 96.0% (0.8) vs 94.4% (1.0) (p=0.1), and 5-yr OS rates of 98.3% (0.6) vs 99.3% (0.4) (p=0.05). Comparing pts randomized pre/post the IV MTX amendment, no advantage to PEG-ASNase intensification was observed with either the oral (5-yr EFS: LRA 96.0% (0.8) vs LRS 93.8% (1.0), p=0.2) or the IV MTX based IM regimens (3-yr EFS: LRA-IV 99.0% (0.6) vs LRS-IV 99.0% (0.6), p=0.9). Using more current COG MRD definitions for low risk ALL (d8 peripheral blood MRD <1% and d29 marrow MRD <0.01%), the 5-yr EFS/OS for SR-Low patients were 96.4% and 98.8%. Treatment-related adverse events were uncommon but occurred more frequently with PEG intensification. As of 12/31/2013 there were 72 relapses, 13 deaths in remission, and 4 second malignancies.Conclusions: Data from AALL0331 show that, using sentinel genetic lesions and MRD response, about 35% of SR B-ALL patients meet LR criteria and are almost certain to be cured using a low intensity regimen comprised of a 3-drug IND and post-IND therapy without intensive consolidation or high dose MTX, an approach that limits the cumulative doses of anthracyclines (75 mg/m2) and alkylating agents (1000 mg/m2). DisclosuresBorowitz:Becton Dickinson Biosciences: Research Funding. Hunger:Sigma Tau Pharmaceuticals: Honoraria; Jazz Pharmaceuticals: Honoraria.

Supportive care in patients with acute leukaemia: historical perspectives.

Although most treatments for leukaemia were ineffective until the middle of the 20th century, it seemed of interest to review some pertinent examples of the evolution of supportive care from its origin to its current use in this disease. Indeed, patients with leukaemia often have associated life-threatening disorders such as severe anaemia, bleeding or serious infections, and improvements in the management of these complications has totally transformed the outcome of the underlying disease. Modern therapy of acute leukaemia began in the 1960s. The major development in the treatment of acute leukaemia came from St. Jude Children’s Research Hospital in Memphis, with Don Pinkel introducing a four-phase treatment plan for acute lymphoblastic leukaemia, which he called “total therapy”1. The principles that were applied took into account the advantage of combination chemotherapy in overcoming initial drug resistance and inhibiting acquired resistance, as well as the superiority of some drugs for inducing remissions and others for maintaining the remissions. All available antileukaemic agents were incorporated into the plan, including vincristine and prednisone as induction therapy, 6-mercaptopurine and methotrexate for maintenance chemotherapy, and then radiotherapy to the central nervous system to prevent meningeal relapse. This schedule resulted in a tremendous increase in the survival rate of children with acute lymphoblastic leukaemia with more than 50% becoming long-term survivors. Concomitantly to the development of more intensive chemotherapies, the need for supportive care increased. Leukaemia and its treatment can cause a number of complications and side effects. Patients receive supportive care to prevent or control these problems and to improve their comfort and quality of life during the leukaemia treatment. Advances in the control of infections and bleeding have been major factors in the improved outcome of leukaemia therapy. Blood transfusions are an important part of haematological care: transfusions of red blood cells may be used to reduce the symptoms of anaemia and platelet transfusions to reduce the risk of serious bleeding. Transfusions have long been associated with some risk to patients. Blood transfusion technology advanced rapidly during World War II, leading to national systems of blood banks. Prior to 1954, blood transfusions were performed with rubber tubes and glass bottles. Progress in technologies and materials led to significant improvements. The development of plastic phlebotomy equipment has made the procedure of plasmapheresis feasible as an alternate form of blood donation. A platelet replacement programme initiated in children with leukaemia in 1958 demonstrated that platelets had an effect on haemorrhages and mortality. In this brief historical review, we examine the major advances in the history of blood transfusion and the impact of transfusions on the treatment of acute leukaemia. Targeted more specifically at the young haematologist and the general educated reader, this review aims to communicate accounts of scientific and medical discovery and practice in this domain. It relies upon chronology as an organising framework, while stressing the importance of themes.

Enhancing the Solubility of 6-Mercaptopurine by Formation of Ionic Cocrystal with Zinc Trifluoromethanesulfonate: Single-Crystal-to-Single-Crystal Transformation

An antitumor drug, 6-mercaptopurine monohydrate (MP·H2O), has a low oral bioavailability due to its poor aqueous solubility. In order to improve its solubility, two ionic cocrystals of 6-mercaptopurine with zinc trifluoromethanesulfonate (Zn(CF3SO3)2), [Zn(MP)2(H2O)2](CF3SO3)2·2H2O (1) and [Zn(MP)2](CF3SO3)2 (2), were prepared. Slow evaporation of the methanol solution containing 1 gave the crystals of [Zn(MP)2(H2O)(MeOH)](CF3SO3)2·H2O (1a). Single-crystal-to-single-crystal transformation occurred under certain conditions, in which 1a transformed to 1 in the open air around 10 °C, and 1 further transformed to [Zn(MP)2(H2O)](CF3SO3)2 (1b) at room temperature (∼25 °C) and low RH%. The structures of 1, 1a, and 1b were determined by single crystal X-ray diffraction, in which MP and Zn(CF3SO3)2 were assembled via coordination bonds and hydrogen bonds, and the coordination geometry of Zn(II) changed from octahedron in 1 and 1a to square pyramid in 1b. After the formation of ionic cocrystals of 1 and 2, both the...

P-107: Effect of exclusive enteral nutrition on the course of CD and intestinal microbiota

P-107: Effect of exclusive enteral nutrition on the course of CD and intestinal microbiota

P-106: The clinical relevance of tacrolimus for ulcerative colitis in children: a national survey of the treatments for pediatric inflammatory bowel disease in Japan

P-106: The clinical relevance of tacrolimus for ulcerative colitis in children: a national survey of the treatments for pediatric inflammatory bowel disease in Japan

PWE-100 Increasing Weight And Body Mass Index Adversely Affect Thioguanine Nucleotide Levels In Inflammatory Bowel Disease

IntroductionInflammatory bowel disease (IBD) often requires long term immunosuppressive therapy with thiopurines such as azathioprine (AZA) or mercaptopurine (MP) and anti-tumour necrosis factor (TNF) agents. Despite the variable response to...